Stable Isotopes in Pharmaceutical Research, Volume 26 (Pharmacochemistry Library)

PREFACE

Stable isotope techniques offer advantages in safety, sensitivity, specificity and economy for many types of pharmaceutical investigations when compared with conventional techniques. Nevertheless, pharmaceutical researchers have been slow to embrace stable isotope techniques. This book was written in the hope that putting together in one place comprehensive reviews of the many applications of stable isotopes, and the background material necessary to understand the applications, will lead to increased usage of stable isotopes in pharmaceutical research. The chapters in this book were contributed by a very talented and diverse group of investigators. The editor wishes to thank these investigators for their excellent contributions. THOMAS R. BROWNE, M.D.

CHAPTER 1

STABLE ISOTOPES: ORIGINS AND SAFETY

PETER D. KLEIN and E. ROSELAND KLEIN Meretek Diagnostics, Inc., Medical Towers Building, 1709 Dryden Road, Suite 1513, Houston, Texas

1. THE DISCOVERY OF STABLE ISOTOPES Approximately 80 years ago, Fredrick Soddy was studying the chemistry of mesothorium and, after a review of the known methods of preparation, concluded that thorium X, mesothorium, and radium formed a chemically inseparable trio. He questioned, " . . . whether some of the common elements may not in reality be a mixture of chemically nonseparable elements in constant proportions differing step-wise by whole units in atomic weight, which would account for the lack of regular relationships between the numerical values of the atomic weights" (1). To this property of multiple radioactive preparations, he gave the name "isotope" to signify their occupancy of the same place in the table of atomic numbers. Three years later, J.J. Thomson examined the positive rays of atmospheric gases and found evidence for two forms of neon at masses 20 and 22. The value accepted at that time for the atomic weight of neon was 20.2, and the inability of Thomson's equipment to achieve unequivocal resolution of mass 20.0 from 20.2 left his postulate, that neon was a mixture of two isotopes, unproved. It was not until after World War I that Aston reported, in a one-paragraph letter to Nature, the finding of two isotopes of neon with masses at 20.0 and 22.0 (determined with a precision of 0.1) (2); within the next year, his manuscript was published (3). He then designed and constructed a new mass spectrometer and embarked on the espousal of the whole number rule. Aston (4) reported that each of the isotopes of lithium, boron, sulfur, chlorine, argon, krypton, tin, xenon, and mercury had a mass number that differed by less than 0.07 from a whole number. This report contained the first account

of an element with a stable isotope (S) relevant to biologic studies. Perhaps even more memorable was Aston's description of his mass spectrometer, "It behaves at times in the most capricious and unaccountable manner.., when by good fortune, all is well, the arrangement is capable of good performances. Thus after a favorable setting of the apparatus, six elements were successfully analyzed in as many working days. On the other hand, after dismantling became imperative and it had to be cleaned and rebuilt, exactly as before as far as any one could tell, no results of any value were obtained during weeks of work" (5). There followed an extensive search among the elements of the atomic table for the existence of other isotopes. Giauque and Johnstone (6), who had examined the atmospheric bands of oxygen, reported in 1929 the finding of an oxygen isotope with an atomic mass of 18; this discovery was followed by the report of a second isotope with a mass of 17 (7). During the same year, studies of carbon arc spectra obtained in a vacuum oven by King and Birge (8, 9) showed the existence of a 13C species. Within that year, Naude measured the absorption spectrum of nitrous oxide in the ultraviolet region and confirmed not only the existence of the oxygen isotopes, but also the presence of an isotope of nitrogen with a mass of 15 (10, 11). There remained a discrepancy between the atomic weight of hydrogen as determined by chemical means (1.00777) and that determined by mass spectroscopy (1.00756). Birge and Menzel (12) postulated that an isotope of hydrogen with an abundance of 1 part in 4,500 parts hydrogen would account for this difference. On the basis of this possibility, Urey et al. (13) were inspired in 1932 to make a limited number of assumptions about the spectral properties of such an isotope and sought evidence for its existence in hydrogen spectra. A line that corresponded to the putative isotope was found only after over-exposure of the principal lines by some 4,000-fold. Convincing proof was obtained when they evaporated 6 L liquid hydrogen and collected the last 2-3 mL as gas; the intensity of the isotopic spectra was increased 5fold, and the final significant gap in the atomic table was closed.

2. THE ENRICHMENT OF STABLE ISOTOPES Little or no attempt was made to obtain enriched fractions of stable isotopes until after the discovery of deuterium. In 1933, Lewis (of the legendary Lewis and Randall, authors of the classic treatise, Thermodynamics) reported that he had achieved an enrichment of heavy water to 35 percent through a series of electrolytic dissociations. In his letter to the Journal of the American

Chemical Society, he concluded, "The separation of any isotope in sufficient quantity to permit investigation.., suggests a wide range of interesting experiments but the isotope of hydrogen is, above all others, interesting to chemists. I believe that it will be so different from common hydrogen that it will be regarded almost as a new element. If this is true the organic chemistry of compounds containing the heavy isotope of hydrogen will be a fascinating study" (14). Later that year, Lewis and MacDonald (15) reported that they had produced 0.3 mL of pure D20 and described its physical properties including density, freezing and boiling points. They concluded, "In the various ways in which water is said to be an abnormal liquid, H2H20 seems to be more abnormal, but the differences between the two become smaller with rising temperature". For many years, deuterium production was based on the electrolysis of water with recombination of the hydrogen and oxygen through combustion. Beginning with the solution from commercial electrolysis cells with an enrichment of 0.005 percent, Urey (16) found that through five stages the enrichments were 1, 2.2, 6.5, 16-20 and 40 percent, respectively. He quoted a price of $15 to $20 per gram for the enriched samples, which in those days prohibited studies that required large volumes at high enrichments. As we shall see, many of the earliest studies were undertaken with enrichments of 2-5 percent. Not until the H2S:H20 dual exchange system was developed in the late 1950s did the price of deuterium drop to $0.20 per gram, thus enabling large-scale studies to be undertaken (17). Urey and coworkers (18) developed fundamental calculations underlying the enrichment process through exchange equilibria in 1935, and then reported in quick succession on ~5N enrichment (19), ~80 enrichment (20, 21) and ~3C preparation (22). Six years later, Nier and Bardeen (23) reported that they had achieved an enrichment of ~3C-methane to 11 percent using a thermal diffusion process set up in the stairwell of their building. Through Nier's design of a gas isotope ratio mass spectrometer (24) and his collaborations with Wood, ~3C was to enable new insights into carbon fixation and the Krebs cycle.

3. THE APPLICATION OF STABLE ISOTOPES AS TRACERS

The development of biochemistry in the late 1930s can be experienced by reading the review articles of Schoenheimer (25), Schoenheimer and Rittenberg (26), and the 1948 recollections of Clarke (27). It was a time of discovery, new concepts and rapid growth. As recounted by Clarke, Urey prepared a quantity of enriched heavy water after which he was awarded a grant from

the Rockefeller Foundation to develop applications for biologic processes. A portion of the grant was designated for the salary of a specialist to be trained to work with heavy water. That person was David Rittenberg, who was commissioned to find interesting biologic applications. He soon encountered Rudolph Schoenheimer, and the two established a formidable alliance. Their work began with a simple synthesis: they hydrogenated linseed oil to produce a deuterium-containing fat to administer to mice. On the basis of the limited amount of food fed to the animals, they expected the deuterium to be liberated promptly by oxidation of the fat to C02 and water. (Their analysis of deuterium-containing fluids was based on a densitometric method and could detect 0.001 percent enrichment.) They were totally surprised by the outcome of the study: first, the total amount of deuterium did not appear in the urine, and second, one-half of the label was recovered from fat depots. In a series of succinct tables, they reported the principles of fatty acid synthesis, storage, oxidation, desaturation, saturation and chain lengthening and shortening. They predicted early in their work that cholesterol was assembled from many units that were two carbons in length. Soon thereafter, the conversion of phenylalanine to tyrosine was demonstrated in the rat, even in the presence of large amounts of dietary tyrosine. With the availability of ~SN in the late 1930s, Schoenheimer et al. (28) studied amino acid and protein metabolism and rapidly demonstrated the incorporation of dietary ammonium citrate into amino acids of body proteins, with the exception of lysine. Moreover, there was interconversion between nitrogen-containing amino acids, such as glycine and the protein amino acids, because the latter were found to contain ~SN when hydrolyzed. When they studied the metabolism of leucine, containing a deuterium-labeled chain and an ~SN amino group, they discovered that leucine isolated from protein had lost 35 percent of its ~SN and that this nitrogen was now found in many other amino acids. They commented on the distinction drawn between exogenous and endogenous nitrogen metabolism, "It is scarcely possible to reconcile our finding with any theory which requires a distinction between these two types of nitrogen. It has been shown that nitrogenous groupings of tissue protein are constantly involved in chemical reactions; peptide linkages open, the amino acids liberated mix with others of the same species of whatever source, diet or tissue. This mixture.., while in the free state takes part in a variety of chemical reactions: some reenter directly into vacant positions left open by the rupture of peptide linkages, other transfer their nitrogen to deaminated molecules to form new amino acids...". The use of ~3C in biologic studies appears to have proceeded at a more measured pace than that of deuterium. The incorporation of ~3C into succinic,

pyruvic and formic acids by bacterial fixation was demonstrated by Nier and coworkers in 1941 (29) and led the way for subsequent demonstrations of the fixation of CO2 during photosynthesis. Olsen et al. (30) also spent considerable effort to synthesize ~3C-glycine, measure its rate of catabolism in the rat, and show the conversion of glycine to glycogen. An examination of Nier's bibliography (31) shows work with a consistent thread of biochemical and clinical applications. Some years ago he described how his entry into stable isotope studies was fortuitous; he and his coworkers just happened to have a ~3C-methane column operating in the stairwell, and they built the first ~3C mass spectrometer. The Iongtime collaboration between Nier and Wood was the result of a chance encounter when Wood visited his brother who was studying medicine at the Mayo Clinic. The eventual exploitation of the stable isotope ~80 to measure CO2 production in free-living subjects, and hence their energy expenditure, was brought about by the collaboration of Nier with Lifson from the Department of Physiology at the University of Minnesota (32). Together they showed that the oxygen of respiratory carbon dioxide was in isotopic equilibrium with the oxygen of body water and that the ~80 content of CO2 could be predicted from the rate of oxygen consumption and the weight of total body water.

4. THE BIOLOGIC CONSEQUENCES OF ENRICHED STABLE ISOTOPE USAGE

The biologic effects of stable isotopic substitution in enzymatic, cellular or physiologic processes can be subdivided into two categories: those that involve deuterium and those that involve all other elements found in an organism. Because of the large mass difference between deuterium and hydrogen, there is a corresponding effect on the chemical-bond reactivity. This same difference helps in the concentration and enrichment of deuterium in virtually limitless quantities. These quantities have enabled extensive investigation of the enrichment level required for toxicologic manifestations. However, the mass differences for isotopes of higher elements are much smaller; their physical properties are more similar, and thus, the enrichment of such isotopes becomes much more expensive. Moreover, the quantities necessary to investigate high levels of exposure become cost-prohibitive. Therefore, toxicity studies of the stable isotopes of biologic interest were undertaken in inverse order of their discovery (~80, discovered in 1929, toxicity determined in 1975; ~3C, discovered in 1929, toxicity determined in 1973; 2H, discovered in 1932 and toxicity determined in 1933). Thus, it was within the same year that deuterium was discovered, that the first studies of its biologic effects started. The zeal with which Lewis pursued this problem is evident in

his letter to the Journal of the American Chemical Society: "Even before I had succeeded in concentrating the isotope of hydrogen, I predicted that H=H=O would not support life and would be lethal to higher organisms. As soon as heavy water became available, experiments to test this idea were begun but it was necessary to choose an experiment which would require the minimum of biological techniques and also very small quantities of water" (33). Lewis chose to use tobacco seeds, germinated in sealed glass capillaries with 0.02 mL ordinary water or pure D20. Seeds in pure D20 did not sprout, but those in 50-percent D20 sprouted in normal time, thus giving the first LDso data for deuterium. Lewis concluded, "1 have long desired to determine the proportions of isotopes in living matter, in order to see whether the extraordinary selective power of living organisms, which is exemplified by their behavior toward optical isomers might lead to a segregation of isotopes in some of the substances that are necessary to growth. The marked biochemical differences between the two isotopes of hydrogen lends a further incentive to this search" (33). Taylor et al. (34) compared the toxicity of 92 percent and 30 percent heavy water in tadpoles, aquarium fish, flat worms and paramecia. All species succumbed in the highest concentration of deuterium within one to three hours, but survived in the 30-percent concentration. Lewis conducted the first mammalian study in which a mouse received 0.66 g pure D20 and showed evidence of thirst, but survived (35). Between 1934 and 1939, 216 papers were published on the biologic effects of deuterium. Most of the papers appeared within the first three years; the last seven were published in 1939 (36). Thus, after the classic studies were completed, it was not until the price of deuterium dropped from $20 to $0.20 per gram that interest was rekindled (see Table 1). Two papers of major importance that summarized the new work were by Katz, a review of chemical and biologic studies (37), and Thomson, a definitive monograph on the biologic effects of deuterium that discusses every consequent aspect of acute, chronic and low-level exposure to deuterium (36). The depth of study is much more attenuated for ~3C and is represented by several publications that resulted from work at the Los Alamos Scientific Laboratory. No evidence of toxicity was found at the highest enrichments attained (60 percent) in two mice (38), nor in mouse embryos cultured in media containing uniformly labeled ~3C-glucose as the sole energy source (39). More recently, Berthold et al. (40) fed a laying hen a ration which contained 50 percent Spirulina platensis grown in an atmosphere of pure ~3CO2. Over the course of 30 days, between 20 and 70 percent of all carcass amino

TABLE 1. Natural Stable Isotopic Content of the Human Body, Daily Consumption of Stable Isotopes, and Quantities of Stable Isotopes Used in Conventional Tracer Studies

Parameter

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acids were replaced with labeled forms. The hen laid 28 eggs during this time, and an analysis of the amino acids in the egg albumen showed two different and distinctive patterns (see Figure 1). Nonessential amino acids, exemplified by glutamine/glutamate, were catabolized and underwent extensive resynthesis. However, essential amino acids, such as phenylalanine, were incorporated intact and appeared as fully labeled isotopomers in the protein. This unexpected consequence of feeding fully ~3C-labeled nutrients has proved to be a valuable tool in identifying essential dietary components. To date, there has been no report of an investigation of whole organism response to 15N-enrichment levels. However, the physical chemistry of 180, has been well reported by Staschewski (41). Only because of the substantial resources of the Stable Isotope Department of the Weizmann Institute, however, was it possible for Samuel and colleagues (42-44) to raise three generations of mice in an atmosphere of 90 percent 1802 and to provide their drinking water as 90 percent H2180. No physiologic or biochemical effects were noted, and the mice reproduced normally through each generation without an increase in infant mortality. There is a large margin of safety in the use of stable isotopes in human studies. The enrichment of total body water with deuterium may be as high as 1 to 2 percent. In the cases of 13C, lSN or 180, the cost of substantial tissue isotope replacement is so prohibitive that research or diagnostic applications are unlikely to be undertaken. Nevertheless, it is useful to have an appreciation of the magnitude of an effect achieved with the introduction of a heavy isotope such as 13C. Biological processes which transform 13C-containing molecules, such as photosynthesis, discriminate slightly against the heavier (and less reactive) 13C in C02. The level of this discrimination can only be detected using the high precision of gas isotope ratio mass spectrometer

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measurements. For example, there are 11,117 molecules of ~3C02 in every 1,000,000 molecules of atmospheric C02. In the process of starch formation in a leaf, between 121 and 284 ~3C02 molecules are left behind for every 1,000,000 molecules of C02 used. The result is a product 1.09-2.55 percent lighter in ~3C than the starting material. Thus, biological systems tend to reject rather than retain the heavier form of isotope, but overall will reflect the isotopic composition of the diet. For example, Boutton et al. (45) studied cows whose diets were either switched from an alfalfa (with an isotopic abundance of -24%o) to a corn (-11%o vs. PDB) or vice versa. The milk produced by these cows quickly reflected the altered isotopic intake and reached equilibrium within 3-4 days. For these reasons, and the more important feature - absence of radiation - the use of these tracers in protected populations has been strongly favored by human investigation review committees. However, this perspective must be provided in an accurate manner to the subject or guardian from whom informed consent is required. The information in Table 1 may be used for this purpose. Listed is the natural abundance for each isotope, calculated as mg/kg of body weight, compared with normal daily intake, and with the amount used in most foreseeable studies. These values demonstrate the absence of any perturbation in body composition when stable isotopes are used. Stable isotopes offer a number of specific advantages to clinical pharmacologists, because they are ideally suited to answer the recurrent types of questions posed in studies. These applications have been the subject of several excellent reviews by Browne (46, 47) who has classified them according to the following types: use in isotope dilution techniques to measure the concentrations of drugs in biological fluids; determination of absorption, bioavailability, and distribution; biotransformation and excretion, including metabolite identification, mechanisms of drug metabolism, and quantitation of drug transformation and elimination. To these applications can be added the determination of compliance and the assessment of unwanted side effects of drugs. Examples of stable isotope applications in pediatric pharmacology are reviewed by Pons in this volume (48). When the applications of stable isotopes are reviewed today, their status remains true to the vision of Rudolph Schoenheimer when almost 60 years ago, he wrote, "The chemical constituents of the living body represent links in a chain of continuous reactions in which apparently all organic substances, even those of the storage material, are involved. It is with this aspect of the dynamic processes of life that the biochemist is especially concerned. The isotopes of those elements which are present in natural organic compounds,

10 presented to the biochemist by the physical chemist will certainly furnish a better insight into the details of this intricate m e c h a n i s m " (25).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

F. Soddy, J. Chem. Soc., 90 (1911) 72. F.W. Aston, Nature, 104 (1919) 334. F.W. Aston, Philos. Mag., 39 (1920) 449. F.W. Aston, Proc. Roy. Soc. Lond., Al15 (1927) 487. F.W. Aston, Mass Spectra and Isotopes, 2nd edn (Edward Arnold, London, 1924) p. 99. W.F. Giauque and H.L. Johnstone, Nature, 123 (1929) 318; J. Am. Chem. Soc., 51 (1929) 1436. W.F. Giauque and H.L. Johnstone, Nature, 123 (1929) 813; J. Am. Chem. Soc., 51 (1929) 3528. A.S. King and R.T. Birge, Nature, 124 (1929) 127; Phys. Rev., 34 (1929) 376. R.T. Birge, Nature, 124 (1929) 182; Phys. Rev., 34 (1929) 379. S.M.Naud~, Phys. Rev., 34 (1929) 1498. S.M. Naude, Phys. Rev., 36 (1930) 333. R.T. Birge and D.H. Menzel, Phys. Rev., 37 (1931) 1669. H.C. Urey, F.G. Brickwedde and G.M. Murphy, Phys. Rev., 40 (1932) 1. G.N. Lewis, J. Am. Chem. Soc., 55 (1933) 1297. G.N. Lewis and R.T. MacDonald, Chem. Phys., 1 (1933) 341. H.C. Urey, Science, 78 (1933) 566. G. Weiss, Chem. Eng. Tech., 30 (1958) 433. H.C. Urey and L.J. Greiff, J. Am. Chem. Soc., 57 (1935) 321. H.C. Urey and A.H. Aten Jr, Phys. Rev., 50 (1936) 575. J.R. Huffman and H.C. Urey, Ind. Eng. Chem., 29 (1937) 531. H.C. Urey, G.B. Pegram and J.R. Huffman, Phys. Rev., 4 (1936) 623. H.C. Urey, A.W.H. Aten Jr and A.S. Keston, Phys. Rev., 4 (1936) 622. A.O. Nier and J. Bardeen, J. Chem. Phys., 9 (1941) 690. A.O. Nier, Rev. Sci. Inst., 11 (1940) 212. R. Schoenheimer, Harvey Lectures XXXII (Academic Press, Amsterdam, 1936-37) p. 122. R. Schoenheimer and D. Rittenberg, Physiol. Rev., 20 (1940) 218. H.T. Clarke, A Symposium on the Use of Isotopes in Biology and Medicine (The University of Wisconsin Press, Madison Wl, 1948) p. 3. R. Schoenheimer, S. Ratner and D. Rittenberg, J. Biol. Chem., 130 (1939) 703. H.G. Wood, C.H. Werkman and A. Hemingway et al., J. Biol. Chem., 139 (1941) 365. N.S. Olsen, A. Hemingway and A.O. Nier, J. Biol. Chem., 148 (1943) 611. T.T. Scolman, W.H. Johnson and O.C. Alfred et al., Int. J. Mass. Spectrom. Ion. Phys., 8 (1972) 241. N. Lifson, G.B. Gordon, M.B. Visscher and A.O. Nier, J. Biol. Chem., 180 (1949) 803. G.N. Lewis, J. Am. Chem. Soc., 4 (1933) 3503. H.S. Taylor, W.W. Swingle and H. Eyring et al., J. Chem. Phys., 1 (1933) 751. G.N. Lewis, Science, 79 (1934) 151.

11 36. J. F. Thomson, Biological Effects of Deuterium, (Macmillan, New York, 1963) p. 133. 37. J.J. Katz, Am. Sci., 47 (1960) 544. 38. C.T. Gregg, J.Y. Hutson and J.R. Prine et al., Life Science, 13 (1973) 775. 39. C.T. Gregg, D. Ott and L. Deaver et al., Proceedings of the Second International Conference on Stable Isotopes, E.R. Klein and P.D. Klein (eds) (US Department of Commerce, Springfield, VA, CONF-751027 NTIS, 1975) p. 64. 40. H.K. Berthold, D.L. Hachey and P.J. Reeds et al., Proc. Nat. Acad. Sci., 88 (1991) 8091. 41. D. Staschewski, Agnew Chem., 13 (1974) 367. 42. D. Samuel, Proceedings of the Second International Conference on Stable Isotopes, E.R. Klein and P.D. Klein (eds) (US Department of Commerce, Springfield, VA, CONF-751027 NTIS 1975) p. 196. 43. D. Samuel, D. Wolf and A. Meshorer et al., Proceedings of the Second International Conference on Stable Isotopes, E.R. Klein and P.D. Klein (eds) (US Department of Commerce, Springfield, VA, CONF-751027 NTIS 1975) 203. 44. D. Wolf, H. Cohen and A. Meshorer et al., Stable Isotopes: Proceedings of the Third International Conference, E.R. Klein and P.D. Klein (eds) (Academic Press, New York, 1979) p. 360. 45. T.W. Boutton, H.F. Tyrell and B.W. Patterson et al., J. Anim. Sci., 66 (1988) 2636. 46. T. R. Browne, J. Clin. Pharmacol., 26 (1986)485. 47. T. R. Browne, Clin. Pharmacokinet., 18 (1990) 423. 48. G. Pons and E. Rey, Stable Isotopes in Pharmaceutical Research, T.R. Browne (ed) (Elsevier Science Amsterdam, 1996) p. 347.

13

CHAPTER 2

ISOTOPE EFFECT: IMPLICATIONS FOR PHARMACEUTICAL INVESTIGATIONS

THOMAS R. BROWNE Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center

1. INTRODUCTION

Any bond involving a heavy isotope and another atom will be stronger than the same bond between the corresponding light isotope and that atom. The greater mass of the heavy isotope results in greater force bonding it to the other atom and a greater energy of activation to break the bond (see Figure 1). In any reaction in which the breaking of this bond is the rate-limiting step, the reaction will proceed slower for the molecule with the heavy isotope due to this "kinetic isotope effect". In addition to kinetic isotope effects there are other ("secondary")isotope effects due to differences in bond length, bond angle, etc., of bonds involving heavy and light isotopes.

2. KINETIC ISOTOPE EFFECT

The physical chemistry of kinetic isotope effect on simple chemical reactions is reviewed in references 1-6. Kinetic isotope effects are modified during enzymatically mediated reactions following a set of rules reviewed in Chapter 15. This section reviews those aspects of the huge theoretical and empirical literature on kinetic isotope effect relevant to the design and performance of pharmaceutical investigations. Kinetic isotope effect is proportional to the mass difference between different isotopes of the same atom. Substitution of deuterium for hydrogen (100 percent mass difference) results in larger isotope effects than substitution of 13C for 12C, or lSN for ~4N (<10 percent difference). A simple chemical reaction

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involving breaking a CmD bond can be 700 percent or more slower than a similar reaction involving breaking a CmH bond (1). However, for enzymatically catalyzed reactions the slowing is usually less than 200 percent and sometimes not detectable for reasons reviewed in Chapter 15. Cleavage of a ~3C~H bond can, at most, be 2.2 percent slower than a ~2C~H (1). Review of the in vitro and in vivo literature on the effect of kinetic isotope effect on drug metabolism leads to the following five rules for stable isotopelabeled drugs: (1) Incorporation of stable isotopes, including deuterium, at sites not involved in the rate-limiting step of a metabolic reaction does not lead to a kinetic isotope effect (7-13). (2) Deuterium placed at a carbon atom involved in the rate-limiting step of a metabolic reaction is likely to lead to a kinetic isotope effect if the carbon is aliphatic (14-26).

15 (3) Deuterium attached to an aromatic carbon atom which is the site of hydroxylation seldom, if ever, leads to an observable kinetic isotope effect in vivo (26-31). (4) If ~3C, ~SN, or another heavy isotope differing from the light one by less than 10 percent in mass is incorporated at the rate-limiting site of metabolism, there may be a small kinetic isotope effect (3 percent or less) which is of the same magnitude as that generally associated with the precision of the measurement and, thus, is barely detectable (7, 12, 32, 33). (5) Potential kinetic isotope effects at minor metabolic pathways can be ignored. Even relatively large deuterium isotope effects at minor metabolic pathways have little effect on total drug clearance (see Chapter 15). Note that using the above rules it is possible to design stable isotopelabeled analogues of drugs whose rate of metabolism will not differ from unlabeled analogues. This is important for most pharmacokinetic studies where the pharmacokinetic values for labeled drugs are assumed to represent the values for unlabeled drugs. On the other hand, the slowing of metabolism of deuterium-labeled drug analogues can be a powerful tool in determining the molecular site(s) and mechanism(s) of a drug's metabolism. This topic is reviewed in detail in Chapter 15. Finally, the potential errors caused by kinetic isotope effect need to be compared with the other potential errors caused by other sources of error in pharmaceutical studies. Analytic errors in the determination of concentrations of stable isotope-labeled drugs in b~dy fluids by gas chromatography (or liquid chromatography) mass spectrometry are generally on the order _+5 percent. The coefficients of variation for multiple determinations of the same pharmacokinetic parameters within, and between, human subjects have not been studied extensively but are probably in the range of 5 to 60 percent (34).

3. OTHER ("SECONDARY")ISOTOPE EFFECTS

The greater strength of bonds involving heavy isotopes can change a number of properties of drug molecules, including bond length, bond angle, polarity, molar volume, electron donation, Van der Walls forces, dipole moment, lipophilicity, enzyme binding kinetics, protein binding and mass spectrometric fragmentation pattern (6, 19, 23, 35-37). These effects are detected principally for deuterium-labeled drugs and are proportional to the number of deuterium labels. At the present time, their pharmacokinetic significance is not completely understood and is an active topic of research. In general, these effects

16 are small and ignored in the design and analysis of clinical pharmacokinetic studies. Differences in physical properties between deuterium-labeled and unlabeled analogues of a drug sometimes allow them to be separated and quantified separately by gas liquid chromatography or high-performance liquid chromatography. This topic is reviewed in Chapter 7.

4. TESTING FOR KINETIC ISOTOPE EFFECT

Because of concerns that kinetic isotope effects may result in different pharmacokinetic values for labeled and unlabeled drug analogues, it has been customary to test stable isotope-labeled drug analogues for kinetic isotope effect in human volunteers. The presence, or absence, of kinetic isotope effects on the pharmacokinetic parameters of labeled drugs can be determined by simultaneous administration of the labeled and the unlabeled forms of the drug and simultaneous measurement of the pharmacokinetic

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17 parameters to be studied (see e.g. references 8-14, 27, 30, 33) (see also Figure

2). At this point in time, there is overwhelming data verifying the five rules listed in Section 2 for kinetic isotope effect. An in vivo test for kinetic isotope effect is not necessary if stable isotope labels are placed at sites not involved in major metabolic reactions or if 13C or lSN labeling is used. An in vivo test for kinetic isotope effect probably is indicated if deuterium is placed on an aliphatic carbon atom involved in the rate-limiting step of a major metabolic reaction. Such a test probably is not indicated if deuterium is placed on an aromatic carbon atom which is the site of metabolic hydroxylation. Potential isotope effects on minor metabolic pathways (and also potential secondary isotope effects) do not require an in vivo study.

ACKNOWLEDGEMENT Supported by the United States Department of Veterans Affairs.

REFERENCES 1. L. Melander and W.H. Saunders, Reaction Rates of Isotopic Molecules (WileyInterscience, New York, 1980). 2. F.H. Westheimer, Chem. Rev., 61 (1961) 265. 3. R.B. Wiberg, Chem. Rev., 55 (1955) 713. 4. P. Paneth, Comput. Chem., 19 (1995) 11. 5. P. Paneth, Comput. Chem., 19 (1995) 231. 6. A. Van Langenhove, J. Clin. Pharmacol., 26 (1986) 383. 7. T.R. Browne, C.E. Van Langenhove and C.E. Costello et al., Clin. Pharmacol. Ther., 29 (1981)511. 8. M.A. Goldberg, J. Gal and A.K. Cho et al., Ann. Neurol., 5 (1979) 121. 9. R.E. McMahon and H.R. Sullivan, Res. Commun. Chem. Pathol. Pharmacol., 14 (1976) 631. 10. J. Osterloh and L. Bertilsson, Life Science, 23 (1978) 83. 11. G.E. Von Unruh, B.C. Jancik and F. Hoffman, Biomed. Mass. Spectrum, 7 (1980) 164. 12. G.E. Von Unruh, K.M. Koth and M. Eichelbaum, in Proceedings of the Third International Conference on Stable Isotopes (Academic Press, New York, 1979). 13. R.L. Wolen, B.D. Obermeyer and E.A. Ziese et al., in T.A. Baille (ed), Stable Isotopes: Applications in Pharmacology, Toxicity and Clinical Research (University Park Press, London, 1978). 14. G.H. Draffan, in T.A. Baille (ed), Stable Isotopes: Applications in Pharmacology, Toxicity and Clinical Research (University Park Press, London, 1978). 15. C. Elison, H.M. Elliott and M. Look et al., J. Med. Chem., 6 (1963) 237. 16. C. Elison, H. Rapoport and R. Laursen et al., Science, 134 (1961) 1078. 17. A.B. Foster, M. Jarmon and J.D. Stevens et al., Chemico-Biol. Interact., 9 (1974) 327.

18 18. W.A. Garland, S.D. Nelson and H.A. Susame, Biochem. Biophys. Res. Commun., 72 (1976)539. 19. M.G. Horning, J. Nowlin and J.-P. Boussma et al., in Proceedings of the Third International Conference on Stable Isotopes (Academic Press, New York, 1979). 20. J.P. Jones, K.R. Korzekwa and A.E. Reftie et al., J. Am. Chem. Soc., 108 (1986) 7074; and correction J. Am. Chem. Soc., 110 (1980) 2018. 21. A. Mural, C. Chen and R.H.K. Kishimoto, J. Biol. Chem., 252 (1977) 352. 22. S.D. Nelson, L. R. Pohl and W.F. Trager, J. Med. Chem., 18 (1975) 1082. 23. C.J. Parli and R.E. McMahon, Drug Metab. Dispo., 1 (1973) 337. 24. K. Sugiyami and W.F. Trager, Biochemistry, 25 (1986) 7336. 25. M. Tanale, D. Yasuda and S. Levalley et al., Life Science, 8 (1969) 1123. 26. I.W. Taylor, C. Ionnides and P. Sarva et al., Biochem. Pharmacol., 32 (1983) 641. 27. T.R. Browne, G.K. Szabo and H. Davoudi et al., Neurology, 44 (1994) 2410. 28. J.P. Dinnocenzo, S.B. Karki and J.P. Jones, J. Am. Chem. Soc., 115 (1993) 7111. 29. K. Mamada, Drug Metabol. Dispo., 14 (1986) 509. 30. J.M. Perel, P.G. Dayton and C.L. Tauriello et al., J. Med. Chem., 10 (1967) 371. 31. J.E. Tomaszewski, D.M. Jerina and J.W. Daly, Biochemistry, 14 (1975) 2024. 32. T.R. Browne, A. Van Langenhove and C.E. Costello et al., J. Clin. Pharmacol., 22 (1982) 309. 33. J.R. Curlin, R.N. Walker and R.O. Davies et al., J. Pharm. Sci., 69 (1980) 1111. 34. A.A. Alvares, A. Kappas and J.L. Eiseman et al., Clin. Pharmacol. Ther., 26 (1979) 407. 35. W.W. Cleland, M.J. O'Leary and D.B. Northrop, in Proceedings of the Sixth Annual Harry Steenboch Symposium (University Park Press, London, 1976). 36. J.L. Brazier, in T.A. Baille (ed), Synthesis and Applications of Isotopically Labeled Compounds (Elsevier, Amsterdam, 1989). 37. J.B. Falconner, J.L. Brazier and B. Tests et al., in T.A. Baille (ed), Synthesis and Applications of Isotopically Labeled Compounds (Elsevier, Amsterdam, 1989).

19

CHAPTER 3

FUNDAMENTALS OF MASS SPECTROMETRY

DAVID A. WILLIAMS Massachusetts College of Pharmacy and Allied Health Sciences, Boston, MA 02115

1. INTRODUCTION

1.1. Overview of Mass Spectroscopy (1, 2) A mass spectrometer (MS) produces charged molecules (ions) and ion fragments and separates these the ions according to their mass to charge ratio. From this data one can infer the analyte's molecular weight and structure. Figure 1 shows a schematic representation of the major components of the MS including the inlet system, ion source, mass analyzer, detector, signal processor and vacuum system.

1. I. 1. Inlet system

The inlet system allows for the introduction of the analyte into the MS without disrupting the high vacuum existing within the MS. In biomedical mass spectrometry, it is necessary to inlet compounds of widely varying physical characteristics: gases, liquids, solids, low or high molecular-weight compounds, thermally stable or labile compounds, ionic and polar (difficult to volatilize) and nonpolar (easier to volatilize) compounds. To accomplish this, several types of inlet systems have been developed. The reservoir inlet permits volatile and thermally stable liquids and gases to be bled into the ion source from a vessel of higher pressure. A heated direct insertion probe is used to insert less volatile solid and liquid analytes directly into the ionization chamber for vaporization. The use of a separation system such as the gas or liquid chromatograph

20

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Figure 1. Schematic of the mass spectrometer. or capillary electrophoresis as the inlet to the MS allows for the mass analysis of complex mixtures of analytes, extending the utility of mass spectrometry for solving biomedical problems. Although studies on drug metabolism, pharmacokinetics, and drug monitoring have been traditionally conducted with a gas chromatograph coupled to a mass spectrometer (GC-MS), a MS interfaced with a liquid chromatograph (LC-MS) has become a mature method for the direct (underivatized) analysis of nonvolatile polar or ionic substances and high molecular weight biomolecules (1). Recent advances in the development of inlet systems that ionize these nonvolatile biomolecules without the need for vaporization prior to ionization have extended the range of compounds that can be analyzed with a MS. 1.1.2. The ion source

The ion source is the heart of the MS and where the analyte is converted into energized ions by bombardment with a high-energy electron beam (electron ionization, El), by interaction with other ions (chemical ionization, CI), by bombardment with a beam of high energy atoms (fast atom bombardment, FAB), by bombardment with a beam of high energy laser light (matrix-assisted laser desorption ionization, MALDI), or by the production of gas phase ions from ions in solution (thermal spray, TS; electrospray, ES; atmospheric pressure ionization, API). The charged molecular ion (charged analyte molecule

21 or parent ion) is unstable, breaking the weaker covalent bonds to form product ions (fragment or daughter ions). The generated ions are drawn into the mass analyzer by an electrical field. The molecular weight of the analyte is inferred from the mass of the molecular ion, and the structure and identity of the analyte is inferred from the pattern of fragment ions (see below). 1.1.3. The mass analyzer The mass analyzer separates the molecular and fragment ions according to their mass-to-charge (m/z) ratio (typically the molecular weight of the fragment if singly charged). The most commonly used mass analyzers are: (1) the magnetic sector analyzer which disperses all of the ions as a function of their m/z ratio in a magnetic field, (2) the quadrupole mass filter analyzer which transmits ions from the ion source within a small range of m/z ratios along an unstable oscillating electrical field, or (3) the ion trap mass analyzer which stores the ions from the ion source within a ring electrode and selectively ejects them from an unstable oscillating electrical field. 1.1.4. The detector The detector converts the beam of ions exiting from the mass analyzer into an electrical signal that can be measured, amplified and recorded by a recording device or digitalized by an analog to digital converter. The electron multiplier is a commonly used device for amplifying the signal because of its rapid response time. The array detector is an array of sensing elements similar to the electron multiplier, which can detect a range of ions as they fall upon the sensors, as opposed to the serial transmission of ions (one after the other) for the electron multiplier. 1.1.5. The signal processor~data system The signal processor is a computer which rapidly collects, digitizes, normalizes and processes ion current data from the detector along with background substraction. The signal processor converts the output into a mass spectrum and the key data for mass spectrometric analysis. The mass spectrum is a plot of the intensity of ions at each m/z value within a range of m/z values (see Figure 3-9). If the molecular ions are sufficiently stable to reach the detector, a molecular ion peak can be observed. Its recognition is important because it usually represents the molecular ion, from which the analyte's molecular weight is determined. The ions with smaller

22 m/z values represent fragment ions. The m/z values of fragment ions can be used to make inferences about the structure of the fragment and the analyte. The overall pattern of molecular and fragment ions (the mass spectrum) is unique for each analyte. The identity of an analyte can be determined by manual or computerized comparison of the analyte's mass spectrum with spectra contained in libraries. The interpretation of the mass spectrum is reviewed in more detail at the end of this chapter. The signal processor/data system computer stores the collected data in a spectral library. The computer also functions to control and monitor instrumentation values (source temperature, scan rate, voltages, etc.) for consistency of data collection. 1.1.6. The vacuum system The vacuum system maintains a very low pressure within the instrument, which minimizes collisions between the ions, neutral particles, and the walls of the instrument, allowing the ions to move through the instrument in a well-defined path. 1.2. Stable Isotopes and Mass Spectrometry Compounds containing stable isotope labels behave the same as their unlabeled analogues during mass spectroscopy, except that the m/z values of the ions derived from stable isotope labeled compounds will be higher (by the number of added neutrons) in the measured mass spectrum. This property has lead to three uses for stable isotopes. First, stable isotopes are used as internal standards for quantitative analysis by mass spectroscopy. Second, stable isotopes are used as markers to help identify compounds derived from a specific molecule of interest in complex mixtures. For example, drug administration as an equal mixture of unlabeled and a single labeled analogue will produce distinctive paired ion peaks in a mass spectrum indicating peaks derived from the test drug, when urine is analyzed. Third, stable isotope labeling has been used to distinguish a drug derived from different sources in pharmacokinetic studies. 1.3. Recent Advances in Biomedical Mass Spectrometry Since its discovery in 1912, mass spectroscopy has been limited mostly to the study of volatile small organic molecules or their derivatives (<2000 MW)

23 because of limitations of vaporizing relatively nonvolatile, polar and ionic, and/or heat labile biomolecules. Analytes with sufficient volatility and thermal stability to survive gas chromatography conditions are well suited to mass analysis by the principle of vaporization prior to ionization with the traditional El or chemical ionization techniques (3). The fundamental problem for bio-analytes that are nonvolatile, ionic, high molecular weight and/or thermally unstable and whose separation requires HPLC technology has been to transform these biomolecules from dilute solutions of ions at ambient temperatures into gaseous ions in the MS without degradation (4). This has lead to the coupling of HPLC separation techniques of normal phase, reverse phase, ion exchange, ion pair or ion chromatographies with a MS as the detector. The rapid growth of LC-MS as a mature technique for the mass analysis of these bio-analytes has come as a result of the improved technology in the development of several inlet systems that serve to transform ions in solution to gaseous ions. These inlet systems both desolvate and ionize these nonvolatile and thermolabile analytes without the need for vaporization prior to ionization (Figure 2) (1, 3, 5), and include the thermospray interface (TS), the electrospray (ES) and atmospheric pressure type interfaces (API), direct liquid introduction interface (DLI), and the continuous flow fast atom bombardment Ionic

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24 interface (CF-FAB). The major difficulties for the LC-MS system are handling the large volume of vapors from the desolvation of the liquid column eluants, the presence of nonvolatile buffers or salts and other nonionic substances, and the ionization of nonvolatile and thermally labile analytes, while at the same time maintaining a low vacuum for the MS (5, 6). The development of these interfaces has made the structural analysis of drug-conjugates (e.g. glucuronides, sulfates), proteins, oligonucleotides, oligosaccharides, and other high molecular weight biosubstances more accessible without the need for elaborate sample preparation and derivatization (4, 5, 7). Also, recent advances in microbore and capillary LC technology have facilitated the direct introduction of the entire eluant from the LC to the MS without the need for splitters. It may be feasible with microbore HPLC to obtain El mass spectra for the analyte from direct liquid introduction techniques. Also, these same interfaces and ionization techniques are being applied to the analysis of biopolymers with capillary electrophoresis (4). The LC-MS system is reviewed in greater detail in Chapter 4. The material below will discuss each component of the MS and the interpretation of mass spectroscopic data in greater detail. The various options for each component will be presented with their advantages and disadvantages. Recent advances will be emphasized.

2. THE INLET SYSTEM

2.1. Reservoir Inlet and Direct Insertion Probe Inlet

See above. 2.2. G C - M S Inlet

The direct coupling of a GC with a MS (GC-MS) has provided one of the most powerful and versatile tools to the bio-analytical scientist for the analysis of complex bio-organic and biochemical mixtures (3). Thus, mass spectra can be collected for the identification of all the compounds eluding from the GC column. The fundamental problem with coupling a GC with a MS is dealing with the volume of gas flow as it enters the heated inlet and the maintenance of a low vacuum. With capillary GC columns, the flow rate is low enough that the entire effluent from the column can be fed directly into the ionization chamber, but for packed columns with larger gas volumes, a post-column jet separator within the inlet chamber is needed to separate the lighter carrier

25 gas (helium) from the heavier vaporized analyte. The jet separator functions to increase the momentum of the heavier vaporized analyte molecules across a narrow gap into a capillary tube (the skimmer) that is connected to the ion source. The lighter carrier gas molecules readily diffuse away from the more heavier analyte molecules into the vacuum pumps, thereby enriching the vaporized analyte as it passes from the separator into the capillary.

2.3. L C-MS INLETS (see Table 1) Over the past 20 years, the LC-MS inlet system has undergone a rapid evolution in its design, from the moving belt to atmospheric pressure inlet systems. As with GC-MS, mass spectra can be collected for the identification of all compounds eluding from the LC column.

2.3.1. Moving-belt interface The once popular moving-belt interface is appropriate only for analytes with good volatility, low polarity. Generally, this interface is not compatible with highly aqueous LC eluants. This interface consists of a continuously moving belt that transports the LC eluant (typically <2 mL/min normal phase and <0.5 mL/min reversed phase) from the LC column to the ion source. During its transport, the eluant is evaporated under heat and reduced pressure, and the resulting analyte residue is flash evaporated from the tip of the movingbelt interface for El, chemical ionization or fast atom bombardment. On its return trip, the belt is heated and washed, ready for the collection of fresh LC eluant. Although the moving belt interface is better suited to normal phase LC, its use in reverse phase LC has been largely replaced with thermospray, electrospray or atmospheric pressure interfaces (see below).

2.3.2. Direct liquid introduction interface With this interface, the LC column eluant (typically <501~L/min) is forced through a small orifice nebulizing the eluant into a fine mist that is readily desolvated under reduced pressure in the ion source (1,4). During the evaporation process, the vaporized solvent molecules are ionized with an electron source, and in turn ionize the vaporized analyte molecules via a soft ionization process called, solvent-mediated chemical ionization. The ionized solvent molecules act as the reagent gas in chemical ionization. Because of problems with the membrane, the small eluant volumes, and the need for the analytes

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27 to be vaporized and thermally stable, this technique is being replaced by the newer thermospray, electrospray and API interfaces.

2.3.3. Thermospray ( TS) interface The TS interface has been effective for solving practical everyday bio-analytical problems requiring LC-MS (5, 8). Because of the potential for thermal degradation of heat-labile bio-analytes in the flash heater tube, TS can be a problem, and the likely reason for its replacement with electrospray and atmospheric pressure ion spray interfaces. This interface is both an inlet and ionizing interface that create ions via electrolyte-mediated chemical ionization during the course of solvent evaporation without an external ionization source. The TS interface allows the direct introduction of up to 2 mL/min of the eluant from the LC column into the MS. The function of this interface is to flash vaporize the LC eluant which assists in nebulizing the remaining liquid into a mist of fine droplets. It is during the rapid desolvation of the mist that the analyte molecules in the concentrated droplets become ionized due to the presence of a volatile buffer (i.e. ammonium acetate) in the LC eluant. The solvent vapor and other vaporized or nonvaporized nonionic substances in the eluant are drawn into the vacuum pump. This interface is applicable to polar nonvolatile analyte molecules that are thermally stable in aqueous/mixed aqueous LC mobile phases, with sensitivity in the low picogram range. Nonvolatile buffers (i.e. phosphates) and salts are not compatible with this interface. Although atmospheric pressure ionization interfaces are competing with thermospray interfaces, the latter have the advantage of larger flow volumes.

2.3.4. Continuous-flow or dynamic fast-atom bombardment interface ( CF-FAB) The CF-FAB interface has been a popular type of interface for the analysis of nonvolatile thermally labile ionic bio-analytes, such as peptide, proteins, oligonucleotides and oligosaccharides (9). With this interface, the LC eluant (typically 5-15 ~L/min) is mixed with a FAB matrix nonvolatile solvent, usually glycerin either in the mobile phase or added post-column, and forced through a narrow bore silica capillary onto the FAB target. The volatile solvents evaporate leaving a thin uniform film of glycerol on the surface of the FAB target which is then subject to bombardment from the FAB source. The ions are generated by bombardment of the continuously renewed liquid film with a

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2.3.5. Particle-beam (PB) interface The PB interface consists of a nebulizer, a desolvation chamber and a momentum separator that are connected to the ion source (5, 8, 10). The principle is analogous to the jet separator used with GC-MS to separate the solvents in the mobile phase from the analyte. With the PB interface, the liquid eluant is first converted into a mist of fine droplets which is desolvated as it is swept into the momentum separator. The separator increases the momentum of the more heavier analyte particles across a narrow gap into a heated capillary tube (the skimmer) for transport to the ion source, causing the lighter solvent molecules to diffuse away from the heavier analyte particles into the vacuum pumps. As a result of this process, a particle beam of analyte molecules that is nearly devoid of solvent enters the ion source. As the particle beam collides with the heated target in the ion source, the analyte molecules are flash vaporized prior to ionization by either El or CI. Because of the need for thermal vaporization, PB has been used mostly for volatile analytes and with LC eluants that are comprised largely of organic solvents.

29 3. THE ION SOURCE

3.1. Electron Ionization (El) El has been the most widely used ionization technique in mass spectrometry because of its extensive ion fragmentation for structural analysis of analytes and its reproducibility. Organic molecules that have been vaporized at high temperatures under a low vacuum (about 10 -s Torr) enter the ion source chamber and are bombarded by an electron beam. This causes molecules to lose an electron and generate a positively charged radical-cation (M-i-), the molecular ion, whose mass is the molecular weight of the original molecule. The low vacuum of the ion source minimizes collisions between the ions and neutral molecules preventing ion-molecule reactions. The highly energized molecular ion decomposes unimolecularly in a predictable fashion into fragment ions according to the strength of the different covalent bonds of which the analyte is composed (Eqs. 1 and 2). The fragment ions will continue to fragment until stable fragment ions are formed. The radical character of the molecular ion contributes to rearrangement reactions and the formation of unusual fragment ions. The fragment ions are characteristic of the original molecule and can be used to reconstruct the original molecule. Collectively, the combination of the molecular ion and the fragment ions constitute the mass spectrum of the analyte and can be used as a fingerprint for comparison with mass spectral libraries (Figure 4). El-MS is usually used only to detect positive ions. The negative ions and neutral fragments are pumped away. M + e--,

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111) (8). commonly referred to as a soft or mild ionization technique. For CI to occur, the analyte molecule must be in the gas state, at a lower vacuum than for El (about 10 -1 to 10 -2 Torr), and the presence of a reagent gas. The reagent gas (e.g. methane, ammonia or in some cases solvent molecules) is in large excess in the ion source and undergoes El to produce reactant ions that can abstract a proton from the vaporized analyte to generate a quasi-molecular ion ([M + H] § (Eq. 5). Because the proton transfer reaction is a mild ionization process and occurs with lower energy than El, the quasi-molecular ion ([M + H] § is not highly energized and therefore, fragment ions are either of low abundance or absent altogether. The extent of fragmentation of the quasimolecular ion depends upon its internal energy gained from the ability of the vaporized molecule to abstract the proton from the reagent gas. For example, more fragmentation of the quasi-molecular ion occurs with methane as the reagent gas than with ammonia.

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(6)

as negative chemical ionization (NCI). Depending upon its internal energy, the negative molecular ion can decompose into fragment ions, similar to that of a positive CI spectrum. Depending upon the type of electron capturing functional group(s), NCI can reach sensitivity below the femtomole level. Therefore, derivatization of the analyte with fluorinated reagents is a common technique for enhancing the sensitivity for trace analysis. Mass spectra recorded in the negative-ion mode are often complementary to the results from positive-ion spectra and is less subject to interferences from impurities that do not produce stable negative ions. For CI, FAB and APCI, negative-ion spectra have become routine and highly useful. Most instruments currently manufactured have negative-ion capability as an option.

3.3. Fast-Atom Bombardment (FAB) Ionization Both El and CI require sample vaporization prior to ionization. With highly polar and ionic nonvolatile biomolecules vaporization prior to ionization may lead to their thermal degradation without the production of a useful mass spectrum. Therefore, the need to volatilize these relatively nonvolatile and thermally unstable compounds without the analyte having to be volatilized

32 in an inlet system prior to ionization, led to the development of desorption ionization techniques (8). Desorption ionization relies upon the localized rapid vaporization of the analyte from a high energy source to produce gaseous ions that are expelled (desorbed) from the surface of the target into the mass analyzer. The gaseous ions are formed directly by focusing a high energy beam of xenon or argon atoms (fast atom bombardment, FAB) at the analyte that is attached to a solid target surface or in a liquid matrix. These gaseous ions expel (desorb) themselves from the solid surface of the beam target or liquid matrix forming a blanket of gaseous ions at the surface interface. These ions are swept into the mass analyzer generating a mass spectrum, similar to that from CI. FAB depends upon ion-molecule interactions for the generation of a quasi-molecular ion and a low abundance of fragment ions with unimolecular ion fragmentation occurring at the weaker covalent bonds. Therefore, FAB shares many characteristics with CI-MS. If an electrolyte is present, an adduct-molecular ion from the electrolyte cation and molecular ion is observed in the spectrum, e.g. [M + NH4]+ from ammonium acetate. Thus, with FAB techniques, the molecular weights of biomolecules greater than 10,000 Daltons have been desorbed. With CF-FAB, the analyte is mixed with glycerin and subjected to a beam of high energy xenon atoms which ionizes the analyte, causing the analyte ions to be expelled from the surface of the glycerin. The nonvolatile glycerin not only provides a matrix for the analyte but also helps to dissipate the thermal energy of the xenon atoms protecting the analyte from thermal degradation. The glycerin is also ionized by the beam of atoms, contributing to the formation of the quasi-molecular ion via solvent-mediated chemical ionization of the analyte from proton transfer reactions between the analyte and the ionized glycerin. The attachment of ionized glycerin molecules to molecular and fragment ions contributes to the complexity of the mass spectrum. The coupling of LC with FAB has extended mass spectrometry to the analysis of peptides, proteins and other thermolabile and ionic substances. The appearance of fragment ions from peptides provides information to confirm the amino acid sequence of the peptide. Matrix-assisted laser desorption ionization (MALDI) is similar to FAB except that the gaseous ions are produced by the laser desorption ionization process. This technique has become a popular method for the mass analysis of peptides to confirm its amino acid sequence. A pulsed high energy laser beam irradiates the solid analyte on the laser target surface producing molecular ions from thermally labile and relatively nonvolatile biomolecules without the problem of the formation of adduct ions from the liquid matrix or from volatile electrolytes common to FAB techniques. The solid analyte is dispersed in a

33 radiation-absorbing matrix (i.e. a fine metal powder) in order to limit thermal degradation to the analyte. Unlike the other desorption ionization techniques, MALDI is not a 'soft' ionization technique as with CF-FAB and does not rely upon ion-transfer reactions for the generation of the ions in the mass spectrum. The mechanism of ion generation remains a mystery. This type of desorption ionization is tolerant of nonvolatile buffers, salts and other substances. MALDI is coupled with the time-of-flight mass analyzer for the production of the mass spectrum.

3.4. Thermospray Ionization (TSI) TSI was introduced for converting ions in solution into gaseous ions for the mass analysis of ionic analytes (10). This method is similar to the direct introduction of a liquid in vacuo into the heated inlet chamber for desolvation prior to the sample entering the ion source. A solution of the analyte containing ammonium acetate (a volatile electrolyte)is forced through a heated capillary tube and is nebulized into a mist of fine droplets. A heated filament or electrical discharge may be used to facilitate ionization of the analyte and solvent molecules in the mist in the absence of a volatile electrolyte. As the droplets decrease in size from evaporation, the volatile electrolyte ammonium acetate yields gaseous ammonium and acetate ions which ionize the analyte molecules through an electrolyte-mediated chemical ionization (charge exchange) mechanism (6). This causes the droplets to develop an excessive charge and the eventual expulsion (desorption) of the ionized analyte molecule from the surface of the droplet into the mass analyzer. TSI is a mild ionization process and only ions indicative of molecular weight are present and structurally informative fragment ions are minimal (Figure 5). Consequently, the TSI mass spectra are similar to that from CI and lack the detail that make El spectra so useful for identification purposes. As a result of the presence of volatile electrolytes, an electrolyte adduct-molecular ion (for example [M + NH4] § with ammonium acetate) as well as the quasi-molecular ion ([M + H] § are found in the mass spectrum. Because of the large volume of vaporized solvent and water, extra large vacuum pumps are needed to draw off the solvent and water in order to maintain the vacuum in the inlet and mass analyzer.

3.5. Atmospheric Pressure Ionization (API) The capability of ionizing nonvolatile polar and ionic molecules at atmospheric pressure without high temperatures, has extended the applicability

34 le~L

ee._ 4) 0 C m

1TT

M

j coax H o ~ q

I

, ,

194

J """:! .......

"0

:

\,

!tl4

C:

<

370 (M-NH4)e

S4_

4)

>

e.--

m 4)

, . , . .

353

m.m ~

le_

m/z Figure 5. Thermospray spectrum of 4-methylumbelliferyl glucuronide (MW 352) (20). of MS to the analysis of drug-conjugate metabolites, peptides, proteins, oligonucleotides and other biomolecules. API is a soft ionization technique for the nonthermal ionization of the molecule at atmospheric pressures by spraying the analyte solution into an electrical field (5, 11). The techniques are called electrospray ionization, ionspray ionization, or atmospheric pressure chemical ionization, which can be coupled to magnetic sector, quadrupole mass filter analyzers, ion trap mass analyzers or time-of-flight mass analyzers.

3.6. Electrospray (ES) Ionization ES ionization depends upon the production of gaseous ions from ions in solution for a broad range of molecules, making it widely used for interfacing of LC to MS. ES is a soft ionization process, but unlike TSI, the process is without any thermal input in the ionization process (5, 11, 12). In ES, the analyte solution emerges from the capillary as a mist of fine droplets into a strong electrical field. The nebulization of the eluant into fine droplets is

35 assisted by a secondary or make-up gas flow (pneumatic nebulization). As the charged droplets decrease in size because of solvent evaporation, the charged analyte ions are expelled from the surface of the droplet. Uncharged nonvolatile material is swept away from the inlet to the mass analyzer by the secondary gas flow. Up to this point, the process is occurring at atmospheric pressure. The charged analyte ions are then drawn through a small orifice into the low pressure of the mass analyzer. As a result of the soft ionization process, fragmentation is usually absent and only molecular weight information is available. Peptide, proteins and oligonucleotides form multiply charged ions that give rise to clusters of peaks carrying slightly different numbers of charges. The multiply charged ions are produced primarily as a result of proton attachment to available basic sites in the molecules. MS separates ions according to their m/z ratio rather than mass. For example, an ion with a mass of 10,000 Daltons with 10 charges will be recorded at 'mass' 1000, reducing the mass range required from the analyzer. The addition of different volatile mobile phase buffers may facilitate structural recognition of fragments through changes in cluster ion formation. Because ES is readily coupled to LC-MS and CE-MS, these techniques are newer methods for obtaining molecule weight information for biomolecules (4). ES and MALDI are complementary techniques for the mass analysis of biomolecules. However, the quality of information from ES exceeds that from MALDI with better resolution and sensitivity. The application of ES in LC-ES-MS requires flow rates less than 10 i~L/min which could require the use of a post-column flow splitter with traditional or minibore columns >2 mm i.d. However, direct coupling with complete transfer of the injected volume into the MS can be achieved with microbore/nanobore LC (<1 mm i.d.) because of the low flow rates for these columns, as well as increased sensitivity.

3. 7. Ionspray (IS) Ionization IS ionization differs from ES in that a secondary flow of gas surrounding the capillary increases the misting of larger sample solutions, 40-50 I~L (12). With flow rates >50 ~L/min and columns >2 mm i.d., a post-column flow splitter may be required. IS spectra are usually devoid of structural information since they mostly contain quasi-molecular ion [M + H] § and cluster ions. IP is more practical than ES because of the higher flow rates and no splitting or low post-column flow-split ratios.

36

3.8. Atmospheric Pressure Chemical Ionization (APCl) APCI is the chemical ionization of analytes in an ion source operated at atmospheric pressure (5, 11). For atmospheric pressure chemical ionization to occur, a heated nebulizer converts the liquid eluant from the column into a mist of fine droplets and vaporized solvent molecules. The mist is swept into the vicinity of the ion source and ionized by a corona discharge, generating positive and negative ions. The ionization of the analyte molecules is achieved by mechanisms similar to those with conventional CI, except that the protonated solvent and water molecules serve as the reagent gas (solventmediated ionization). APCI can operate in either the positive- or negative-ion mode. In the positive-ion mode, the formation of positive ions occurs from proton transfer with the solvent ions, adduct formation or charge-exchange reactions between the buffer and the analyte. Whereas in the negative mode, the negative ions are formed from proton abstraction, electron capture, or anion attachment reactions. APCI is run with conventional bore HPLC columns at nominal flow rates of 2 mL/min and the use of volatile or nonvolatile buffers are permitted. This technique appears to be more sensitive than TS.

4. THE MASS ANALYZER

See Table 2.

4.1. Magnetic Sector Analyzer The magnetic field of the magnetic sector analyzer is scanned causing the positively charged ions to accelerate and follow different circular paths according to their m/z ratios. For any one magnetic field strength, those ions with a given m/z ratio and equal energy will follow the path with the correct radius and arrive at the detector, whereas, those ions with an incorrect radius will be deflected to the sides of the analyzer and not arrive at the detector. By scanning the magnetic field, a complete mass spectrum can be obtained. The magnetic sector analyzer can be either a unit resolution single focusing or high resolution double focusing analyzer. The latter utilizes a combination of electromagnetic and magnetic fields to obtain m/z ratios with masses calculated to 3 or 4 decimal places. The mass analyzer is maintained at an internal pressure of about 10 -5 Torr, which keeps the ion from colliding with itself or with other ions or molecules in the system.

37 TABLE 2. Experimental Options in Mass Spectrometry

Ionization techniques electron ionization chemical ionization negative chemical ionization fast atom bombardment matrix assisted laser desorption thermospray ionization electrospray/ionspray ionization atmospheric pressure chemical ionization Mass analyzers magnetic sector scanning quadrupole mass filter ion trap time-of-flight MS/MS (collision activated decomposition) single- and multiple-ion scanning

4.2. Quadrupole Mass Filter Analyzer The quadrupole mass filter is composed of four circular rods that serve as electrodes using a combination of dc voltages and oscillating radio frequencies to filter ions within a limited range of m/z ratios. For any one dc voltage and radio frequency, only those ions with a given m/z ratio will follow a stable path and arrive at the detector, whereas, the other ions will develop an unstable path and collide with the rods without arriving at the detector. When the voltages to the rods are scanned at a fixed radio frequency, the m/z range of ions is varied allowing all the ions to traverse the length of the analyzer to the detector, thus, an entire mass spectrum can be recorded. The quadrupole MS is more rugged, less expensive with faster scan times than the magnetic sector analyzer, except that it resolves ions that differ in mass by one unit. Its range has been extended to 3000-4000 m/z.

4.3. Ion-Trap Mass Analyzer The quadrupole ion-trap mass analyzer utilizers similar principles to the quadrupole mass filter except that ions from the ion source are trapped for several seconds within a circular electromagnetic field before being expelled from the trap to the detector (13). The trap consists of a central ring electrode and

38 a pair of end-cap electrodes that control the entrance and exit of the ions. The ions from the ion source enter the ion trap through the upper end cap, and circulate in a stable orbit within the cavity of the ring electrode. Increasing the voltage to the ring electrode causes the orbits of the more heavier ions to become stabilized, while those for the lighter ions become destabilized, and are expelled from the ring electrode cavity through electrostatically controlled slits in the lower end cap into the detector. The ion trap is a powerful mass analyzer for the storage of ions over a wide mass range with excellent detection limits. This analyzer has been used as both an ion source and ion trap with El, CI, MALDI, and as an ion trap from external ion sources, API, ES and TS. The ion trap achieves its high sensitivity through ion storage and integration of the ion signal over an extended period of time to allow the detection of relatively strong signals from a weak ion beam. Thus, ion traps are especially useful for the trace analysis of analytes. Ion-trap spectrometers are more compact and less costly than the quadrupole instruments.

4.4. Time-of-Flight Mass Analyzer The time-of-flight mass spectrometers are relatively simple, inexpensive instruments that can analyze ions of very large and broad mass ranges with high sensitivity, reasonable resolution and mass accuracy (14, 15). This mass analyzer has become a widely used tool for the structural analysis of biological macromolecules such as proteins, carbohydrates and oligonucleotides. The time-of-flight mass analyzer utilizes the principle of the time it takes for an ion to cover a fixed distance from the ion source to the detector along a tube free of any electrical or magnetic control. After an initial acceleration from the ion source, the burst of ions will drift along the field-free tube according to their m/z ratios, and arrive at the detector at different time intervals. The velocity of an ion is inversely proportional to the square root of its mass. Therefore, small ions will travel much faster than heavy ions. This analyzer is more suited to desorption ionization methods, such as the pulsed matrixassisted laser desorption ionization method (MALDI), than from ion sources that generate ions continuously (e.g. El, CI, TS, ES).

5. THE DETECTOR, SIGNAL PROCESSOR/DATA SYSTEM, AND VACUUM SYSTEM See above.

39

5.1. Tandem M S - M S MS/MS instrumentation has found increasing use in LC-MS in conjunction with the soft ionization techniques used with TSI, FAB and API because their mass spectra usually show the quasi-molecular ion and are devoid of much fragmentation, therefore, minimal structural information. However, tandem MS affords fragmentation spectra for selected individual ions that allow full characterization for the identification of the analyte in question with improved sensitivity as compared with conventional MS (16). Most MS/MS systems consist of two mass analyzers arranged in tandem separated by a collision cell (16). A soft ionization source of the first MS produces predominately singly charged ions that selectively transmits an analyte ion of a specified mass (m/z) into the collision cell, where these fastmoving ions collide with neutral gas molecules, such as helium or argon. The collision cell induces fragmentation of the selected ion into numerous daughter fragment ions, which are scanned by the second analyzer generating a mass spectrum of the daughter ions (Figure 6). Thus, with MS/MS, a pure

(a)

145

100OCONHCH a

173

[185 k

100 (b)

-~ ~ . @

77

ft.

115

91

Precursor ion

39

29 ] ,

s,,

65 I ,

145 !

1

lOO nl/z

Figure 6. A MS/MS spectrum of carbaryl" (a) the methane CI mass spectrum, and (b) the argon collision daughter fragment ions of the m/z ion at 145.

40 ionic species can be individually separated from other interfering ions such as background ions in the mobile phase or from other unresolved components of the sample, and be structurally identified. Little sample cleanup is required. If the quasi-molecular ion is selected for transmission to the second analyzer, none of the background or other ions will reach the collision cell, unless these ions have a similar m/z ratio. In other words, you do not need to have a completely resolved chromatogram, and only minimal sample cleanup is required if using LC-MS-MS. Tandem MS is a technique that will find wider application in the solution of bio-analytical problems. Figure 6 depicts the results from a LC-CI-MS/MS analysis of the pesticide, carbaryl (17). In Figure 6a, the methane CI-spectrum produced the major fragment at m/z 145, corresponding to protonated a-naphthol. The product ion spectrum (Figure 6b) yielded a large number of fragment ions that confirmed, without question, the aromaticity of the pesticide and the structural confirmation of the pesticide as carbaryl. Tandem MS-MS is reviewed in greater detail in Chapter 5.

6. DATA ANALYSIS

6.1. Quantitative Analysis The basis for obtaining quantitative data is the accurate measurement of the abundance of a selected mass ion. Because of the lack of reproducibility of the ion source for the generation of the selected ion, quantitation is best achieved using an internal standard. The best accuracy is obtained using the analyte that has been isotopically labeled so that there is at least a separation of 3 m/z between the unlabeled and the labeled analyte in the mass spectrum. Deuterium is an excellent source for isotopically labeling the analyte. Also, the isotopic purity of the labeled analyte has to be included in the quantitation of the analyte. To enhance the sensitivity for the measurement of the ions, the data acquisition system can record only the ion current of selected ions characteristic of the analyte (selected ion monitoring, SIM) rather than measuring the ion current for the entire spectrum.

6.2. The Mass Spectrum The mass spectrum of benzoic acid is shown in Figure 7 (2). The x-axis is the m/z ratio. The peaks in the spectrum are normalized to the height of the most abundant or base peak (100 percent), the y-axis. The singly charged molecular (or parent) ion, if present, is typically the highest mass peak in the spectrum,

41

~

105

base peak

122

,o, I

I

C I~-OH / I 5

Mr-122

77

molecular ion 77

~LL,L ..... 11

9. , . , . , - , - . i r , . ,

, ,,'~i .,.~ r , , ' , , i 9 Ioo

MIZ Figure 7. The El mass spectrum of benzoic acid.

unless cluster ions, adduct ions or impurities are present. The lower mass ions (fragment or daughter), arise by decomposition of the molecular ion, either directly or by multi-step pathways (18). Especially for El, the normal routes of fragmentation are well understood and predictable, so that the spectrum of the unknown can be interpreted to reveal the molecular structure. The patterns are quite reproducible, and extensive mass spectral libraries are available for computer or manual search. By analyzing the fragment ions of the analyte, the whole molecule can be reconstructed. Even without a complete reconstruction of the analyte, a surprising amount of information about the structure of the analyte can be obtained. The abundance of any ion is its ability to stabilize a charge, without further bond cleavage from the excess internal energy remaining in the ion. Individual laboratories often assemble their own libraries appropriate for particular projects. Ions of low abundance are associated with each of the larger peaks. These correspond to the natural abundance of isotopes such as 13C, 2H, lSN, 180,

42 34S, 3701 and 8~Br. Because the MS can easily differentiate isotopes, the technique lends itself to studies that utilize the incorporation of stable isotopes into compounds of interest for purposes of quantitation, or for investigations of reaction mechanism or metabolic disposition. When the natural abundance of a stable isotope is high, the distribution pattern for the various ion clusters can be used to calculate the number of those atoms in each cluster. Figure 8 shows the El mass spectrum of pentachlorophenol (2). In the inset are the calculated distributions for peak abundances when 1-5 chlorine atoms are present. One can easily judge how many chlorine atoms are included in the molecular ion and in each of the major fragments.

,1~176176 ..

--

X +2

X

.--L

.

+4

X +4

.I--

-.

t--

-"

X +4 + 8

."

.

.

X +4 + 8

OH CI ~ ~ ' ~

CI

CI ~

CI

Y

9

!~

\"r"

CI

"-I-"I "t ~'I"-I"

1oo

200

l-,-r

I',I-'I

300

MIZ Figure 8. The El mass spectrum of pentachlorophenol. [Inset] the calculated distribution of the isotopic abundances for 1-5 chlorine atoms.

43 As the molecular weight of the analyte increases, the contribution of even the less-abundant stable isotopes becomes significant. For analytes with large molecular weights, such as polypeptides, the contribution of the less-abundant isotopes can become very significant when many atoms are present in the molecule. These factors must be given careful consideration when an analysis is planned so that the determination of the number and location of the labeled atoms will be experimentally feasible. Incorporation of a sufficient number of heavy atoms to shift the labeled peak away from the normal abundance cluster is an obvious advantage. Derivatization of the sample may be necessary in order to achieve volatility or, to minimize or direct, mass spectral fragmentation. Selection of a derivative should take into account both the chromatographic properties of the products and the distribution of ion current in their mass spectra. The mass spectrum of the derivative of choice will have a molecular-weight related ion with good abundance and structurally significant high-mass fragments (which retain the label, if one is used). This assures the uniqueness of the analysis and minimizes interference from low-mass background ions. Figure 9 shows the trimethylsilyl (TMS) and methyl esters of phenyl acetic acid (2). In this case, the methyl ester is clearly preferable; the spectrum of the TMS ester has only a very low abundance molecular-ion, and the base peak corresponds to the TMS group. The abundance of individual ions in the spectra of compounds of interest can be plotted as a function of time, producing a signal that is relatively free

I

I

159

i . ~ . ~ , C-~ 9,1 o 173

9o =.

8 i I I

9,

73 M +"

119

208

M-+" 150

=.

M+, M+

.,.,,

,,,!,i

j"

i,..~L,.,,.,.,,.I.

L L2

j~

' ,-,-,-.,

..,.,_,.,.l.,:,:.i.

9 d

,

.i-,-,-,,--r-,.,.,-i

MIZ

Figure 9. El mass spectra of phenylacetic acid derivatives" (a) the trimethylsilyl ester, and (b) the methyl ester.

44 of interference. This type of trace (a mass chromatogram) can be generated from sets of complete spectra in order to locate components with specific molecular weights or a common structural feature in a complex mixture or to enhance chromatographic resolution. Specific ion signals can be recorded with very high sensitivity by selectively detecting one or a few ions during the entire experiment (single- or multiple-ion monitoring). The former approach is useful for qualitative analysis or for profiling of complex mixtures; the latter approach provides good quantitative results. Exact mass measurements made at high resolution have sufficient accuracy for determination of elemental composition of molecular and fragment ions. This information helps in the elucidation of structure of unknowns and also in the characterization of new compounds that cannot be isolated in quantities necessary for elemental analysis. In those cases in which the El mass spectrum does not include a molecular ion, the elemental composition of the compound may still be determined by employing one of the several soft ionization techniques to assign a composition to the (M + H) § or M+ ion or other appropriate molecular-weight related species. High-resolution measurements may also be necessary when impurities that produce ions isobaric with those of interest are present. Thus, the analyst today has the choice of a wide range of mass spectral capabilities. These are summarized in Table 2. The selection of the method to be employed will be dependent on the information sought, the type of analyte (its volatility or nonvolatility, thermostability or thermolability) to be analyzed, and the level of sophistication (of both operator and instrument). This is clearly the era of biological mass spectrometry, the study of macromolecular science in the overall context of human health and disease (4). The technical advances in creating ions efficiently and reproducibly from nonvolatile polar and labile biopolymeric substances has redefined bio-analytical chemistry. This dynamic field has much to contribute toward the solution of pharmacologic and biochemical problems.

REFERENCES 1. R.J. Anderegg, Mass Spectrometry: An Introduction, in Biomedical Applications of Mass Spectrometry, Vol. 34, Methods of Biochemical Analysis (John Wiley, New York, 1990), pp. 1-89. 2. C.E. Costello, Fundamentals of Present Day Mass Spectrometry, J. Clin. Pharmacol., 26 (1986) 390. 3. W.H. McFadden, Techniques of Combined GC-MS (John Wiley, New York, 1973). 4. A.L. Burlingame, R.K. Boyd and S.J. Gaskell, Mass Spectrometry, Anal. Chem., 66 (1994) 634R.

45 5. W.M.A. Niessen and A.P. Tinke, Liquid Chromatography-Mass Spectrometry: General Principles and Instrumentation, J. Chromatogr. A., 703 (1995) 37. 6. W.M.A. Niessen and J. van der Greef, Liquid Chromatography-Mass Spectrometry (Marcel Dekker, New York, 1992). 7. E. Gelpi, Biomedical and Biochemical Applications of Liquid ChromatographyMass Spectrometry, J. Chromatogr. A., 703 (1995) 59. 8. J.R. Chapman, Mass Spectrometer as an LC Detection Technique, in: A practical Guide to HPLC Detection, D. Parriott (ed) (Academic Press, New York, 1993). 9. R.M. Caprioli (ed), Continuous-Flow Fast Atom Bombardment Mass Spectrometry (John Wiley, New York, 1990). 10. A.L. Yergey, C.G. Edwards, I.A.S. Lewis and M.L. Vestal, Liquid Chromatography/Mass Spectrometry: Techniques and Applications (Plenum Press, New York, 1990). 11. E.C. Huang, T. Wachs, J.J. Conboy and J.D. Henion, Atmospheric Pressure Ionization Mass Spectrometry: Detection of the Separation Sciences, Anal. Chem., 62 (1990) 713A. 12. P. Kebarle and L. Tang, From Ions in Solution to Ions in the Gas Phase: The Mechanism of Electrospray Mass Spectrometry, Anal. Chem., 65 (1993) 972A. 13. S.A. McLuckey, G.J. van Berkel, D.E. Goeringer and G.L. Glish, Ion Trap Mass Spectrometry of Externally Generated Ions, Anal. Chem., 66 (1994) 689A. 14. R.J. Cotter, Time-of-Flight Mass Spectrometry for the Structural Analysis of Biological Molecules, Anal. Chem., 64 (1994) 1027A. 15. M.G. Qian and D.M. Lubman, A Marriage Made in MS, Anal. Chem., 67 (1995) 234A. 16. F.W. McLafferty (ed), Tandem Mass Spectrometry (John Wiley, New York, 1983). 17. T. Cairns and R.A. Baldwin, Pesticide Analysis in Food by MS, Anal. Chem., 67 (1995) 552A. 18. F.W. McLafferty, Interpretation of Mass Spectra, 3rd edn (University Science Books, Mill Valley, California, 1980). 19. D.J. Bell, M.D. Brightweil, W.A. Neville and A. West, Rapid Commun. Mass Spectrom., 4 (1990) 88. 20. D.J. Liberato, C.C. Fenselau, M.L. Vestal and A.L. Yergey, Anal. Chem., 55 (1983) 1741.

47

CHAPTER 4

MASS SPECTROMETRY: LIQUID C H R O M A T O G R A P H Y - MASS SPECTROMETRY

JAMES E. EVANS E. K. Shriver Center, Inc., 200 Trapelo Rd., Waltham, MA 02254

1. INTRODUCTION 1.1. Background

For nearly thirty years, mass spectrometry (MS) has been the premier technique for the detection, structure determination and quantitation of compounds labeled with stable isotopes. High-performance liquid chromatography (HPLC) developed over this same period into the preferred technique for separation and measurement of compounds in biological research. Analysis by HPLC requires much less sample isolation and derivatization and is applicable to a much wider range of compounds than the previously well-established gas chromatography. The on-line combined technique of LC-MS provides the advantages of both and is widely recognized as the most powerful tool available for analysis with high sensitivity and specificity of low concentrations of drugs and their metabolites in biological matrices. The ability of LC-MS to distinguish and measure compounds labeled with stable isotopes with the same analytical prowess makes it the obvious choice when tracer studies with pharmaceuticals are considered. Yet, only a few examples of the application of stable isotope-labeled pharmaceuticals with LC-MS analysis are found in the literature. Reasons for this may include what seems to be the small, albeit increasing, role stable isotopes have in studies of drug disposition and the fact that easy to use high-performance LC-MS instrumentation has only become available over the last five years. LC-MS instrumentation has now developed to the point that it can conveniently solve the difficult bioanalytical problems encountered in such studies. This chapter

48 will describe the operation and capabilities of LC-MS instrumentation and illustrate how it can be best used for stable isotope tracer studies of drug disposition. Since the early 1970s, intense research activity resulted in the development of highly successful approaches for interfacing the rapidly developing technique of HPLC directly to MS. The primary impetus for this was to extend the demonstrated benefits of combined GC-MS to the majority of compounds that are too involatile or thermally labile to pass through a GC column even after derivatization. During this developmental period more than 25 LC-MS interfaces were reported in the literature and about ten of these have been offered commercially (1). Today only five interfaces have survived the competition and are commercially available. These are continuous flow fast atom bombardment (CF/FAB), particle beam (PB), thermospray (TS), electrospray (ES), ionspray (IS) and atmospheric pressure chemical ionization (APCI). LC-MS now makes possible the convenient analysis of a very broad range of compounds in complex matrices often with very high sensitivity and precision. These compounds include polar, involatile, thermally liable and/or high molecular weight analytes such as peptides, proteins, oligosaccharides, polynucleotides, etc. Mass spectrometric analyses of many of these were unimaginable twenty-five years ago. 1.2. L C - M S Interfaces

LC-MS interfaces are conveniently divided into two groups: those that deliver the LC analytes to a conventional ion source for subsequent ionization and those that ionize the analytes and transmit the ions to the mass spectrometer. These are referred to respectively as transport and ionization type interfaces. Figure 1 provides an indication of the amount of developmental and applications effort that is going into each LC-MS interfacing technique. The ionization type interfaces, notably the API interfaces, are currently attracting the most interest and developmental effort. Transport interfaces deliver analytes from the HPLC eluate to a conventional MS ion source where they are ionized and subsequently mass analyzed. They include the direct liquid introduction (DLI), moving belt (MB) interface, PB and CF/FAB interfaces. DLI and MB were the first successful techniques developed for LC-MS interfacing and were available commercially in the late 1970s. The DLI interface, conceptually and physically the simplest of all LC-MS interfaces, functions by spraying the chromatography eluate at a few I~l/min directly into a conventional chemical ionization (CI) ion source. There both the sample and solvent are thermally vaporized and the sample components

49 400 350

mTSP mCFFAB IlaPB i l S P

I-IAPCI i E S I

300 t~ o 250 O. o 200 t_ O

E 150

:3 Z

100 50

1991

1992

1993 1994 1995 Meeting Year Figure 1. Graph of number of presentations concerning different LC-MS interfacing methods presented at the annual American Society of Mass Spectrometry Conferences from 1991 through 1995.

ionized by CI using the vaporized solvent as the reagent gas. Two transport interfaces, MB and PB, deliver solute nearly free of LC solvent to the ion source and have the advantage of allowing conventional electron impact (El) or CI mass spectra to be acquired. PB, MB and DLI interfaces have the limitation that analytes must have sufficient volatility and stability to be thermally vaporized before ionization can occur. While this condition limits the analysis to such compounds, it is a much less severe limitation than is encountered by GC-MS analyses. Indeed compounds that were traditionally considered quite nonvolatile or thermally labile have been successfully analyzed by M B LC-MS with chemical ionization (2, 3) and can provide spectra that are similar to those obtained by direct chemical ionization (DCI). Vaporization of highly nonvolatile or thermally labile biopolymers remains a problem for these interfaces. There is no such requirement for direct volatilization with CF/FAB and its range of application extends to biopolymers with molecular weights

50 up to about 5,000. The transport interfaces discussed in more detail below are currently used for a variety of analyses but are losing popularity in favor of the newer atmospheric pressure ionization interfaces (API). Because the DLI and MB interfaces are little used and not presently commercially available they will not be discussed in detail below. While the popularity of transport interfaces as a group has decreased enormously in favor of the API interfaces, the MB, PB and CF/FAB techniques still retain areas of application in which they are quite useful. LC-MS interfaces that serve as ionization sources include the thermospray interface (TSP) and three API interfaces: ES, IS and APCI. TSP achieved a very high level of popularity and was the dominate LC-MS interface from the mid1980s until recently, especially for drug analysis and development studies. Since the announcement of ES by Fenn and coworkers (4) it, and subsequently APCI and IS, have become the dominate LC-MS interfaces. TSP is now being replaced by those techniques that offer higher sensitivity and are easier to use with a much wider range of compounds. The atmospheric pressure ionization interfaces are having a tremendous impact on the analysis of analytes with a very wide range of properties and are much more universal for analysis of compound types than any of the earlier techniques. Currently a large portion of instrumentation development in LC-MS is directed toward further development of API interfaces.

1.3. Objectives of this Review This chapter will present a practical overview of LC-MS techniques, providing guidelines that will indicate the potential and limitations of each technique for analysis of various sample/compound types and the compatibility of each interface with various types of separation systems. Features of each interface that make it particularly useful or less applicable for stable isotope tracer studies will be noted. The capabilities that are desirable for stable isotope tracer experiments generally include high sensitivity, accuracy, reproducibility and, of course, a capacity of good mass discrimination. Rather than presenting an all inclusive overview of interfacing techniques and the history of their development, this chapter will be limited to those that are available commercially. Because only very limited use of LC-MS has been made for studies with stable isotope tracers, examples used here will usually be drawn from studies in which they were not utilized but that will illustrate the potential of the technique for stable isotope tracer studies. A number of useful recent reviews of LC-MS development and applications have appeared that can provide the reader with further information concern-

51 ing these techniques. One by Burlingame (5) is highly recommended and appears biannually in Analytical Chemistry, and covering the major developments in mass spectrometry over the preceding two-year period. Because LC-MS interfacing has been one of the most active areas of development in mass spectrometry, much of this review concerns LC-MS. A good recent review of the general principles and instrumentation is by Niessen and Tinke (6). Several reviews of the application of LC-MS in pharmaceutical or biomedical research are worth note (7-9). Other reviews related to specific interfaces will be noted in the discussion of each technique.

2. LC-MS INTERFACING METHODS 2.1. Transport Interfaces 2.1.1. Particle beam L C-MS The PB interface is now well established and popular as a user-friendly and versatile LC-MS interface (Figure 2). The jet separator developed by Ryhage (10) for interfacing packed column GC-MS served as a model for development of the PB interface. Takeuchi et al. (1 1) reported the use of a device similar to a GC-MS glass jet separator to interface micro column HPLC to MS with limited success. Willoughby and Browner (12) were then largely responsible Two Stage Momentum Separator

He in

Nebulizer ----

LC Solvent

Heated Desolvation Chambel

/'

~

Ion Source i lli

~1

I'

Vacuum Pumps

Figure 2.

Schematic representation of a particle beam LC-MS interface.

52 for developing an understanding of many of the critical factors for achieving high enrichment and recovery of solutes from liquid flow streams. The insight gained from their work led to today's commercial PB interfaces. Most mass spectrometer manufacturers now offer PB interfaces for their instruments instead of the once commonly offered MB interfaces. Because PB delivers the concentrated LC analytes nearly free from solvent (enrichment of about 10s) to the ion source, it is highly versatile and has been used with most types of mass spectrometers and with a wide array of ionization sources including El, CI, FAB, laser desorption, high temperature surface ionization and chemical reaction interface mass spectrometry (CRIMS, discussed in Chapter 6 of this volume). The PB LC-MS interface is the only one currently commercially available that allows the acquisition of El and CI mass spectra that are identical to those obtained by conventional GC-MS or direct probe inlets. These El spectra are useful for structure elucidation based on established rules and can be searched against library reference spectra. PB also has few limitations for analysis of different compound types and chromatographic solvents beyond those of the ionization source used. Ease of use, one of the advantages of this technique, results from its simple design and operation. It is the most popular of the transport-type interfaces and the only one that is currently undergoing active development. A recent detailed review of the development, operation and use of this interface by Creaser and Stygall (13) is highly recommended. Figure 2 is a schematic representation of the PB interface that will serve to illustrate its operation. It functions solely by aerodynamic means, enriching solute from an LC flow-stream and transporting it directly to a conventional MS ion source. Several important and distinct steps are involved in this process. The first is the conversion of the LC solvent into an aerosol of small uniform droplets by nebulization in a stream of helium. This aerosol then passes through a desolvation chamber that is maintained slightly above ambient temperature, where much of the solvent evaporates and residues from the dissolved analytes form particles. The resulting suspension of solute particles in solvent vapor and helium nebulizing gas is collimated by passing through a small orifice into the first pumped chamber (about 103 Pa) of the 2-stage momentum separator where much of the gasses are pumped away. The beam of more massive particles continues on through an in-line skimmer into the second chamber (about 100 Pa) where the process is repeated. Finally, the collimated particle beam continues its line-of-site path through a transfer tube into the ion source of the MS. The design and operation of the nebulizer and the diameter of the sampling orifice, the temperature of the desolvation chamber and the spacing of the skimmers in the momentum separator are

53 critical for high transmission of solute containing little solvent to the mass spectrometer ion source (12). For highest transmission of analyte the particles should retain some solvent to increase their mass. Huang and coworkers (14, 15) investigated the interaction between mobile phase composition, flow rate, helium nebulizer gas pressure and desolvation chamber temperature on the optimization of two commercial PB interfaces from Hewlett-Packard and Extrel. They found that these interfaces behaved quite differently with respect to those variables and that adjustment of nebulizer flow and desolvation chamber temperature for different solvent compositions was critical to obtain maximum sensitivity. One commercial PB interface that departs significantly in design from others is the Vestec Universal Interface (16). It incorporates a heated thermospray vaporizer in place of the pneumatic nebulizer for more complete vaporization of the solvent and a membrane separator that removes much of the solvent vapor prior to the momentum separator. Helium is admitted separately to serve as a transport gas to carry the aerosol through the membrane separator and the momentum separator. This device has been reported to achieve a much more complete removal of the solvent than other PB designs, and to be the only PB interface suitable for use with LC-CRIMS where the usual levels of residual solvent would swamp the microwave reaction interface (17). Problems with low transfer efficiencies (18), nonlinear response and chromatographic band-broadening have been widely reported with L C - P B - M S interfaces. While these problems are of considerable consequence they do not preclude accurate quantitative analysis when the proper measures are taken. They do indicate that a better understanding of the processes of nebulization, desolvation and momentum separation needs to be gained for future design modifications if PB is to gain wider popularity. As discussed below, a number of reports have demonstrated that accurate and reliable data can be obtained with L C - P B - M S without difficulty. Nonlinear calibration curves have been of major concern in the use of PB for quantitative analysis. Response generally varies exponentially with analyte concentration for reasons that are not presently well understood. This results in poor detection limits and a limited dynamic range. Factors, including particle size and particle charging, have been investigated as the source of this problem without clearly identifying its basis. Investigations have demonstrated that some measures can be taken to considerably improve both response and linearity. One measure is to add a carrier compound (often ammonium acetate) to the chromatographic solvent. Apffel and Perry (18) investigated this carrier effect with a number of analytes and additives and found that the effect was more pronounced with some combinations than

54

others. In general, this effect is substantial and can produce, in many cases, considerable signal enhancement and linear calibration curves. This ammonium acetate carrier effect is not observed with the Vestec Universal Interface, presumably because the membrane separator removes most of the ammonium acetate before the momentum separator. The other effective approach to correction of this problem has been the use of co-eluting stable isotope-labeled internal standards. This also results in linear calibration curves and in most cases considerable signal enhancement. A good discussion of the problems with nonlinear calibration curves and poor analyte yield is presented by Creaser and Stygall (13). The PB interface works well for the analysis of compounds in a midrange of volatility and with considerable thermal stability. A problem occurs with less massive, or more volatile, analytes because they are vaporized and pumped away with the helium and solvent vapor and not transmitted as particles to the mass spectrometer. Another limitation arises with compounds of low volatility or low thermal stability. When these particles arrive in the ion source they must be thermally vaporized before ionization by gas phase techniques such as CI and El. This vaporization occurs by collision with the heated wall of the ion source and provides a major limitation to the PB analysis of these compounds. This, together with the limit on higher volatility compounds, results in a volatility range for analytes that is somewhat restricted. Richardson and Browner (19) demonstrated that much higher than usual ion source temperatures or laser vaporization leads to much higher sensitivity with much less thermal decomposition when thermally labile/low volatility compounds are analyzed. Ionization methods that do not require thermal vaporization have been used in combination with the PB interface and have shown promise for the analysis of compounds with low volatility, poor thermal stability or higher molecular weight. FAB using a PB interface has been demonstrated (20, 21) and provided good spectral quality and high sensitivity without the flow rate limitation or solvent load experienced with continuous flow FAB. PB-massive particle impact ionization-MS (22) is a somewhat similar technique to FAB and uses massive glycerol cluster impact to ionize the sample in place of FAB. This approach shows promise for the analysis of compounds beyond the molecular weight range of FAB because it produces multiply charged ions similar to the API methods such as ES. Early reports of attempts to perform matrix-assisted laser desorption/ionization (MALDI) directly on matrix containing particles in the ion source were presented and shown to be promising (19, 23). PB-hyperthermal surface ionization-MS that produces ions by collision of a supersonic molecular beam of analyte particles with a platinum, tungsten

55 or rhenium surface has been developed using a modified PB interface (24). The developers reported ionization efficiencies ten times higher than normal El ionization for polycyclic aromatic hydrocarbons using this technique. PBchemical reaction interface-MS (LC-PB-CRIMS) has been reported for the selective detection and measurement of stable isotopes of carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorous, selenium, bromine and chlorine in organic compounds (17). The LC-PB-CRIMS technique has shown considerable promise and is covered in Chapter 6 of this volume. Furthermore, developments of ionization techniques that take advantage of PB's ability to deliver highly enriched analyte from an LC flow stream are expected. Several recent applications demonstrate quantitation of pharmaceuticals in biological matrices using LC-PB-MS. These are selected to demonstrate that nonlinearity and other problems do not preclude high quality quantitative analyses with good sensitivity. Many of these applications used electron capture negative ion chemical ionization (NCI) to achieve high sensitivity. Girault et al. (25) reported a LC-PB-NCI/MS assay for the measurement of BN50727 (a platelet activating factor antagonist) in human plasma and urine. They used a simple solid-liquid extraction procedure after addition of a chemical analog internal standard prior to injection. A quantification limit of 1 ng/ml and very good reproducibility were reported. The procedure was used to obtain preliminary pharmacokinetic data. The same group also developed a similar method for another platelet activating factor antagonist with similar success (26). The measurement of the antibiotic tylosin in bovine muscle by LC-PB-NCI/MS was reported to be useful as a confirmatory technique for residues in animal products (27). Similar assays for spiramycin (28) and chloramphenicol (29) residues have been reported by the same group. Celma (30) reported a method for measurement of an aza alkyl lysophospholipid in rat plasma after a single liquid-liquid extraction using a polymeric reversed phase column and LC-PB-EI/MS. He was able to make measurements in the range from 25 ng/ml to 5 i~g/ml with good accuracy and precision and applied the procedure in pharmacokinetic studies. The immunosuppressant FK506 and its metabolites were determined in blood and urine by Christians et al. (31) using LC-PB-NCI/MS. They achieved a 25 pg limit of detection for standard solutions and a limit of quantification of 250 pg for blood (CV 11.3 percent at 5 ng). An excellent example of the use of LC-PB-MS for stable isotope tracer metabolism studies has appeared from Markey's group (32). They reported quantitative studies on the conversion of [13C6]-L-tryptophan to [13C6]-L-kynurenine in interferon-~/stimulated human monocytes and poke weed mitogen stimulated gerbil lung and brain slices. The effect of whole body stimulation

56 with interferon--y on plasma levels of endogenous of L-kynurenine was assessed. After addition of 180- or 2H-labeled internal standards to biological samples, they used a simple combined extractive derivatization to form electron capturing pentafluorobenzyl derivatives of tryptophan and kynurenine. These were separated on either a 1.1 or a 2.1 mm i.d. normal phase silica column using gradient elution with toluene-ethyl acetate mixtures at 0.4ml/min flow rate. LC-PB-NCI/MS was performed to obtain standard curves that were linear over the range of 1-250 ng/sample with limits of detection from 10-50pg injected. Excellent recovery and reproducibility values were reported. Recently this group reported improvement of the derivatization procedure and inclusion of a number of additional tryptophan metabolites in the analysis (33). 2.1.2. Continuous flow FAB

The introduction of fast atom bombardment mass spectrometry (FAB-MS) by Barber et al. (34) caused a revolution in biochemistry and mass spectrometry. FAB-MS is also used here to refer to the similar technique of liquid secondary ion emission-MS (liquid-SIMS or LSIMS). It made possible for the first time the MS analysis of many compounds that were polar, thermally labile and/or of higher molecular weight. Before FAB-MS, it was necessary to convert such compounds to volatile and thermally stable derivatives, if possible, for analysis by gas phase ionization techniques or to use field desorption ionization, which was only marginally successful with a select group of compounds. Because FAB produces gas phase ions by bombardment with high energy atoms of the surface of a solution of analytes in a low volatility matrix, it eliminates the need for thermal vaporization. FAB ionization causes little fragmentation and produces primarly pseudomolecular ions even with very labile compounds. This made possible the analysis of previously unapproachable compounds. These included a large variety of biological compounds such as peptides, oligosaccharides, drug conjugates, etc. This capability generated a tremendous interest among biologists and resulted in intense research efforts to further develop FAB-MS, and subsequently many other new MS methods, for the analysis of such compounds. One of the main disadvantages of FAB is the intense matrix ions that obscure portions of the spectra, especially at low mass. Another disadvantage that makes mixture analysis difficult is ion suppression that occurs when better responding components of mixtures suppress ion production from other components. A second phase of this revolution began with attempts to combine FAB

57 Frit

Fused Silica Capillary

/

LC i n _ _ ~ , ~ 4 ~ l ~ .

.~

Wick// Fast Atom

Ii AnMiSzer

""'"'-....

Beam//" ""'"'""

Figure 3. Schematic representation of a continuous flow fast atom bombardment probe.

with a separation technique (HPLC). The first LC-FAB-MS techniques used a moving belt transport interface (35). The fast atom beam was focused directly onto the belt where the residues were ionized. Addition of matrix to the belt was difficult and often had little or negative effect on the resulting spectra. Moving belt FAB was difficult to use and did not provide the necessary sensitivity or the soft ionization of probe FAB to make it a method that was useful for practical analyses. Continuous flow FAB (CF/FAB, also termed frit FAB or dynamic FAB) was introduced by Ito et al. (36) as a much simpler and more successful approach to on-line LC-FAB-MS. Figure 3 illustrates a contemporary device that is not very different from the one they used. Mobile phase containing a few percent glycerol (to serve as the FAB matrix) flows through a capillary tube onto a stainless steel frit at the tip of the FAB probe. The volatile components of the solvent evaporate leaving the analyte dissolved in the remaining glycerol matrix on the surface of the frit. There it is the target for the fast atom beam. Ito et al. (36) used nano-scale chromatography with a packed capillary column that ran through the FAB probe and terminated at the frit. Today's CF/FAB interfaces differ from this original design only by having a heated probe tip to avoid evaporative cooling and freezing of solvent and the addition of a wick to absorb excess solvent and matrix. Caprioli et al. (37) reported a modified design that allowed direct introduction of samples either from an external HPLC stream or reaction vessel or by direct injection. This design differed significantly from Ito's in that the flow from an unpacked capillary went directly onto a copper target at the probe tip and used no frit. With the capillary serving as a transfer line, analysis of flow streams from a variety of sources including HPLC columns, capillary electrophoresis, direct injections of samples, on-line monitoring of incubations and in vivo microdialysis were

58 reported. A book edited by Caprioli (38) summarizes many aspects of the development and application of CF/FAB. CF/FAB is now a well-established technique that has proved useful for analysis of a wide variety of compounds. It provides high sensitivity and, consequently, selectivity for the analysis of compounds that have amphiphilic character and/or a strong ionic functional group. Other compounds that do not have this character are also successfully analyzed but usually with lower sensitivity. The design of CF/FAB interfaces seems to be quite stable and not to have undergone much development in the last five years. Most commercial interfaces available today are of the frit-FAB design and use a 50 i~m i.d. fused silica capillary to transfer the analyte-containing solvent to the probe. The flow rate is limited to about 5-10 i~l/min by the vacuum requirements of the mass spectrometer. Because of this it is necessary to split the flow from HPLC columns with a larger i.d. than about 0.5 mm. The requirement for matrix in the solvent has been met either by addition pre-column to the LC mobile phase or post column via a coaxial delivery system that mixes the matrix with the mobile phase at the probe tip. Reports have appeared to demonstrate that addition of a viscous matrix such as glycerol to the solvents used for chromatography has a detrimental effect on chromatographic efficiency (39, 40). This effect is of considerable consequence at levels above about 5 percent matrix in the chromatographic mobile phase. It is now normal practice to include from 1 to 5 percent matrix in the chromatographic mobile phase. This level of matrix is sufficient to provide optimal FAB results but does not impair chromatographic efficiency to a significant degree. Cesium and massive cluster ion sources have been used in place of FAB and provide improved sensitivity because these beams can be focused to provide high intensity on the target. The massive cluster ion source offers the additional advantage of producing multiply charged ions from higher molecular weight compounds (similar to electrospray) allowing them to be detected by analyzers that have an upper m/z limit of less than 2,000. CF/FAB has very significant advantages over static FAB in addition to the ability to couple on-line to LC. These include increased sensitivity, the ability to effectively subtract background from spectra and much less ion suppression of compounds in mixtures. The increase in sensitivity is often on the order of a 100-fold or more. It results from both reduced ion suppression and a much reduced matrix background. The ability to reliably subtract background allows one to obtain clean spectra of compounds even in mass ranges that are dominated by matrix ions. Reduced ion suppression improves the capabilities for performing quantitative analysis.

59 The quantitative capabilities for CF/FAB have proved to be quite good. Detection limits are often in the low picomole range with linear ranges greater than two orders of magnitude. These capabilities plus the ability to use a wide variety of mobile phase compositions (from 100 percent organic to 100 percent aqueous) make LC-CF/FAB-MS a very useful technique for analysis of many compounds. Compounds that have a polar ionic group and a fairly hydrophobic moiety produce the highest sensitivity. Examples of these include: bile acids; drug conjugates with glucuronic acid, glutathione and sulfates; and other compounds that can be classified as ionic surfactants. Other compounds such as peptides, oligosaccharides, phospholipids, steroids, etc., have also been analyzed very successfully by these techniques but usually with somewhat less sensitivity. A number of reports demonstrating the qualitative and quantitative capabilities of LC-CF/FAB-MS analysis systems have appeared. These were reviewed through 1991 by Caprioli and Suter (41) and applications in forensic analysis were recently reviewed by Sato et al. (42). Some recent examples of quantitation using this technique illustrate its capabilities for metabolite identification and measurement. An elegant automated system for analysis of diethylstilbesterol (DES) isomers in urine was described by Davoli et al. (43). Samples with added stable isotope-labeled DES internal standards were extracted on an immunoaffinity column. The eluate was concentrated on a C-18 cartridge, chromatographed on a 3 mm i.d. C-18 HPLC column with 0.8 percent glycerol matrix included in the mobile phase. The HPLC eluate (200 l~l/min) was split with about 1 percent going to CF/FAB-MS where spectra were acquired from m/z 40-400. Selected ion plot peak areas were used for measurements. The total process was under computer control with a cycle time of 28 min. They reported detection limits of 2 ng/ml of sample for both the cis and trans isomers of DES with 4.6 and 4.8 percent standard errors respectively. Evans et al. (44) reported a method for separation and measurement of urinary bile acids using gradient elution, micro-HPLC-negative ion CF/FAB-MS. They reported detection limits in the pg injected range for a number of bile acids with 1:10 post column splitting. The simultaneous structure-activity determination of disulfiram photolysis products by micro-HPLC-CF/FAB combined with an aldehyde dehydrogenase inhibition assay has been reported (45). The flow from the column was split with 5 percent going to the MS and 95 percent to the inhibition assay. Using this technique Evans et al. were able to closely couple the mass spectral identification to the inhibitory activity of the products. These reports along with many others illustrate the potential of LC-CF/FAB for drug disposition studies with stable isotope-labeled corn-

60 pounds. This potential lies in its capabilities for separation, structure identification and sensitive detection and measurement of a wide variety of compounds, with its best applications being with polar conjugates and peptides.

2.2. Ion Source Interfaces 2.2.1. Thermospray The TSP LC-MS interface was until recently the most used of all LC-MS interfacing techniques and has probably solved more practical analytical problems than any other. This is especially true in the pharmaceutical industry where it has played a very important role in studies of drug disposition. It is now being displaced by the newer API techniques. This trend is expected to continue because most of the compounds TSP analyzed best are now better analyzed, with greater ease, by the API techniques. For this reason this discussion of TSP will be brief. TSP was invented and developed largely through the efforts of Vestal and coworkers (46). Reviews of TSP that deal in considerable detail with its development and application have appeared (47-49). A schematic representation of a TSP interface is shown in Figure 4. It operates by rapidly heating the LC solvent (to 200-300 ~ in the heated entrance tube) forming an aerosol that sprays directly into the heated source. There under reduced pressure, the droplets rapidly desolvate and ionization takes place by solvent-mediated chemical ionization processes. The solvent must contain ammonium acetate or another volatile buffer as a source of ions for true thermospray ionization to occur. For some difficult analytes ionization can be promoted by the use of an external energy source such as an electron beam or discharge source

Sampling cone

MS

Vapirizer LC in-*.

.. '

J

m

; Vacuum Pump

:

Fili~~amen t

Repeller

Figure 4. Schematic representation of a thermospray LC-MS interface.

61

to promote ionization of the solvent and subsequently the sample. This mode of operation is often termed assisted thermospray. Controversy is still abundant over the details of TSP ionization. The interface operates well at 1-2 ml/min flow rates that are compatible with normal 4.6 mm HPLC columns and with reverse phase solvents that contain 20-80 percent water, as long as nonvolatile components are absent. This is a major convenience since it allows operation with many already established reversed phase HPLC separations without modification. TSP has achieved considerable success with polar compounds that are of relatively low molecular weight and of appreciable volatility. The initial heating process is quite severe and causes the thermal decomposition of many labile analytes, limiting its usefulness for these. Otherwise, it is a soft ionization technique in that little fragmentation is present and ammonium adducts of the molecular ions are usually strong ions in the positive ion mode. Operation requires the fine optimization of the vaporizer and source body temperatures, both of which are very sensitive to changes in solvent flow rate or composition (gradient operation is very difficult) often resulting in unstable operation over time. This optimization is compound dependent and differs even for compounds that are relatively similar, making analysis for unknowns or mixtures uncertain. Its quantitative capabilities can be good in cases where stable isotopomer or closely related internal standards are used. In an exemplary report, LC-TSP-MS was used for stable isotope tracer studies to determine the steady state pharmacokinetics of carbamazepine and its epoxide in patient blood samples (50). Moor et al. used [lSN, 13C]carbamazepine and [~SN, ~3C]-carbamazepine epoxide as tracer compounds with their d4 isotopomers as internal standards. Following a single extraction, samples were analyzed by reversed phase HPLC-TSP-MS. The good sensitivity and reproducibility for these compounds allowed for the precise determination of each of these analytes in 0.25 ml pediatric blood samples. The reader is referred to the reviews mentioned above and those by Baillie (7), Blair (8) and Burlingame et al. (5) for further examples of the application of LC-TSP-MS. It will probably continue to be used for some time to carry out established assays but will most likely be replaced by the new API techniques in the future. 2.2.2. Atmospheric pressure ionization (API)

The API techniques are the most used and intensely developed of all LC-MS techniques today. The announcement of electrospray (ES) ionization by Fenn and coworkers (4) began the revolution that has resulted in the further devel-

62 opment of ES, and the derived technique IS, into very robust analytical tools for analysis of a very wide range of analytes. What was most exciting in their early reports (for a review of these see Ref. 51) was the demonstration that ES could generate multiply-charged ions with very high efficiency from intact proteins having molecular weights up to 40,000 and that the high charge states on these ions allowed them to be analyzed using relatively inexpensive quadrupole instruments with m/z limits of less than 2,000. The third API technique, APCI, was first reported by Horning et al. (52); (reviewed by Carroll et al. (53)) as both a GC-MS and LC-MS interface but its potential was not fully appreciated until recently. These three API ion source/LC interfaces have much in common in design. They are now the most used and rapidly developing techniques for LC/MS today. This popularity seems to be especially high in studies of drug disposition (8). The abilities of ES and IS for the analysis high molecular weight biopolymers (e.g. peptides, proteins, oligosaccharides, polynucleotides, etc.) as well as small molecules opened up a whole new world for mass spectrometry in biology. The impact of these developments is now becoming apparent and new areas of application are rapidly developing. 2.2.2.1. Electrospray and ionspray In ES ionization, nebulization of the liquid flow stream occurs solely by electrostatic means in a strong electrostatic field. Multiply-charged analyte ions then result from residual charge left on solute molecules after evaporation of the solvent. Figure 5 is a schematic representation that will serve to illustrate the operation of all three API interfaces. Many variations on the design shown here are discussed in the reviews mentioned above. In ES, ion production

I

Solution in

-=,l

I Ion Optics

I I

Nebulizer

Atmospheric Pressure

,.

! I I

Mass Analyzer

ITI i!l I I Drying gas

1 st

2 nd

Vacuum Pumps Figure 5. Schematic representation of an electrospray/ionspray LC-MS interface. Also serves as a representation of a atmospheric pressure chemical ionization interface after addition of a corona discharge needle.

63 occurs in two steps. In the first step, solvent flows in through a stainless steel tube (1-5 i~l/min) with its outlet at atmospheric pressure and at several thousand volts potential relative to the surrounding chamber (negative potential for positive ion production, positive potential for negative ion production). An aerosol of highly charged droplets of the analyte-containing solvent is generated by a charge dispersal mechanism. The next stage is evaporation of solvent from the droplets by collision with a dry bath gas that is flowing counter to the direction of ion travel. This serves to exclude large droplets from the mass spectrometer and aid in desolvation of ions as they are drawn electrostatically toward the ion exit. As the neutral solvent evaporates, the concentration of charge on the droplets increases to the point that Raleigh fission occurs, eventually resulting in multiply-charged analyte ions. These are then electrically drawn through pressure reduction stages into the mass analyzer. Recent reviews of the ES ionization process and its applications have appeared (51, 54) and are recommended for a detailed discussion of the processes involved. Not all types of compounds provide a good response with ES and IS ionization (55). Compounds that respond best are those that are ionic in solution. Those that can be ionized through Brensted or Lewis acid/base chemistry also respond well providing (M + H) § or other cation adducts or (M - H)- ions. A sizable majority of the compounds of interest to biologists and pharmacologists are good responders. Neutral, nonpolar compounds give very low ion yields and are not usually detected by ES-MS with useable sensitivity. Van Berkel and associates have devised several chemical derivatization strategies for such compounds to generate "ES-active" forms by derivatization for a variety of functional groups before analysis (56) and an on-line post-separation derivatization for aromatic and highly conjugated compounds (57). These and other efforts have successfully extended the ES advantages of high sensitivity and in some cases selectivity to a broader range of compounds. ES ionization imposes limitations on flow rate, ionic strength and dielectric constant that put restraints on its use as an LC-MS detector. Electrolyte concentrations higher than 10-4N result in poor ion production, unstable operation and limit the flow rate to very low values. Solvents with low dielectric constants (e.g. methanol) give the highest ion yields, while high dielectric constant solvents (e.g. water) require higher ES voltages and result in lower ion production. The problems caused by highly aqueous solvent systems have been overcome to some extent by mixing methanol or acetonitrile with the sample stream (usually at the probe tip) to achieve better ion production and better stability. Still the use of ES as a HPLC detector is difficult and only

64

a few examples of such have appeared. Capillary electrophoresis and capillary isotachophoresis are techniques that give very high separating power and operate at very low flow rates (0-100 nl/min). They are well suited to interfacing with ES-MS by addition of a post capillary make-up solvent. Many examples have appeared demonstrating the utility of these in a wide variety of analyses including peptides and proteins for which they are most popular (58). The ionspray (IS)interface was developed as a solution to the problems of interfacing HPLC to ES. The significant modification is that, instead of nebulization solely by electrostatic means, a pneumatic nebulizer is used to assist in aerosol formation. This overcomes many of the solvent composition limitations of ES and allows flow rates of 200 i~l/min or higher. Other thermal and ultrasonic assisted ES nebulization devices have been used with similar results. While much higher flow rates are allowed with IS, allowing it to be used with conventional HPLC systems, the sensitivity does not increase as a result of a higher rate of sample delivery. Response in ES and IS depends upon analyte concentration and not its rate of sampling. Another advantage of IS is that gradient elution, which is generally not possible with ES, is usually not difficult. The analysis of large biomolecules by ES and IS coupled to MS or MS/MS has been the major impetus behind the development of these interfaces. These efforts have generated an extensive literature that was recently reviewed by Loo et al. (54). Molecules with molecular weights extending to over 100,000 yield mass spectra consisting of a series of multiply-charged (M + nil) n+ ions (n approximates Mr/I,000) differing by one charge. These fall in a m/z range that can be analyzed using mass spectrometers with mass ranges of m/z 2,000 or less. The spectra, consisting of ions of differing charge states, can be easily deconvoluted by MS data systems to spectra showing only the singly charged species. Molecular weights can be determined with an accuracy of about 0.01 percent, making ES and IS the most accurate way to determine molecular weights of large molecules. Structural information for biopolymers can be obtained by collisional activation dissociation (CAD) MS/MS of a selected multiply-charged molecular ion. The resulting product ions also occur in a series of charge states and are highly useful for determining the sequence of biopolymers. For higher molecular weight compounds, the resulting MS/MS spectra are very complex, containing multiple fragment ions, all with multiple-charge states, and assignment of charge state can be impossible. The use of higher resolution instruments, such as magnetic sector or FTICR mass spectrometers, can allow the measurement of one m/z unit separation of 13C isotope ions to determine charge state of individual ions

65 and their atomic mass, allowing better mixture resolution. MS/MS analyses have been used to obtain sequence information on proteins in excess of 65,000 molecular weight. Analysis of small molecules by ES and IS is highly effective for many compound classes. Generally for compounds of less than molecular weight 1,000, little multiple charging is seen unless multiple anionic or cationic functional groups are present. Very little fragmentation is seen even for very labile compounds when the temperature and flow rate of the bath gas are proper (51). Even though the current interest in ES/IS is due to its capabilities for analysis of large molecules, a major area for application of LC-ES/IS-MS is the analysis of small molecules. API techniques seem likely to replace others for most analyses of small molecules by LC-MS. The number of reports of applications of ES and IS LC-MS to pharmaceutical analysis has become quite large and are appearing at an increasing rate. This is an indication of the acceptance of these techniques as near universal interfaces for LC-MS. Only a few examples from this large literature can be mentioned here. The reader is referred to the recent review by Loo et al. (54) for examples of its use for analysis of large molecules and to the review of mass spectrometry in studies of drug disposition and pharmacokinetics by Blair (8) for other examples of its use in analysis of small molecules. In one study, Moseley and Unger (59) evaluated the combination of packed capillary chromatography with ES-MS for the characterization of protein mixtures in the development of pharmaceuticals. They concluded that it performs very favorably compared to SDS/PAGE gel electrophoresis that is traditionally used for this purpose, by providing good chromatographic separation with sensitive detection and accurate molecular weight determination. An excellent example of the measurement capabilities of LC-IS-MS was reported by Murphy et al. (60). They demonstrated the rapid (2.5 min), sensitive (0.075-5.0 ng/ml), and accurate LC-IS-MS/MS determination of xanomeline (a muscarinic receptor agonist) in human plasma. This method was used clinically for determination of the pharmacokinetics of this compound. Another example of quantitation of small molecules by LC-ES-MS was reported by Pacifici et al. (61). They measured levels of morphine and its 3- and 6-glucuronides in serum, achieving detection limits of 10, 100 and 50 ng/ml respectively, and applied the assay in pharmacokinetic studies. 2.2.2.2. A t m o s p h e r i c pressure chemical ionization

An APCI interface physically resembles an ES/IS interface and APCI operation is a possible operating mode with many ES/IS interfaces, but the ionization process appears to be distinctly different (62). The consequential differences

66 between ES/IS and APCI are that a corona discharge needle is placed in the source and high voltages are not used to form an aerosol or charge the droplets in APCI. Unlike ES/IS, APCI is a gas phase process and requires thermal volatilization of the analytes before ionization. The mechanisms involved are the same as in a standard CI ion source: ion molecule reactions, charge transfer and electron capture. APCI uses the chromatographic solvent vapor as the reagent gas. In operation, the solvent is nebulized via a heated pneumatic nebulizer and the aerosol is carried through a heated tube, where the solvent and analytes are evaporated, then to an ionization region where a corona discharge needle generates solvent reagent ions for APCI. As in ES and IS, the neutral volatile solvent is swept away by a counter-current flow of drying gas while the analyte ions are drawn into the mass spectrometer by electrostatic forces. A wide range of polarities of solvents that may also contain nonvolatile buffers can be used with flow rates up to 2 ml/min, making APCI one of the least restrictive on the HPLC system of all LC-MS interfaces. APCI as a gas phase ionization technique is complementary to the solution ionization techniques ES/IS. It is useful for analysis of lower molecular weight compounds that have sufficient thermal stability and volatility. It does not produce the multiply-charged ions seen with ES/IS but gives spectra that are very similar to those obtained with normal CI. As such it has found much use for the LC-MS analysis of pharmaceuticals. Its ease of use, and the lack of restrictions it places on HPLC systems, have led to its broad acceptance as a replacement for the once pervasive TSP interface. As in TSP, the major limitation of APCI for analysis of polar, higher molecular weight compounds derives from the requirement that analytes be thermally vaporized for ionization. A major strength is that it gives a more constant ion yield with diverse types of compounds than the solution ionization techniques. This is because APCI does not require charged analytes in solution to achieve high sensitivity. Its general applicability to quantitative analysis of small molecules makes it a natural choice for many of the analyses performed with pharmaceuticals. Numerous examples can be found of LC-APCI-MS applied to pharmaceutical research. These have been reviewed recently by Bruins (63) and Gelpi (9). One study of the relative performance of APCI, ES and TSP for use in studies of drug metabolism paid particular attention to compound polarities and the resulting sensitivity achieved by each technique (64). They found that the best sensitivities for nonpolar compounds were obtained with APCI but, as expected, it was not as useful for hydrophilic compounds such as glutathione conjugates. TSP was reported to be useable for all compounds studied as was ES. However, ES gave 10-100 times more sensitivity for hydrophilic analytes. The determination of pilocarpine (used to treat glaucoma) in aque-

67 ous humour by LC-APCI-MS was reported by Matsuura et al. (65). They obtained linear calibration curves from 2 ng to 10 ~g/ml of sample and excellent intra- and inter-day precision without the use of internal standards. This provides an example of the high stability (and sensitivity) that can be expected with LC-APCI-MS. A elegant LC-APCI-MS/MS multiple reaction monitoring assay for MK-434 (a 5(x-reductase inhibitor) and its two principal metabolites in plasma was reported (66). Using less than 5-min runs they were able to measure concentrations from 0.5-50 ng/ml with high precision, accuracy and specificity. Fraser et al. (67) compared LC-APCI-MS/MS to GC-MS for the analysis of an inhibitor of acylcoenzyme A cholesterol acyltransferase in plasma. The LC-MS/MS method had the advantage of not requiring the extensive sample clean-up and derivatization of the GC-MS procedure and provided increased sensitivity, selectivity and speed. Many other examples of the usefulness of LC-APCI-MS can be found that are models for development of new assays.

3. CONCLUSIONS AND FUTURE DIRECTIONS

The development of methods for interfacing HPLC directly to MS has proceeded with vigor for the last twenty-five years or so resulting in the mature techniques discussed here. LC-MS now finds routine use and has become an extremely important technique in a number of application areas, including many phases of pharmaceutical development. It is expected that growth in reliance on LC-MS will increase with the increased availability of modern interfaces in laboratories. It remains to be seen what other LC-MS interfacing techniques may be introduced or what improvements may be made to the present ones. The API interfaces exceed the capabilities envisioned by their inventors and they can be expected to be the most useful for solving problems in biology for some time to come. They appear to have achieved a high degree of development, which is not to say that further improvements are unimaginable or unlikely to occur. It is expected that certain apparent limitations will be addressed and improved devices developed. For instance, the API techniques provide high sensitivity by producing nearly total ionization of analytes, but today's interfaces can only direct a small percentage of those ions into the mass spectrometer. Efforts to improve ion transmission are likely to result in an enormous improvement in sensitivity. Similarly, the PB interface only obtains an efficiency of a few percent for transmission of solute to the mass spectrometer ion source. Further developments can be expected to increase this efficiency.

68 The further development of highly popular MALDI techniques for use with LC-MS, possibly with PB or continuous flow probes, seems a likely area of future accomplishment. While other developments will surely occur, it seems unlikely that, with the major goals met or wildly exceeded, there will be the same level of innovation in the next twenty-five years as the first. Much of the developmental work that used to be done in academic laboratories has now moved into those of the instrument manufacturers where further product development can be expected to be more incremental and less innovative. The increasing domination of the API techniques is evident from the reports presented at the annual meeting of the American Society of Mass Spectrometry (ASMS). Figure 1 is an update of a similar graph by Niessen and Tinke (6) and shows the number of papers reporting on the use or development of each LC-MS interfacing technique at each annual meeting. Clearly, ES/IS have come to dominate. At the 1995 conference, of the 464 reports concerning LC-MS interfacing techniques, 360 (84 percent) of them concerned ES/IS. Most of these reports concerned applications of API techniques to biological problems and did not deal with further development of the devices, indicating general satisfaction with their present capabilities. It should be noted that many of these reports concern direct injection of samples into a flow stream and not actual LC-MS experiments. This is particularly true for ES which is often used for characterization of isolated macromolecules. Reports at this conference generally deal with methods in their developmental or early application phases and may not reflect overall use in routine analysis, which probably relies more on well-established methods. Indeed some LC-MS interfacing techniques that are not or are poorly represented here may still be the best available in particular situations. This author feels this way about the now little used moving-belt interface for complex lipid analysis (2, 3, 68, 69). No ASMS papers dealt with MB-MS in 1995. A variety of liquid mobile phase separation techniques have been directly interfaced to mass spectrometry including: capillary liquid chromatography (70), capillary electrophoresis and isotachophoresis (58), normal column HPLC, (pseudo)electrochromatography (71), ion chromatography (72) and thin layer chromatography (TLC-MS) (73). Many of these have solvent composition or flow rate requirements that make them especially suited to use with one or more of the LC-MS interfaces mentioned above. Table 1 presents a generalization of the operating parameters that apply to each interface and limit its applicability as a interface for chromatography-MS. Apparently almost any conceivable liquid-based separation technique has been interfaced to MS using one of these interfaces, without serious operational compromise for either the separation or mass spectrometry systems. The routine use of corn-

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70 bined liquid separation-MS techniques is now practical and convenient for most bioorganic compounds. In the future we can expect the use of these techniques to vastly expand as the availability of lower cost, easy-to-use instrumentation increases.

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73

CHAPTER 5

STABLE ISOTOPES IN PHARMACEUTICAL RESEARCH: TANDEM MASS SPECTROMETRY

CECILIA BASIC ~, SUSAN F. SILVERTON 2 and RICHARD A. YOST 3 1Department of Chemistry, Villanova University, Villanova, PA 19085-1699; 2Department of Oral Medicine, School of Dental Medicine, 525 Levy Research Building, 4010 Locust St., University of Pennsylvania, Philadelphia, PA 19104-6002; 3Department of Chemistry, University of Florida, Gainesville, FL 32611-7200

1. INTRODUCTION

Tandem mass spectrometry (MS/MS) is a powerful analytical tool which not only allows the quantitative analyses of complex biological mixtures but also provides a high degree of molecular specificity. In contrast to the technologies of high-performance liquid chromatography (HPLC), gas chromatography/ mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) which have been adapted relatively easily into pharmaceutical research, MS/MS has been under utilized. Part of this under utilization is due to the instrument cost, and part to the inherent individuality of each new application which requires appreciable research investment. Where, then, has MS/MS been used successfully, and in particular, where has its use in concert with stable isotopes significantly advanced the field of pharmaceutical research? To address this question, a series of illustrative examples in which stable isotopes have been used in conjunction with MS/MS methods will be presented. Prior to this, however, a general introduction to MS/MS will be provided, including descriptions of the more common types of MS/MS instruments, summaries of their relative performance capabilities, descriptions of the types of tandem scan methods available, and the qualitative and quantitative information these scan methods can provide. The chapter is designed so that readers who are more familiar with MS/MS may proceed directly to the

74 series of illustrative examples, while others may benefit from the material presented in the first part of the chapter. It should be stressed that the overview of MS/MS is by no means exhaustive, nor is the presentation of the stable isotope applications. Rather, the chapter is designed to familiarize the reader with the instruments and terminology encountered in MS/MS and to highlight the power of MS/MS when used in conjunction with stable isotopes. Readers are encouraged to seek more detailed information in the references provided or in the additional reading list.

2. TANDEM MASS S P E C T R O M E T R Y - AN-OVERVIEW

Two types of scan methods can be employed in conventional MS analyses: normal (full-scan) MS and selected-ion monitoring (SIM). In full-scan MS, the mass-to-charge (m/z) ratios of either all the ions (or a range of ions formed in the ion source) are recorded. For relatively pure compounds undergoing sufficient fragmentation in the source, a normal mass spectrum can provide molecular weight (MW) information as well as ion structural information vis-a-vis the m/z ratios of the fragment ions. In SIM, only ions of a single m/z ratio (or a selected few m/z ratios) are transmitted and detected, providing improved sensitivity due to the increased signal-to-noise (S/N) ratio obtained by dwelling only on the ion signal of interest rather than on the background ions. SIM is of value in the quantitation of targeted compounds (1). The ability to obtain unambiguous MW and ion structural information from normal mass spectra becomes limited in the analysis of impure compounds or complex mixtures, since the normal mass spectrum of a mixture cannot definitively establish which ions are molecular ions (e.g. M § or M-) nor can it establish any links between a given molecular ion and its associated fragment ions. While SIM can serve to select a specific targeted ion for analysis, it does not provide any structural information. Normal MS also has limitations in analyses using "softer" ionization methods, such as fast atom bombardment (FAB; see Appendix). Many softer ionization methods used in biomolecular analyses result in the formation of a single ion characteristic of the compound of interest, most commonly the protonated MH § or deprotonated [ M - H ] - molecular ion. Normal FAB/MS can provide accurate MW information, but little or no structural information due to the lack of fragment ions in the mass spectrum. MS/MS analyses involve the initial separation, or mass-selection, of an ion of interest from the ensemble of ions formed in the source, the dissociation of this precursor (or parent) ion in a collision region of the mass spectrometer,

75 and the subsequent mass-analysis of the resulting product (or daughter) ions. Dissociation is usually achieved via collision-induced dissociation (CID, or collisionally-activated dissociation, CAD) with a neutral target gas. The two stages of mass-analysis, separated by a dissociation step, allow the ability to establish relationships between a given ion and its associated fragment ions and provides enhanced fragmentation to gain detailed ion structural information. Thus, MS/MS provides a greater degree of selectivity and molecular specificity than that found in conventional MS methods (2).

2.1. Tandem Mass Spectrometers Tandem mass spectrometers can be broadly divided into two main types: (i) those in which the mass-selection, dissociation, and mass-analysis steps are performed in different spatial regions of the mass spectrometer (tandemin-space); and (ii) those in which the steps are performed within the same region of the mass spectrometer but at sequentially controlled times (tandemin-time). Tandem-in-space instruments require at least two coupled mass analyzers separated by a collision region (Figure 1), while tandem-in-time methods are performed with a single mass-analyzing device which can trap and store ions. Both ion cyclotron resonance (ICR) and quadrupole ion-trap mass spectrometers (QITMS) are tandem-in-time instruments. While these

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Q B E BE EB

Figure 1. Schematic of a tandem-in-space mass spectrometer illustrating mass-selection, collision-induced dissociation (CID) and subsequent mass-analysis of fragment ions. (Q, quadrupole mass filter; B, magnetic sector and E, electric sector.)

76 ion-trapping mass spectrometers have been successfully used to analyze biological molecules (3, 4), they have not seen widespread use in routine MS/MS analyses and as such will not be discussed further. Interested readers are referred to several reviews of both ICR (3, 5, 6) and QITMS (4, 7, 8) instruments. Three tandem-in-space mass spectrometers have found widespread use in pharmacological studies: triple quadrupole (QlqQ2), double-focussing sector (BE and EB), and hybrid sector (BEqQ and EBqQ) mass spectrometers. The performance capabilities of these instruments are dictated in part by the operating principles of the different mass analyzers, i.e. the way in which quadrupole mass filters (Q) (9-11), and magnetic (B) and electric (E) sectors (9, 12, 13) separate ions of differing m/z ratios, and by differences in the design and operation of rf-only quadrupole collision cells (q) versus the collision cells used in sector mass spectrometers. The performance characteristics of the different tandem mass spectrometers can be assessed based on (9): (i) the maximum m/z ratio which can be measured; (ii) the resolving power of the instrument and the concomitant ability to perform accurate mass measurements; (iii) the ability to rapidly switch from one MS/MS scan mode to another; (iv) the ability to perform high- versus low-energy CID; (v) the ease with which the MS can be interfaced to alternate ionization methods and auxiliary GC and LC equipment; and (vi) the cost of the instrument. The performance capabilities of Q~qQ2, BE and EB, and hybrid mass spectrometers are summarized in Table 1 (3) and discussed in greater detail below.

2.1.1. Triple quadrupole mass spectrometers Triple quadrupole mass spectrometers (QlqQ2, Figure 2) make use of quadrupole mass filters (Q) to perform the first and second stages of mass-analysis and an rf-only quadrupole collision cell (q). (Most commercial instruments employ hexapole or octopole collision cells.) Mass-analysis in quadrupole mass filters is based on the inherent stability, or instability, of an ion's trajectory as it traverses the length of the two-dimensional quadrupolar field formed by applying alternating radio-frequency (rf) and direct current (dc) potentials to a set of four parallel, hyperbolic (or circular) rods. Ions entering the mass filter must have kinetic energies of less than 100 eV in order to experience enough cycles of the rf to establish stability/instability and thus provide adequate mass resolution. It is this feature that dictates the use of low-energy CID on quadrupole-based tandem instruments. MS/MS analyses using lowenergy CID on a Q~qQ2 instrument was first demonstrated by Yost and Enke (14).

77 TABLE 1. Performance Characteristics of Common Tandem-in-space Mass Spectrometers a'b

Mass analyzer

Maximum m/z ( D a )

Resolving power

Rapid scanning?

High- vs. low-energy CID

Higherorder MS/MS?

Triple quadrupole Double-focussing sector, BE or EB Hybrid sector BE or EB Q Multiple sector

<2,000 c <3,000 d

Low High

Yes No

Low High

No No

<3,000 d <2,000 ~ <3,000 d

High Low High

No Yes No

High Low High

Yes

Yes

aAdapted from (3). bperformance characteristics based on commercially available mass spectrometers. CQuadrupole mass analyzers with upper m/z ratios of 4,000 Da are available but as yet are not in widespread use. dThis is based on the range obtained using a full acceleration voltage. At reduced accelerating voltages the mass can be extended up to 20,000 Da with an accompanying loss in sensitivity.

01

ion source

first mass analyzer

q

collision cell

G~

second mass analyzer

detector

Figure 2. Schematic of a triple quadrupole mass spectrometer, QlqQ2. (Q1, Q2, first and second quadrupole mass filters; q, rf-only quadrupole, octapole or hexapole collision cell.)

Quadrupole mass filters can analyze ions with m/z ratios of up to 2,000 Daltons (Da) with unit mass resolution (AM = 1) over the mass range of the analyzer. The ion sources of quadrupole instruments operate at, or near, ground potential and, as such, are easily coupled to almost any ionization method, with facile coupling to both GC and LC. The linear scanning capabilities of quadrupoles allow rapid switching between MS/MS scan modes, since both positive and negative ions are transmitted with the same rf and dc fields. Thus, only the dc offsets of the quadrupoles and ion transmission lenses

78 need to be changed in order to switch from positive- to negative-ion analyses (11). QlqQ2 mass spectrometers enjoy widespread popularity due to their ability to rapidly switch between MS/MS scan modes. However, their mass range, resolving power, and mass accuracy are less than those which can be obtained with double-focussing sector mass spectrometers.

2.1.2. Double-focussing mass spectrometers Double-focussing mass spectrometers make use of both magnetic and electric fields to separate ions of differing momentum-to-charge and kinetic-energyto-charge ratios, respectively, and come in two geometries: forward (EB) and reverse (BE, Figure 3). In order to perform mass-analysis using electric and magnetic fields, ions must be accelerated through the magnetic (B) and electric sectors (E) with kiloelectron volt (keV) energies. It is this feature that dictates the use of high-energy CID on sector-based instruments. CID can be performed in the small, rectangular, collision cells located in the first fieldfree region (FFR1) before the first sector, or in the second field-free region (FFR2) located between the sectors (Figure 3). The use of high-energy CID for ion structural studies was first demonstrated by McLafferty et al. (15); subsequent applications to the analysis of complex mixtures was demonstrated by Kondrat and Cooks (16). collision

cell 2

E

,,

El

FFR2

U

FFR1 collision

cell I

i----

r'F detector

F 7 ion source

Figure 3. Schematic of a double-focussing sector mass spectrometer of the forward EB geometry. In a reverse geometry instrument, B precedes E. (B, magnetic sector; E, electric sector; FFR1 and FFR2, field-free regions 1 and 2, respectively.)

79 The mass range of B sectors is dictated by the magnetic field strength, the ion accelerating voltage (V = 4-10 kV), and radius of the magnetic sector (r_< 1 m). Typical maximum m/z ratios range from 3,000 at full accelerating voltage, to no greater than 20,000 using decreased accelerating voltages (9). The scan speed of modern, laminated magnetic sectors is comparable to that of quadrupole mass filters, while older magnetic sectors may require scan times on the order of seconds to compensate for magnet hysteresis. Since the polarity of the magnet must be changed in order to switch from positiveto negative-ion detection, it is not possible to perform positive- and negativeion analyses "on the fly". The coupling of alternate ionization methods or chromatographic systems is often complicated by the need to operate the ion source at the requisite 4-10 kV acceleration voltages. EB and BE mass spectrometers can achieve resolving powers as high as 100,000 (9), where the resolving power is the value of M/AM measured for two peaks, M and M + &M, of equal height for which the valley between the peaks is equal to 10 percent of the peak height. The practical importance of this high-resolution capability is a function of the mass of the compounds of interest. For example, two compounds with a nominal mass of 200 u, e.g. C15H2+o and 013H120 2, + with exact masses of 200,32 and 200,24 differing by 0,08 u would require a resolving power of 2,500 to theoretically separate the two ions. If the same AM separated two ions at a nominal mass of 1,000 u, then a resolving power of 12,500 would be required. While resolutions of this magnitude are easily achieved, increasing resolution requires considerable operator effort and results in a substantial decrease in the ion transmission efficiency. The high-resolution capabilities of EB and BE mass spectrometers are therefore most applicable in the analysis of compounds with MW < 200 u (11). The high-resolution capabilities of EB and BE instruments also allow accurate mass measurements (1-10 ppm) to determine elemental compositions. However, these measurements also lose their practical value at MW > 500 u due to the improved mass accuracies required at higher mass (e.g. _+0.001 u requires 10 ppm at m/z 100 and 2 ppm at m/z 500) and the increasing number of elemental compositions possible at a higher mass (17). Three- and four-sector mass spectrometers of EBE, BEB, EBEB, and BEEB geometries have been developed to overcome some of the resolution problems encountered when MS/MS scans are performed on BE and EB instruments (see discussion below) (18, 19). These multiple sector instruments offer a variety of additional and improved performance capabilities not available with BE and EB mass spectrometers (Table 1). In the discussion of the MS/MS scan modes below and in the sample studies in the second part of the chapter,

80 the multiple sector EBEB and BEEB instruments shall be considered to be two double-focussing instruments separated by a collision region. More detailed discussions of the capabilities of multiple sectors are available elsewhere (18, 19). 2.1.3. Hybrid sector mass spectrometers

Hybrid sector mass spectrometers have the geometry EBqQ or BEqQ (Figure 4) and thus combine the high-energy CID, high-resolution performance capabilities of double-focussing sectors with the low-energy CID, unit mass resolution capabilities of quadrupoles. Moreover, high-resolution mass-selection of the precursor ions with EB or BE is possible for low-energy CID in q. Control of the hybrid configuration is somewhat more demanding than other tandem instruments and switching between scanning modes is less facile than with a QlqQ2 given the need to coordinate the nonlinear scanning of the B sector with the linear scanning of the quadrupole mass filter. However, the flexibility

collision cell 2

B

FNt2

U

r- F m m m

FFR1

collision cell I E

_'-1

F Ion source

---

1 1

deceleration lenses

Q

I_1

detector

Figure 4. Schematic of a hybrid sector mass spectrometer of the BEqQ geometry. (B, magnetic sector; E, electric sector; FFR1 and FFR2, field-free regions 1 and 2, respectively; q, rf-only quadrupole collision cell; Q, quadrupole mass filter.)

81 of hybrid mass spectrometers have made them extremely valuable in ion structural studies (20, 21).

2.2. Collision-Induced Dissociation and Metastable Ion Decomposition As noted, MS/MS analyses make use of either high- or low-energy CID (or both) based on the type of tandem mass spectrometer employed. In CID, the ion of interest is accelerated with kinetic energies (E~ab) between 5 and 100 eV (low-energy CID) or between 1 and 10 keV (high-energy CID) and collided with an inert target gas (T = He, N2, Ar, or Xe) in the collision region of the mass spectrometer. Detailed studies into the dynamics of low- (22) and high-energy (23) CID reveal that fragmentation of an ion (e.g. M § proceeds via a twostep mechanism involving an initial endothermic activation step to form an internally excited molecular ion (M § followed by a separate, exothermic, unimolecular dissociation reaction to form fragment ions (F~) and neutrals (Ni), M + + T--, M § + T M§

F~ + N~.

(1) (2)

Differences in the initial activation step for low- versus high-energy CID can lead to differences in the nature and relative intensities of the resulting fragment ions (22-25). Indeed, high-energy CID spectra of biopolymers can often provide structural information not seen in the low-energy spectra (26), although these differences may arise in part from differences in the internal energy left in the ions from the ionization techniques typically employed on sector instruments (i.e. FAB). Fragment ion intensities and yields are also a function of the nature and pressure of the collision gas employed, as are the transmission efficiencies of both the collision cell and the second mass analyzer. Thus, to obtain reproducible tandem mass spectra on a given mass spectrometer, accurate control of the collision gas conditions and E~ab is required. The pressure of the collision gas is the most difficult parameter to compare between instruments due to differences in the type and location of the various pressure sensing devices. For this reason, the pressure of the collision gas is often controlled by measuring the attenuation of the precursor ion beam as the gas is added to the collision cell. Typical precursor ion beam attenuations range between 10 and 50 percent, although attenuations as high as 90 percent can be used to attain multiple collision conditions on sector instruments (15, 27).

82 Fragmentation efficiencies on a Q~qQ2 range from 15 to 65 percent (14) due to the high focussing capabilities of rf-only quadrupole collision cells. Also, because the length of q is on the order of 20cm, CID on Q~qQ2 instruments typically occurs under multiple collision conditions. In contrast to this, CID on sector mass spectrometers typically occurs under single collision conditions due to the higher E.ab and the smaller collision cells (approximately 20 x 1 mm) employed, resulting in CID efficiencies of <10 percent (16). While MS/MS analyses are typically performed using CID, analyses can also be based on unimolecular, also referred to as metastable ion, decompositions. A metastable ion is an ion which leaves the source intact but which fragments in some region of the mass spectrometer without the aid of a collision gas before it reaches the detector, i.e. the ion undergoes unimolecular dissociation. Metastable ion studies are used extensively in the determination of ion structures and to study reaction mechanisms of small organic ions (28). 2.3. MS~MS Scan Methods In addition to normal full-scan MS and SIM, it is possible to operate any of the above mass spectrometers in four MS/MS modes depending on whether the mass of the precursor ion, the product ion, or the neutral fragment is fixed by the experimental parameters. The four modes are: (i) product ion scans; (ii) precursor ion scans; (iii) neutral loss scans; and (iv) selectedreaction monitoring (SRM, Figure 5). These MS/MS scan modes can be used to selectively monitor either the fragment ions or the neutral losses following CID and can be used to obtain detailed ion structural information and/or screen for classes of compounds present in complex mixtures. The following is a description of how each of the four scan modes can be performed on the above tandem instruments. These descriptions are provided as references when reading the MS/MS experimental sections of the sample studies presented in the second part of the chapter. Only a few of the possible methods for performing product, precursor, and neutral loss scans using EB, BE and hybrid EBqQ and BEqQ mass spectrometers are presented here. More comprehensive presentations of the range of scan methods possible on EB and BE sectors (9, 13, 29, 30) and hybrid sectors (20, 31, 32) are available. 2.3.1. Product ion scans Product ion scans are the most frequently used MS/MS scan mode and involve selecting the precursor ion of interest, performing CID in a collision region of the mass spectrometer, and then mass-analyzing the resulting

83

a) product ion scan ~ J

>.

collision gas

b) precursor ion scan

C ...... /

C

, -T=

r

C r

I

----~

Ill

I

collision gas

c) neutral loss scan

Ill

~f

collision gas

d) selected reaction monitoring

r

'v'

~ I I ~

I

J

collision gas

Figure 5. Schematic of four MS/MS scan modes" (a) product ion scan; (b) precursor ion scan; (c) neutral loss scan; and (d) selected-reaction monitoring (SRM) (2).

84 product ions (Figure 5a). A product ion spectrum is thus a record of all the fragment (or product) ions arising from a single precursor ion formed in the source and can provide detailed structural information for the ion. Product ion scans are acquired on Q~qQ2 instruments by mass-selecting the precursor ion with Q~, performing CID in q, and then mass-analyzing the product ions by scanning Q2. Because quadrupole mass filters separate ions based on their m/z ratios (9), the result is a plot of the intensity versus the m/z ratio of the product ions, with unit mass resolution over the range of the product ion spectrum (2, 11). The most convenient method of obtaining a product ion spectrum on an EB instrument is to perform a B/E l i n k e d - s c a n in which both E and B are scanned together so that the ratio of the magnetic and electric field strengths, B/E, is held constant (9). A B/E linked-scan monitors the product ions formed in FFR1 before E (Figure 3). The resulting spectrum has reasonable product ion resolution, but poor precursor ion selection since mass-selection of the precursor ion with a mass-analyzer does not takes place. Moreover, linkedscans often result in the formation of artifact peaks. The construction of a complete ion intensity map (9) can identify the origin of these peaks, but can be costly in terms of both time and sample (11). B/E linked-scans can also be performed on reverse geometry BE instruments to study the decomposition reactions occurring in FFRI. However, product ion scans are more commonly acquired on BE instruments by massselecting the precursor ion with B, performing CID in the collision cell in FFR2 (Figure 3), and then mass-analyzing the resulting product ions by scanning E downwards from the value corresponding to the voltage at which the precursor ion is transmitted. In contrast to quadrupole mass filters, E sectors separate ions based on differences in the kinetic energy-to-charge ratio of the ions (9, 12). Thus, the result of a product ion scan performed by massselecting the ion with B, is a plot of the relative intensity versus the kinetic energy-to-charge ratio of the product ions. In order to obtain a product ion mass spectrum, the kinetic energy-to-charge ratios of the fragments are converted to their corresponding m/z ratios using: Fi = xMpEi yEp

(3)

where Mp and F~ are the masses, x and y are the charges, and Ep and E~ are the transmission voltages of the precursor and product ions, respectively. Mass-selection with B and the subsequent conversion of the product ion kinetic energy spectrum to the mass spectrum is referred to as m a s s -

85

analyzed-ion-kinetic-energy spectrometry, or MIKES. MIKES analyses cannot be performed on EB instruments and mark the key difference between BE and EB mass spectrometers. MIKES scans on BE instruments can be carried out simply and rapidly; however, because of the kinetic energy released during the fragmentation in FFR2, the resolution of the product ion spectrum is often less than 200 (11). While MIKES spectra do not suffer from the artifact peaks noted in B/E linked-scans, the low translational energies of the lower mass fragment ions can lead to poorer transmission efficiencies through E (11). EBEB, and BEEB multiple sector mass spectrometers overcome the poor product ion resolution found on BE instruments and the poor precursor ion resolution of EB instruments (18, 19). Multiple sector EBEB and BEEB instruments allow product ion scans to be performed using high-resolution precursor mass-selection with (EB)~ or (BE)~, CID in FFR3 between B~ and E2, or E~ and E2, and high-resolution product ion detection with (EB)2. The cost, however, is a loss of several orders of magnitude in transmission efficiency and thus overall sensitivity (18, 19). EBqQ and BEqQ hybrid mass spectrometers can also improve on the low resolution attained for precursor ion selection in B/E linked-scans and the poor product ion resolution in MIKES. Product ion mass spectra can be acquired on hybrid instruments by mass-selecting the precursor ion with EB or BE, performing low-energy CID in q, and then mass-analyzing the product ions with Q. The use of a double-focussing mass spectrometer allows high-resolution mass-selection of the precursor ion and since a quadrupole mass filter is used in the final mass-analysis, unit mass resolution is obtained in the product ion spectrum (11, 31, 32). It is also possible to acquire product ion spectra of the fragment ions formed during high-energy CID in FFR1 or FFR2 of hybrid instruments. The ability to perform both high- and low-energy CID analyses is of particular importance in the MS analyses of biological molecules with MW > 1000 u since molecular ions of this size often have poor fragmentation efficiencies under low-energy CID conditions (20, 26). On a BEqQ instrument, the precursor ion can be massselected with B, undergo CID in FFR2 and then E and Q can be scanned in unison in a Q/E linked-scan mode. A Q/E linked-scan results in improved resolution of the product ion spectrum compared to that obtained on a simple BE instrument; however, this is accompanied by a subsequent loss in sensitivity and the need to scan a series of offset potentials (11 ). Alternative methods of performing high-energy CID studies on hybrid instruments are discussed elsewhere (11, 32). Another powerful capability of hybrid mass spectrometers is the ability to

86 perform sequential product ions scans, or MS/MS/MS analyses. On a BEqQ instrument, an ion can be mass-selected with B and undergo CID in FFR2; a resulting product ion can then be mass-selected with E, dissociated in q, and finally mass-analyzed by Q. Such a scan provides structural information about a fragment ion formed from a mass-selected precursor ion. MS/MS/MS product ion scans can also be performed on the fragment ions formed in the FFR1 of BEqQ and EBqQ mass spectrometers (20, 31, 32). Note that MS/MS/MS analyses, and indeed M S n analyses, are most easily performed on tandem-in-time ion-trapping mass spectrometers (3-8). 2.3.2. Precursor ion scans

Precursor ion scans are performed by passing ions with a range of m/z ratios into a collision region of the mass spectrometer and then setting the final mass analyzer to transmit product ions of a single m/z ratio (Figure 5b). The resulting spectrum contains those precursor ions which fragmented to form the product ion of interest. To perform a precursor ion scan, the m/z ratio of the common fragment ion must first be determined, typically by acquiring product ion spectra of each of the precursor ions of interest or by identifying the common fragment ion from pure samples of the compounds. Since classes of compounds often fragment by loss of a specific functional group, precursor ions scans can be used for the rapid screening of classes of compounds present in complex mixtures (2, 32). Precursor ion scans are acquired on Q~qQ2 mass spectrometers by scanning Q~ over the range of m/z ratios of interest, performing CID in q, and setting Q2 to transmit the targeted product ion (2). The resulting precursor ion spectrum will have unit mass resolution. Precursor ions can be acquired on both EB and BE mass spectrometers using CID in FFR1 by performing a linkedscan in which the ratio B2/E is kept constant, where the value of B2/E is determined by the mass of the product ion of interest (9). Some peak broadening does occur due to the release of translational energy during fragmentation; however, the precursor ions are usually identified with sufficient accuracy (9). In addition, precursor ion spectra can be obtained on BE instruments by scanning B over the m/z ratio range of interest, performing CID in FFR2 and then setting E to transmit only those ions with kinetic energy-to-charge ratios corresponding to that of the product ion. Precursor ion spectra arising from low-energy CID in q can be acquired on both EBqQ and BEqQ hybrid mass spectrometers by using E as a kinetic energy filter (i.e. its value is fixed to transmit ions with kinetic energies equal to the full accelerating voltage), scanning B over the range of m/z ratios of

87 interest, and setting Q to transmit the product ion of interest. These scans result in the precursor ion resolutions on the order of 1,000 and unit mass resolution of the product ion (20, 31, 32).

2.3.3. Neutral loss scans

Neutral loss scans are performed by scanning both the first and the second mass-analyzers in a linked-scan relationship defined by the fixed neutral mass difference and performing CID in a collision region of the mass spectrometer (Figure 5). The resulting mass spectrum contains all the precursor ions which fragmented by loss of the given neutral (2). Once again, the identity of the characteristic neutral loss must be determined from previous product ion scans. Neutral loss scans can also be used to rapidly screen for classes of compounds present in a complex mixture (2, 32). The facility with which neutral loss scans can be performed is a function of the ability to synchronously scan two mass analyzers with a constant offset corresponding to the mass of the neutral. Since quadrupole mass filters have linear scanning capabilities, neutral loss scans are easily performed on Q~qQ2 instruments (2). The nonlinear scan laws of B and E sectors makes neutral loss scans more difficult to perform (9). Neutral loss scans using CID in FFR1 can be performed on both BE and EB mass spectrometers by scanning E and B such that the left-hand-side of the relation

B2(1 - E2/E1) 2V1 (E2/E~)2 = r2 N,

(4)

is held constant throughout the scan, where E1 is the electric sector setting required to transmit the precursor ions, B2 and E2 are the magnetic and electric settings required to transmit the product ions, r is the radius of the magnet, V is the accelerating voltage and N~ is the neutral loss. These neutral loss scans have resolutions on the order of 800 (9). Neutral loss scans can be acquired on hybrid EBqQ and BEqQ instruments by using E as a kinetic energy filter, performing CID in q, and scanning B and Q in unison at a constant neutral offset. It is also possible to perform consecutive product-neutral loss scans, neutral loss-precursor scans, and consecutive neutral loss scans on hybrid instruments (32).

88

2.3.4. Selected-reaction monitoring In SRM, a particular fragmentation reaction can be monitored by selecting a precursor ion, allowing it to undergo CID, and then setting the second mass analyzer to transmit a specific product ion (Figure 5d). SRM scans result in increased sensitivity since the first and second mass analyzers are transmitting ions of only a single m/z ratio. The reader can infer the number of ways in which SRM scans can be performed on the various MS/MS instruments based on the above descriptions of the various scan modes.

2.4. MS~MS Analysis Scheme for Identifying Drug Metabolites MS/MS methods lead to an increased selectivity over that found in conventional MS, and thus lead to a greater degree of molecular specificity. MS/MS scan methods can be used alone or in concert to characterize and quantitate targeted compounds or to identify unknowns in complex biological matrices. Figure 6 presents a MS/MS analysis scheme for identifying drug metabolites in a biological extract (33, 34). The scheme is based on the observation that drug metabolites often retain structural features found in the parent drug (33) and as such normal MS and product ion scans of the pure drug can be used to identify characteristic neutral losses or product ions likely to arise following CID of the drug metabolites (Steps 1 and 2). Neutral loss and/or precursor ion scans can then be used to screen the biological extract for the drug metabolites (Step 3) and identify the m/z ratios of the potential metabolites. The product ion spectra of these ions then lead to the structure of the metabolite (Step 4). This MS/MS scheme is analogous to the radioisotope labels used in traditional methods of metabolite identification. The MS/MS analysis scheme in Figure 6 was used to identify the sites of metabolism of the antiepileptic drug zonisamide (34). A QlqQ= mass spectrometer with methane positive-ion chemical ionization (CI) (35) was employed and no chromatographic separation of the urine extracts was required prior to MS analysis. The normal mass spectrum of pure zonisamide (Figure 7a) shows an abundant MH § ion at m/z 213, as well as [M + 29] + (m/z 241) and [M + 41] + (m/z 253) methane adduct ions and an [M + 18] + (m/z 230) ammonium adduct ion (35). The product ion spectrum of the MH § ion shows a major ion at m/z 132 (representing the ring structure of the molecule) arising from the neutral loss of the sulfonamide (81 u) group (Figure 7b). The neutral loss spectrum of a urine extract obtained with a Q1, Q2 offset of 81 u shows four prominent precursor ions at m/z 213 (MH§ 229, 259 and 299 (Figure 8), identifying three candidate metabolite ions which underwent

89 Step1: .

.

.

.

.

.

Acquire normal mass spectrum of pure drug.

Step2:

1

Acquire product ion spectra for the principal ions observed in step 1.

Step3:

/

",,, Acquire neutral loss spectra of extract based on characterisUc neutral loss found in step 2.

Acquire precursor ion spectra of extract based on the most abundant product ion found in step 2.

Step4:

1

Acquire product ion spectra for those candidate metabolite ions Identified in step 3.

Step5:

1

I Repeatsteps 3 and 4 for other characteristic product ions or neutral l o s s e s Identified in step 2.

Figure 6. MS/MS analysis scheme for the identification of drug metabolites (33).

a neutral loss of 81 u upon CID in q. The precursor ion scan of the urine extract in which Q2 was set to transmit the product ion at m/z 132 shows prominent ions at m/z 213 (MH+), 215, 229, 241 ([M + 29]+), and 255 (Figure 8b). Product ion spectra of the ions at m/z 215, 229, 255, 259 and 299 in the urine extracts were then acquired to assign structures to these candidate metabolite ions. The resulting zonisamide metabolites are presented in Figure 9. The structures of metabolites A (MW = 228) and B (MW = 258) were assigned based on the neutral loss and product ion spectra, while metabolites C (MW = 228) and D (MW = 254) were assigned from the precursor and product ion spectra.

90 MH+ 213

a. zonisamide 100O

II

80.

W

e~

60

._= o

.> _m .e 40

[M + H20] +

20

120 g8

0

L,,=

9

,,

230 136

I !

,

,

149 II "t

.,It,

100

50

.It

.

,,...

,.

.

,,

l, i

150

! 'i

200

!

300

250

m/z 100-

_c.; 80.

O

II II 0

H-.----S---- NH2

"== e0: 0 ,4..,;

.E 4)

.>_. _m 8 4O

-81

MH + 213

20

0

|

50

~

77

104 120

!

Jh

!

100

9

I

~ I 9

149 9

!

w"

"' ' '

"

150

;

I'

200

"

"

"

';

I

250

"

"

"

' "

I

300

m/z Figure 7. M S spectra of pure z o n i s a m i d e : (a) n o r m a l p o s i t i v e CI mass s p e c t r u m ; and (b) p r o d u c t ion s p e c t r u m o f the M H + ion (m/z 213) of z o n i s a m i d e (34).

a.

100-

udne

extract

91

259

229

80.

v)

:

60

0 lu 213 299

2o" I

I 0

9

,

9

t, ,!

9

150

9

9

9

170

9

.|

,

,

190 '

9

,,i,, I

9

9

2t0

,

9

I,I,, , , ,

9

230

9

9

9

250

,

270

9

,,i, ,t, 9

9

9

290

m/z b.

urine

213

extract

100-

80o

"~:

60-

e~

x5

_> Ig

~ 40

241 255

20" 229 132 0

9

100

.

9

,

120

9

9I

,

150 I ,

140

,

~

,

,

160

9

.

~

i

'.

180

,

,

,

200

,

,

lw,.ll. 220

.. 240

,...,I 260

.... 280

, 300

m/z

Figure 8. MS/MS spectra of urine extract" (a) neutral loss spectrum (81 u); and (b) precursor ion scan in which Q2 was set to transmit the product ion at m/z 132 (34).

92 O

!!

CH~--~-NH 2

O

N

O

zonisamide or

i~~

- 81

m/z 132

Io'. II ~'~" "O "N

ell-

OH

NH2 O

II -CH2-- --'--N\ H O

A

C

0

II

CH3-'-'C

I- ~ - 8 1

H

\N /

m/z 132

O

II

Io+H

I

!il o

/C_._CH3

-""'

l.-c.=o "N

-S---N

II

0

"

H

OH B

D

Figure 9. Metabolites of the antiepileptic drug zonisamide identified using the MS/MS analysis scheme in Figure 6 (34).

It can be seen that metabolites A and B retain the intact, unmetabolized sulfonamide portion of the parent drug and thus undergo facile neutral loss of 81 while metabolites C and D retain the intact, unmetabolized ring structures of the parent drug and thus form a prominent m/z 132 fragment ion. The neutral loss and precursor ion scans thus reflect different sites of metabolism and, therefore, provide complimentary metabolite information. It is interesting to note that it was possible to distinguish between the two isomeric monohydroxy metabolites A and C because metabolism occurred on different parts of the parent drug. No structures were assigned to the ions at m/z 215 and 299 because no plausible metabolite structures could be deduced from their product ion spectra (34). This study illustrates how the molecular specificity inherent in MS/MS

93 methods can lead to the assignment of metabolite structures from a biological extract without isotopic labeling. MS/MS analyses in conjunction with stable isotopes leads to an even greater degree of molecular specificity; the remainder of this chapter is dedicated to presenting a series of illustrative examples of MS/MS analyses using stable isotopes.

3. M S / M S ANALYSES USING STABLE ISOTOPES

Given the wide flexibility of MS/MS instruments and techniques, we are limited to describing only a few selected examples of MS/MS utilizing stable isotopes representing several different approaches for quantitation, identification, and characterization of compounds of pharmacological interest. The examples fall into two main categories: (i) quantitation using stable isotope dilution MS/MS; and (ii) the use of stable isotope tracers in MS/MS analyses for identification and structure elucidation. The primary purpose of the following sample studies is to illustrate the range of MS/MS applications in pharmacological research. As such, the summaries are focussed on the MS/MS methods employed and the contributions of the MS results to the overall conclusions of the study. Details of the MS methods have been presented in italics with specific terms or MS/MS methodologies described in the first part of the Chapter. Since many of the studies use fast atom bombardment (FAB), a summary of this ionization method has been presented in the Appendix. A list of additional reading including other MS/MS stable isotope applications has also been provided.

3.1. Quantitation Using Isotope Dilution MS~MS The selectivity of two stages of mass-analysis in MS/MS is of particular value in quantitating trace components in complex biological samples. Alternating MS/MS scans (product, precursor, neutral loss or SRM, Figure 5) permit monitoring of the analyte and its isotopically labeled internal standard for quantitation. In isotope dilution MS/MS, quantitation is achieved by measuring the ion intensities (or areas) corresponding to the unlabeled compound and that of a known amount of labeled internal standard. The ratios can be determined from the product, precursor, neutral loss, or SRM spectra and then compared to an appropriately constructed standard curve. The use of ratio measurements increases the precision of quantitation in that it accounts for changes in the ion intensities due to changes in the extraction efficiency, variability in injection volumes, adsorption losses, and any chemi-

94 cal interferences. It also allows quantitation of the targeted compound independent of volume. The following examples illustrate the use of isotope dilution MS/MS to quantitate targeted compounds in a series of biological matrices. Results of the quantitative studies were used for the diagnosis and rapid screening of disease states (36-38), for tracking targeted compounds in tissue (39-42), and for pharmacokinetics studies (43). 3.1.1. Carnitines in urine and plasma: diagnosis of carnitine deficiency

L-Carnitine (3-hydroxy-4-N-trimethylammonium butyrate) serves as a carrier of acyl groups across the mitochondrial inner membrane and is essential for the transport and metabolism of fatty acids (36, 37). Genetic disorders of fatty acid or amino acid catabolism may result in accumulation of abnormal, disease-specific, carnitines with the concomitant depletion of carnitine stores. Since carnitine is used in vivo as a scavenger of accumulated toxic metabolites, the depletion of carnitine can lead to sudden death (37). As such, a rapid and reliable method for the routine assay of carnitines in biological fluids was required for early diagnosis of carnitine deficiency (36). To this end, total and free carnitine in urine and free and short-chain carnitines in plasma were assayed using isotope dilution in combination MS/MS. The carnitines were butylated prior to MS/MS analysis to increase the intensities of the FAB ionization signals. The results were then compared to those obtained using the standard method of radioenzymatic assay (REA) (36). A Q 7qQ2 mass spectrometer and FAB ionization were employed. Product ion scans of the MH § ions of butylated synthetic carnitine (m/z 218) and the butylated internal standard, D3-carnitine (m/z 221), were performed using Ar collision gas at a pressure of 0.2 to 0.5 • 10-6 Torr and an E/ab of 15 e V. The MH + product ion spectra revealed a common fragment ion at m/z 103. The butylated carnitine levels in the urine and plasma extracts were therefore determined from the ratios of the nondeuterated and deuterated MH § ions in the precursor ion spectra obtained by scanning the Q7 from m/z 200 to 240 and setting Q2 to detect the ion at m/z 103.

Figure 10 shows the m/z 103 precursor ion spectra of butylated carnitine and butylated carnitine plus the isotopically labeled internal standard in a urine sample. Calibration curves for carnitine in urine constructed from the precursor ion spectra showed excellent linearity over the ranges 0-1500 nmol/ml

95 1oo

1oo

b.

o~ 215

220 m/z

225

215

220

225

m/z

Figure 10. Precursor ion spectra of (a) butylated carnitine (m/z 218), and (b) butylated carnitine plus the butylated isotopically labeled internal standard D3-carnitine (m/z 221). The product ion at m/z 103 was monitored (36). Adapted with permission from Elsevier Science.

(R = 0.999), and 0-20 nmol/ml (R = 0.998). The calibration range for free carnitine in plasma using MS/MS was 0-200 nmol/ml. Similar coefficients of variation (<4 percent) were found for measures of carnitine using REA and MS/MS. The concentrations of free and total carnitine in urine samples taken from 26 patients correlated well with those measured using REA, i.e. correlation coefficients of 0.939 and 0.918 were obtained for free and total carnitine over the range 0-100 nmol/ml and coefficients of 0.989 and 0.929, respectively, were obtained over the range 100-10,000 nmol/ml. The limit of detection of the MS/MS analysis was <8 pmol of carnitine added to the FAB probe tip and better than 1 nmol/ml of carnitine could be detected in urine or plasma. Overall, FAB/MS/MS had comparable accuracy, precision, specificity, and sensitivity to those found with REA but required less analysis time (<2 min per sample) and, thus, allowed higher sample throughput than REA (36).

3.1.2. Amino acids in blood spots: screening for phenylketonuria Isotope dilution MS/MS has been used for the rapid diagnosis of phenylketonuria (PKU) and as a second line method to screen for possible false positives generated by standard fluorometric methods (38). Levels of phenylalanine (Phe) and tyrosine (Tyr) in small volumes of plasma (100 i~1) and whole blood (~11 i~1, extracted from dried blood spots) were measured using MS/MS following extraction and derivatization.

96 A Q TqQ2 mass spectrometer with a liquid secondary ion source (LSlMS or FAB, see Appendix) was employed. Product ion spectra were obtained using Ar collision gas (precursor beam attenuation of 50 percent) and an E/ab of 30 e V. The MH + ions of the butyl ester derivatives of synthetic Phe (m/z 222) and Tyr (m/z 238) gave rise to product ions 102 u less than the corresponding precursor ions. Quantitation of the blood spot extracts was therefore based on the ratios of the MH § ions of butylated Phe and Ds-Phe (m/z 227) and Tyr and D4-Tyr (m/z 242) in the neutral loss spectra obtained by simultaneously scanning Q7 from m/z 125 to 300 and Q2 from m/z 23 to 198.

Typical concentrations of Phe and Tyr in the blood spots were on the order of 50-150 i~mol/I, with estimated lower limits of 3 and 10 i~mol/I, for Phe and Tyr, respectively, well below physiologically normal ranges. The precision of the assay of blood spots, determined for the same sample over a one month period, was 8.8 percent for Phe, 9.7 percent for Tyr and 11.5 percent for the Phe/Tyr ratio. Quantitative analysis of Phe and Tyr in the blood spots found that the Phe/Tyr ratio of the control group was 0.71 _+ 0.22 (range 0.2-1.4, n = 55) while that of the PKU cases was elevated to 7.8 _ 6.5 (range 2.6-22, n = 8). The MS/MS Phe/Tyr ratios successfully identified five false positives from previous fluorometric assays and could be used as a rapid method for the diagnosis of phenylketonuria (38). For these analyses, even higher sensitivities might have been achieved using SRM since SRM would have required the scanning of only two ions as opposed to the 175 u range scanned here. 3.1.3. Opioid peptides in human pituitary

The enkephalins are opioid peptides which play a role in chronic pain syndromes and in psychiatric disorders (39). The pentapeptide methionine enkephalin (ME, tyrosine-glycine-glycine-phenylalanine-methionine, Tyr-GlyGly-Phe-Met) was targeted for analysis in tissue from human pituitaries as an initial compound in the investigation of mechanisms of pain and pain relief (39, 40). In an effort to identify the analytical method with the highest molecular specificity, internal standards of Ds-ME and MS/MS were used to measure the endogenous ME content of individual post-mortem pituitaries. The results were compared to those obtained from radioimmunoassay (RIA), and radioreceptor assay (RRA) (39). MS~MS spectra of the HPLC purified extracts of ME and Ds-ME were acquired using an EB double-focussing sector mass spectrometer with a

97 FAB ionization source. SRM scans were performed using alternating B/E linked-scans to monitor the product ions at m/z 425 and 429 arising from the metastable decompositions of the MH + ions of ME (m/z 574) and Ds-ME (m/z 579). Quantitation was carried out using the ratios of the ME and Ds-ME MH § ions from both the normal and SRM mass spectra.

The four analysis methods yielded the following ME results: RRA, 7.9 +_ 1.9 ~g/g tissue; RIA, 1.8 +_ 0.7 ~g/g tissue; normal MS, 2.7 _+ 0.6 ~g/g tissue; and SRM, 3.0 _ 0.8 I~g/g tissue. RRA showed an overestimation of ME, probably due to the nonspecificity of the receptor. The three other methods were not significantly different in estimating the ME concentrations. However, the degree of molecular specificity of the methods varies, i.e. 120 different pentapeptides will have exactly the same amino acid composition, accurate mass and composition as ME. As such, normal MS may not unambiguously quantify ME in a given extract. However, SRM overcomes this limitation due to the higher level of molecular specificity provided by the link between the MH § ion and the targeted product ion (39). 3.1.4. Tryptolines in brain tissue: endogenous levels and artifactual formation

Tryptolines (TLNs) are plant alkaloids which can be formed in mammalian tissue under physiological conditions from the condensation of endogenous indoleamines and aldehydes from dietary sources. The presence (or absence) of tryptolines in the brain is of interest due to their possible link with chronic alcoholism (41, 42). GC/MS and GC/MS/MS methods using isotope dilution were developed in an effort to identify and quantify tryptoline (TLN), methyltryptoline (MTLN), 5-hydroxytryptoline (5-HTLN) and 5-hydroxymethyltryptoline (5-HMTLN) in whole and dissected portions of rat brain tissue (42). These analyses were confounded by the possible ex vivo formation of TLNs from indolethylamines and aldehydes during sample preparation. To assess this possibility, the analyses were performed upon addition of an D4-1abeled indolethylamine internal control to check for the artificial formation of TLNs during the analytical procedures. Derivatization of the extracted tissue TLNs to their heptafluorobutyryl derivatives (HFB-TLN) was performed prior to MS analysis to take advantage of the added sensitivity offered by electron capture (EC) negative ion CI (35). MS/MS analyses were carried out on a Q TqQ2 mass spectrometer using EC negative ion CI with methane moderating gas (35). SRM scans were acquired by mass-selecting the [M-HF]- ions with Q7 and monitoring the

98 m/z 179 product ion with Q2. ClD was performed using N2 gas at a pressure of 1 x 10-9 Torr and a collision energy of 20 e V. Quantitation was based on the ratio of the [M-HF-] ions of the derivatized TLNs to the those of the respective internal standards De-TLN, Ds-MTLN, Ds-HTLN and Ds-5-HMTLN seen in the SRM spectra. The indolethylamine internal controls were D4-tryptamine and D4-5-hydroxytryptamine.

MS/MS analysis of whole rat brain tissue indicated that tryptolines are normally present in brain at concentrations of 1.0-3.0 ng/g for TLN and 0.24.4 ng/g for MTLN. Analysis of the dissected regions of brain tissue indicated that TLN has concentrations ranging from below baseline to 1.27 ng/g with the highest levels in the cortex, hippocampus and cerebellum and the lowest levels in the hypothalamus, brainstem and striatum. The distribution of MTLN paralleled that of TLN, with ranges from 0.44-0.90 ng/g. In 95 percent of the whole brain samples, no evidence of in vitro metabolism of TLN and MTLN was noted, i.e. no D4-1abeled peaks were found in the SRM spectra. To further test this, TLN and MTLN levels were measured in tissue samples that had been allowed to sit at room temperature for up to 30 rain; no evidence of D4-TLN or D4-MTLN was found. Analyses of 5-HTLN and 5-HMTLN were accompanied by large quantities of their D4-analogues, suggesting that HTLN and HMTLN formation occurs during sample preparation. In these analyses, GC/MS/MS using SRM often resulted in an improved signal-to-noise ratio over that found in GC/MS-SIM quantitation. This, coupled with the improved molecular specificity of SRM resulted in the first unambiguous confirmation of the presence of MTLN in rat brain (42). 3.1.5. Carbamazepine in plasma: pharmacokinetics of mono- versus polytherapy

This study differs somewhat from the above examples in that the isotopically labeled compound does not act as the internal standard. Rather, the stable isotope form of the drug is used to differentiate a single administered dose from the endogenous levels of the drug in patients on chronic therapy. Quantitation of the isotopically labeled species is then achieved using MS/MS and an unlabeled internal standard. The fate of carbamazepine (CBZ) and its metabolite carbamazepine-epoxide (CBZ-EP) in human subjects using this anticonvulsant alone or in combination with other anticonvulsants was explored (43) in patients on chronic therapy. To better understand the drug interactions occurring during polytherapy, changes in the CBZ and CBZ-EP concentrations during mono- and polythera-

99 pies with a series of anticonvulsant drugs were measured in plasma using LC. Changes in the pharmacokinetics of CBZ during co-administration of valproate (VPA) were then determined using MS/MS and a stable isotope form of the drug D4-CBZ. The use of stable isotope-labeled drugs permits the concentration of the drug to be followed "beneath" the steady state of unlabeled drug, i.e. the patients need not be "cleared" of the drug in order to perform the study. Patients were administered a single dose of D4-CBZ and blood samples withdrawn at timed intervals after administration. CBZ and D4-CBZ plasma concentrations were determined as a function of time using 2-methylcarbamazepine as an internal standard.

MS/MS analyses of the plasma extracts were performed using a Q lqQ2 mass spectrometer with methane positive ion CI (35). CID was performed with NEX at a pressure o f 3 x 10-s Torr and an E/ab of 18 to 20 e V. SRM of the MH § ions of CBZ (m/z 237), D4-CBZ (m/z 241), and 2-methylcarbamazepine (m/z 251) were performed using the most abundant product ions at m/z 194, 198 and 208, respectively. The ratios of the product ion intensities to that of the internal standard were used for quantitation.

The equivalence of CBZ and D4-CBZ kinetics were confirmed from the plasma samples of patients who received a 50/50 dose of CBZ and D4-CBZ. The pharmokinetic studies showed that the half life (tl/2) of CBZ in patients on chronic CBZ therapy decreased by about one-half when compared to the control subjects. Co-administration of VPA resulted in a similar decrease in tl/2 (20 hr). The maximum concentration of D4-CBZ following a 200 mg dose tended to decrease for chronic CBZ monotherapy (2.1 ~g/ml) and for co-VPA (1.4 ~g/ml) when compared to the controls (2.4 ~g/ml). The time of maximum D4-CBZ concentration also decreased from 5.3 hr for controls to 2.3 hr for both chronic CBZ and co-VPA therapies. Steady-state concentrations were maintained during the pharmokinetic studies. Changes in the CBZ-EP to CBZ ratio of patients receiving concurrent therapy measured by LC indicate an increase in the metabolite CBZ-EP with polytherapy. During chronic therapy with other anticonvulsants, CBZ-EP levels were significantly increased; however, increased toxicity was not evident (43). This study illustrates the use of quantitative MS/MS methods to acquire pharmacokinetics data.

100

3.2. Identification and Structure Elucidation Using Isotope Tracers and MS/MS

One of the traditional uses of MS/MS is the elucidation of molecular structure, both for low MW biological compounds and for biological polymers. The incorporation of isotopic labels into the molecules provides a tracer or tag to (i) track a targeted compound through its metabolic pathways and subsequent sample work-up; or (ii) identify fragmentation sites in an effort to elucidate structure and/or fragmentation mechanisms. The isotopic labels are identified by shifts in the observed m/z ratios in the respective MS/MS spectra or by the presence of a "twin peak" in the spectra acquired for samples containing both the labeled and unlabeled sample. The following examples illustrate the use of stable isotope tracers to confirm suspected drug metabolites (44), to elucidate the mechanism of conjugation (45) and drug action (46, 47), to differentiate structurally similar biomolecules (48, 49) and to elucidate fragmentation pathways (48, 50, 51). 3.2.1. Identifying glutathione conjugates of N-methylformamide

N-methylformamide (NMF) is an industrial solvent and antineoplastic agent which undergoes oxidative metabolism to form toxic intermediates in the liver. The formation of glutathione (GSH) and other related S-linked conjugates from a reactive metabolite of NMF aids in the disposition of these toxic intermediates by excretion in the urine and bile. Characterization of these Slinked conjugates in biological matrices would provide insight into the structure of the short-lived metabolic intermediates of NMF (44). However, the analysis of NMF conjugates has been complicated by the lability, reactivity, and low concentrations of these compounds. MS/MS analysis of GSH conjugates was carried out in an effort to determine if the "twin peak" method could aid in the identification of GSH and related NMF conjugates and to determine if neutral loss scans would be an effective method for screening samples which might contain NMF conjugates. NMF and D3-NMF were injected into mice and bile collected from the animals post treatment. Prior to MS/MS analysis, the bile extracts were derivatized to benzoxylcarbonyl methyl esters and purified with HPLC (44). MS/MS analyses were performed using a EBqQ hybrid mass spectrometer with a FAB ionization source. Product ion mass spectra of the MH § ions of the benzoxylcarbonyl methyl esters derivatives of synthetic GSH-NMF and D3-GSH-NMF (m/z 527 and 530, respectively) were ob-

101 tained by mass-selecting the precursor ions with EB and then performing CID in q with Ar collision gas at 2 • 10-8 Torr and an E/ab of 25 eV. The MH + ions gave rise to a common neutral loss of 89 u. Neutral loss mass spectra of the bile extracts were then obtained by simultaneously scanning B over the range m/z 100 to 700 and Q over the range m/z 11 to 611.

Figure 11a shows the FAB/MS spectrum of a derivatized extract of bile. The twin peaks at m/z 527 and 530 observed in this spectrum were not seen in the spectrum of a drug-free bile sample. These peaks were therefore tentatively assigned to the MH § ions of GSH-NMF and D3-GSH-NMF. To confirm this, the neutral loss mass spectrum of the bile extract was acquired to identify those components of the sample which gave rise to a neutral loss of 89 u. The neutral loss spectrum also shows two prominent peaks at m/z 527 and 530 (Figure 11b) confirming the identity of the MH § ions of GSH-NMF and D3-GSH-NMF. The spectrum also suggests that only one type of S-linked conjugate is formed during NMF metabolism and that neutral loss scans may be useful in screening samples for the presence of NMF conjugates (44). This study illustrates that the inclusion of the D3-NMF stable isotope resulted in an easily identifiable twin peak in both the normal MS and neutral loss MS/MS spectra which documented the occurrence of an expected metabolite.

3.2.2. Mechanism of glutathione conjugation of cyclophosphamide and phosphoramide

MS/MS was used in conjunction with stable isotopes to compare the in vivo degradation of the therapeutic alkylating agents cyclophosphamide and phosphoramide mustard via enzyme catalyzed GSH conjugation (45). GSH conjugation of cyclophosphamide can occur either by direct displacement of CI (Figure 12a) or via the formation of a cyclic aziridinium intermediate (Figure 12b). Similar pathways are possible for phosphoramide. It is possible to determine which of the two mechanisms is occurring by D-labeling the terminal /3-methylene groups in the mustard moiety. If conjugation were to take place via CI displacement, then the isotope labels would remain on the original/3-methylene groups. If conjugation were to take place via an aziridinium ion, then the isotopes would be randomized between the two methylene groups. To determine which of the two possible degradation pathways occurs, MS/MS analyses of the mono-GSH derivative of cyclophosphamide and the di-GSH-derivative of phosphoramide and their D4-analogues were performed. The GSH conjugates were formed either by immobilized microsomal transfer-

102 100

~

.

*

5o

MN"

369*

0 100

200

300

527, / 530

400 m/z

500

600

700

MH* 527\ /530

100" bll

(/) l.

._= ._~ 50 (i)

dl L loo

200

300

400

soo

600

700

m/z

Figure 11. FAB mass spectra of derivatized bile extract containing equimolar amounts of NMF and D3-NMF. (a) Normal, full-scan mass spectrum showing the twin peaks at m/z 527 and 530 attributed to the MH § ions of GSH-NMF and D3-GSH-NMF, respectively. The peaks labeled with asterisks are glycerol matrix ions; and (b) The neutral loss (89 u) MS/MS spectrum of the same bile extract confirming the identity of the m/z 527 and 530 ions (44). Adapted with permission from Elsevier Science.

103 m/z 213

H

/ ~ N~!__N~"-- CD2Cl

\

o/

\

H

rn/z 215

,c;-;i-/ --N N

\

H

co,c,

f-co,c,

..... of-~

CO2CI

~ H

_o/

7~

/--N

SG

N/'-'I-CD2

GSH

co=c,

,

c~

GSH

SG

CD2C I

HO

m/z213 SG .... ] /

Figure 12. Alternate mechanisms of GSH conjugation of cyclophosphamide: (a) direct displacement of CI" or (b) formation of a cyclic aziridinium intermediate (45).

ase, by microsomes, or in aqueous solution and the reaction products purified by HPLC prior to analysis (45).

An EBEB mass spectrometer with a FAB ionization source was employed. Product ion mass spectra were obtained by mass-selecting the MH § ion of the 35CI, 72C isotope of GSH-cyclophosphamide (m/z 532) and D4-GSH-cyclophosphamide (m/z 536) with (EB)I, performing CID in the FFR between B7 and E2, and then mass-analyzing the product ions using a B/E linked-scan of (EB)2. He was used as a collision gas (precursor beam attenuation of 80 percent) with a collision energy of 6keV. Product ion spectra of the MH § ions of di-GSH-derivative of phosphoramide (m/z 763) and D4-di-GSH-phosphoramide (m/z 767) and their principal fragment ions at m/z 684 and 688, respectively, were obtained under the same collision conditions.

104 The product ion spectra of the MH + ion of GSH-cyclophosphamide showed only one fragment at m/z 211 while that of D4-GSH-cyclophosphamide showed one D2-1abeled fragment at m/z 213. These fragments indicate that the D labels are located on the /3-methylene group and are consistent with direct CI displacement (Figure 12a). In contrast, fragmentation of the m/z 684 ion arising from the MH § ion of di-GSH-phosphoramide yielded fragment ions at m/z 363 and 321, in keeping with conjugation through the formation of a cyclic aziridinium intermediate. Similarly, the product ion spectra of the D4-1abeled analogue showed doublets at m/z 321 and 323, and at m/z 365 and 367, consistent with aziridinium formation and some distribution of the D isotopes between the two methylene groups. Thus, MS/MS proved to be a rapid method for determining the differing mechanism of GSH conjugation in cyclophosphamide and phosphoramide mustard. The high-resolution capability of the EBEB sector instrument allowed selection of precursor ions containing only one 3sCI and C12 isotope and allowed the deconvolution of the 37CI, 13C and D patterns in the product ion spectra (45). 3.2.3. Mechanism of antitumor reactions of neocarzinostatin Neocarzinostatin (NCS)is an antitumor agent which results in DNA damage. The mechanism of action of this antibiotic protein is thought to be resident in a labile chromophore (NCS-chrom), which is the first natural compound found with an epoxybicyclo-diyne-ene ring system (46) (Figure 13). Activation of NCS-chrom in the presence of DNA requires a thiol (e.g. glutathione, GSH) or sodium borohydride (NaBH4). Once activated, NCS-chrom abstracts a hydrogen atom from the C-5' position of deoxyribose of thymidine in DNA. NCS-chrom is irreversibly inactivated upon addition of thiol or NaBH4 in the absence of DNA. FAB/MS, FAB/MS/MS and reverse isotope labeling were utilized in conjunction with nuclear magnetic resonance (NMR) to characterize both the activated and inactivated NCS-chrom species in an effort to determine the mechanism of NCS action on DNA (46). A EBEB with a FAB ionization source was employed to study the HPLC purified NCS-chrom reaction products. Low-resolution (1,000) product ion mass spectra were obtained by mass-selecting the precursor ions with (EB)7, performing ClD in the FFR between B7 and E2, and then mass-analyzing the product ions using a B/E linked-scan of (EB)2. (No CID conditions were provided.) Accurate mass measurement were obtained using high resolution (10,000).

105 CH 3

R

\

O,

CH3~(

OH"

)

"

NHCH3 OH NCS-chrom A,

NCS-chromB,

r..-o~ R= LO,~=O .OH R= L. OH

Figure 13. Structure of NCS-chrom A and B (46).

FAB/MS mass spectra of the reaction products of GSH activated NCS-chrom following reaction with DNA and that of GSH-inactivated NCS-chrom both contained peaks at m/z 969 and 991, and at m/z 943 and 965. These spectra suggest that the inactivated NCS-chrom species is structurally similar to the activated form. The ion at m/z 969 corresponds to the MH § ion of a species incorporating NCS-chrom A (Figure 13), one GSH molecule and two hydrogen atoms while the ion at m/z 991 is the corresponding sodiated adduct. The ion at m/z 943 is attributed to the decarbonylation product of the former material (NCS-chrom B, Figure 13), with m/z 965 ion corresponding to the sodiated adduct. The FAB/MS spectra of GSH activated NCS-chrom with DNA showed no difference in D2 enrichment when either water or D20 were used a solvents, indicating that D atoms from the exchangeable mercapto hydrogens of GSH are not incorporated into the spent drug. As such, NaBH4 was used as the activating (or inactivating) agent for the remaining studies. Because of the

106 difficulty in synthesizing labeled NCS-chrom, a reverse D-labeling method was employed. This strategy was based on previous studies which had shown that the use of NaBD4 leads to the incorporation of three D atoms at the C12, C-2, and C-6 positions of the epoxybicyclo diyne-ene ring (47). By using NaBD4, it is possible to determine if hydrogens from DNA are being incorporated into the inactivated drug since inactivated NCS-chrom should bear three nonexchangeable D atoms, while the activated drug that has abstracted hydrogen from DNA should have one or more of these three nonexchangeable D atoms replaced by a hydrogen (46). The FAB/MS spectrum of native NCS-chrom showed an MH § ion at m/z 660 while those of inactivated NCS-chrom in NaBHJH20/CH3OH, NaBH4/D20/ CH3OD, NaBDJH20/CH3OH and NaBD4/D20/CH3OD showed MH § ions at m/z 450, 451,452, and 453, respectively. The FAB/MS spectrum of activated NCSchrom in NaBDJD20 with DNA showed a MH § ion at m/z 451. Accurate mass measurements of the MH § ions of NCS-chrom, inactivated NCS-chrom in NaBD4/H20/CH3OH, and activated NCS-chrom with DNA resulted in the following exact masses and corresponding elemental compositions: 660.2054 (calculated 660.2081), C35H34012N;452.1892 (calculated 452.1890), C22H26D209N; and 451.1828 (calculated 451.1827), C22H27DO9N. These high-resolution results clearly indicate that the activated NCS-chrom is compositionally the same as the inactivated form from NaBD4, except for differing numbers of D atoms. This is supported by the presence of similar fragment ions (at m/z 160 and 86 and at m/z 211, 212, 213 and 214, respectively) in the product ion spectra of the four inactivated NCS-chrom MH § ions and by the corresponding NMR spectra (46). Figure 14a shows the product ion spectrum of the MH § ion of NCS-chrom A (m/z 660.2). The prominent fragment ion at m/z 215 is attributed to the naphthoate group. In contrast, the product ion spectra of the MH § ion of NCSchrom inactivated in NaBHJH20/CH3OH (m/z 450.2) (Figure 14b) as no m/z 215 fragment ion. Based on this, the structure of the activated NCS-chrom is postulated to be [1 NCS-chrom A + 4 H - 1 naphthoate group], i.e. the aminogalactose and cyclic carbonate ring remain intact. The NMR spectra support this proposed structure and also conclude that one exchangeable proton is added to the open epoxide ring, while the three remaining nonexchangeable H atoms are bound to carbons C-2, C-6 and C-12 of the epoxybicyclo ring, in agreement with previous assignments for NCS-chrom product generated by reaction with thiol (47). Based on the mass shifts measured of the MH § ions in the FAB/MS spectra of inactivated NCS-chrom using different combinations of NaBH4, NaBD4, and deuterated and nondeuterated solvents, one of the three carbon-bound

107 CHi a.

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4

/il~" . i / / ' " " 'i "" '"' '" (' ';' .-i'.:. r" "r!l:, "l" ',',', 'l:,' ," ,' ~' i',' i.,. i.I./',',',' i', ",', ', i ' 1 , 450 so ~00 ~so 200 2so 3oo 3so 400

Figure 14. Product ion spectra of the MH § ions of (a) NCS-chrom A, and (b) the product obtained from NCS-chrom inactivated by NABH,/H=O/CH3OH. Asterisks indicate matrix-related cluster ions (46). Reproduced with permission from the American Chemical Society.

h y d r o g e n s is c o m i n g f r o m the h y d r o x y functional g r o u p of the solvent w h i l e the others are c o m i n g directly f r o m the NaBH4. A n o t h e r i m p o r t a n t conclusion d r a w n f r o m the FAB/MS data is that t w o of three i n c o r p o r a t e d C-bound D a t o m s are replaced by h y d r o g e n a t o m s upon reaction w i t h DNA, i.e. inactivated NCS-chrom in NaBD4/D20/CH3OD has an MH § at m/z 453 w h i l e activated NCS-chrom in N a B D , / D 2 0 w i t h DNA has an MH § at m/z 451. NMR analyses conclude that the t w o DNA-related C-bound h y d r o g e n a t o m s are localized on C-2 and C-6, w h i l e the third is added directly to C-12 f r o m b o r o h y -

108 dride. Because the reaction of NSC-chrom with DNA leads to the sugar oxidation at C-5' of thymidine, one of the DNA hydrogens which is incorporated into NCS-chrom must come from the 5'-H of thylidylate DNA (46). This study elegantly illustrates the ability of accurate mass measurements, isotopic labeling, and MS/MS analyses, in conjunction with NMR, to elucidate the structure of a drug intermediate and in so doing to gain further insight into the mechanism of drug action. 3.2.4. Structure of monohydroxy unsaturated fatty acids

Hydroxy-substituted unsaturated fatty acids derived from arachidonic acid are potent biological modulators of inflammation. Product ion spectra for 12 underivatized monohydroxy unsaturated fatty acids derived from oleic, linoleic, linolenic and arachidonic acids were obtained in an effort to determine if low-energy CID would give rise to structurally informative fragment ions, i.e. ions indicative of the hydroxy substituent in relation to the sites of unsaturation which could be used to identify these acids in biological samples (48). D2-1abeled analogues of selected fatty acids, formed from the exchangeable hydroxyl and carboxyl protons using deuterated solvents, were used to confirm the postulated fragmentation pathways. Negative ion, continuous flow FAB ionization (see Appendix) of the synthetic unsaturated fatty acids was performed on a Q TqQ2 mass spectrometer. Product ion spectra were collected for nondeuterated and deuterated forms of the unsaturated fatty acids by performing ClD of the [M - H]- ions with A r collision gas added to a beam attenuation of 70 to 80 percent and an E/ab of 30 e V.

All of the [M - H]- carboxylate anions of the monohydroxy unsaturated fatty acids gave rise to product ions due to the loss of H20 ([M-H-18]-) and some showed ions due to the subsequent loss of CO2 ([M-H-18-44]-). In addition, structurally significant product ions formed via fragmentation of the carboncarbon bond adjacent to the hydroxy group (c~-hydroxy) were seen for those unsaturated fatty acids containing either an allylic or vinylic bond adjacent to the hydroxy group, i.e. these e-hydroxy fragmentations were not seen when neither an allylic or vinylic bond was present. For example, the product ion spectra of the [M - H]- ion of 5(+_)-hydroxy-6, 8, 11, 14-eicosatetraenoic acid (5-HETE, m/z 319) shows two product ions from a-hydroxy fragmentation of the C-5 to C-6 bond at m/z 115 and 203; however, no product ions due to

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100 203

115

5-HETE

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115 OH

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~

[M-H-H20-CO 2] 257

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150

200

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Figure 15. Low-energy CID product ion spectrum of 5-HETE showing the [ M - H]-, [M - H - 18]-, and fragment ions due to c~-hydroxy fragmentation (m/z 115 and 203, respectively) (48).

fragmentation of the C-4 to C-5 bond are observed (Figure 15). The product ion at m/z 203 is attributed to charge-driven vinylic fragmentation involving initial rearrangement of the conjugated diene double bonds via a 1, 6 hydrogen shift, followed by charge site rearrangement from the carboxy to the hydroxy group. Fragmentation, therefore, results in the formation of a neutral aldehyde containing the carboxy terminal group and a negatively charged alkene at m/z 203. Formation of the 5-HETE product ion at m/z 115 is then attributed to the subsequent loss of proton from the carboxy group from the neutral aldehyde. This proposed mechanism is supported by the fact that the product ion spectrum of D2-5-HETE also contains fragment ions at m/z 115 and 203, i.e. the D atom of the hydroxy group is initially contained in the neutral aldehyde fragment and subsequently lost from the carboxy group. In all, five mechanisms were proposed for the formation of the structurally informative fragment ions arising from e-hydroxy fragmentation of the 12 hydroxy substituted unsaturated fatty acids studied (48). In addition to the observation of these structurally informative fragment ions, it was found that the [M - H]- ions from hydroxy-substituted saturated

110 fatty acids (e.g. 12-hydroxystearic acid) and from unsaturated fatty acids with no hydroxyl substituents (e.g. arachidonic acid) did not give rise to any structurally informative fragment ions under low-energy CID conditions. This finding is contrary to previous high-energy CID studies which found many informative fragment ions indicative of the locations of both the hydroxy constituents and double bonds (49). These findings illustrate the differing degrees of ion structural information available from low- versus high-energy CID methods. 3.2.5. Fragmentation of angiotensin III

The following study illustrates the use of isotopically labeling and both product ion and MS/MS/MS spectra to determine the sequence-specific fragment ions of several small peptides, and to determine the genesis of "internal" fragment ions which do not correspond to the well known A-, X-, B- and Ytype peptide fragment ions (50). In particular, the angiotensin III (AIII, Arg-ValTyr-Ile-His-Pro-Phe) and its 1802-1abeled analogue with the carboxyl oxygens were tagged and studied. The study employed a BEqQ hybrid mass spectrometer and FAB ionization. Product ion spectra of synthetic AIII were obtained by mass-selecting the precursor ions with BE and performing low-energy CID in q using 0.2 to 0.5 mtorr of Ar and collision energies between 15 and 30eV. MS~MS~MS scans were obtained by mass-selecting the MH § ion of AIII (m/z 931) with B (Ar collision gas in FFR2 at a 50 percent precursor ion beam attenuation and an E/ab of 8keV), selecting the first-generation product ion (m/z 784) with E, performing low-energy ClD in q, and then mass-analyzing the second-generation product ions with Q.

Figure 16a shows the low-energy CID product ion spectrum of the MH § ion of AIII. The spectrum shows characteristic peptide fragment ions due to backbone cleavage of either the carbon-carbonyl (A- and X-type) or the amide bonds (B- and Y-type) (51) with satellite peaks 17 u lower, characteristic of the presence of an Arg residue (50). There is also a prominent fragment ion at m/z 784 which does not correspond to one of these common types of peptide fragments. The low-energy CID spectrum of the MH § ion of ~802-AIII showed a prominent fragment at m/z 786 (Figure 16b) indicating a loss of only one carboxyl oxygen. Thus, formation of the m/z 784 was attributed to loss of the terminal Phe residue with rearrangement to retain one of the carboxyl oxygens, resulting in a fragment ion which is the equivalent of the

111 100

a.

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784 931 9

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800

784

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.

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.

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m/z Figure 16. Low-energy CID spectra of (a) the MH § ion of angiotensin III (m/z 931); (b) the MH § ion of 1802-1abeled analogue (m/z 935); and (c) MS/MS/MS spectrum resulting from mass-selection and high-energy CID of the MH § ion of AIII (m/z 931) followed by selection and subsequent low-energy CID of the m/z 784 fragment ion (50). Reproduced with permission from John Wiley.

protonated hexapeptide Arg-VaI-Tyr-Ile-His-Pro. The MS/MS/MS product ion spectrum of m/z 784 (Figure 16c) shows only one significant fragment ion at m/z 687, corresponding to the loss of the terminal amino acid, Pro. Thus, the first-generation product ion at m/z 784 fragments in a similar manner to that of the MH § ion. The formation of these "internal" fragment ions is attributed,

112 therefore, to the formation of an intermediate zwitterion due to interaction between the terminal carboxy oxygens and the oxygen of the first peptide bond (50). The MS/MS/MS scanning capability of the hybrid mass spectrometer thus allowed the facile elucidation of the mechanism of formation of these "internal" peptide fragment ions.

4. S U M M A R Y

MS/MS is a versatile method for quantifying and characterizing compounds of pharmacological interest. When used in conjunction with stable isotope methods, an even greater degree of molecular specificity is attained. Advantages of these combined methodologies include their versatility (analyses range from quantitation, to pharmacokinetics, to structure elucidation), the small requisite sample sizes (e.g. 11 i~1 dried blood spots), the speed of the analyses (min vs. hr), and the inherent selectivity which allows for the detection of trace components in complex biological matrices. Thus, MS/MS offers an attractive compliment to more conventional pharmacological methods of analysis.

5. APPENDIX

5.1. Fast Atom Bombardment Ionization

Fast atom bombardment (FAB) is a method of producing intact molecular ions of compounds present in the liquid phase (52). FAB is performed by mixing the sample of interest in a liquid matrix, depositing it on a sample probe and then bombarding the sample under vacuum with a beam of atoms of high kinetic energy (8-10 keV). Common incident beams include Ar or Xe. If the sample is bombarded with a beam of high-energy ions (e.g. Cs § ions), then the method is referred to as liquid secondary ion mass spectrometry, or LSIMS. Typically, 1 ~1 of the sample solution is combined with 3-5 i~1 of the matrix and 2 i~1 of this solution is subjected to bombardment. Positive ion FAB typically leads to the formation of MH § or multiply protonated [M + nil] n§ ions. Negative-ion FAB leads to the formation of [M - H]- ions. Often, the molecular ion is the only ion in the spectrum indicative of the compound of interest. Thus, FAB is a facile method for determining the MW of biological compounds. The judicious choice of a matrix is critical in obtaining good quality FAB

113 spectra. It is not unheard of for a sample to produce a strong signal with one matrix and no signal at all with another. Classical matrices include gylcerol, thioglycerol and nitrobenzyl alcohol. The liquid matrix serves two main purposes: (i) it keeps the sample in the liquid phase as it is being introduced into the vacuum chamber; and (ii) it reduces damage to the sample from the bombarding beam by constantly diffusing fresh sample to the surface. Detailed discussions of FAB matrices have been presented (53, 54). The principal drawback of FAB analyses is the often high chemical-noise background formed by the clustering and fragmentation of the sample matrix. The sample also has limited lifetime upon continued bombardment. Also, FAB analyses can suffer from suppressed sample ionization due to the migration of the sample away from the liquid/vacuum interface. Continuous flow FAB sources (CF-FAB) were developed to overcome the limitations of conventional FAB sources by providing a continuous flow of sample matrix into the ion source via a sample introduction probe. The continuous introduction of liquid allows the use of much less viscous and more volatile sample carriers such as 5 percent glycerol in water, methanol, or acetonitrile. Typically, 0.5 to 1 ~1 of sample is injected into the flowing solvent stream and introduced to the bombarding atom, or ion beam, in the ion source. CF-FAB analyses has decreased ion suppression effects, deceased background noise due to matrix ions and thus overall improved limits of detection. A more detailed discussion of CF-FAB sources and methods is available (55).

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6.1. Additional Reading D.J. Ashworth, W.M. Baird, C. Chang, J.D. Ciupek, K.L. Busch and R.G. Cooks, Chemical Modification of Nucleic Acids. Methylation of Calf Thymus DNA Investigated by Mass Spectrometry and Liquid Chromatography, Biomed. Environ. Mass Spectrom., 12 (1985) 309. M.J. Bennett, P.M. Coates, D.E. Hale, D.S. Millington, N. Pollitt, P. Rinaldo, C.R. Roe and K. Tenaka, Analysis of Abnormal Urinary Metabolites in the Newborn Period in Medium-Chain AcyI-CoA Dehydrogenase Deficiency, J. Inherit. Metab. Dis., 13 (1990) 707. C. Chang, D.J. Ashworth, I. Isern-Flecha, X-Y. Jiang and R.G. Cooks, Modification of Calf Thymus DNA by Methyl Methanesulfonate. Quantitative Determination of 7Methyldeoxyguanosine by Mass Spectrometry, Chem. Biol. Interact., 57 (1986) 295. R.B. Cole, C.R. Guenat and S.J. Gaskell, Effect of Experimental Conditions on the Daughter Ion Spectra Derived from Tandem Mass Spectrometry of Steroid Glucuronidides, Anal. Chem., 59 (1987) 1139. A. Dell, Mass Spectroscopy of Carbohydrates, Adv. Carbohydr. Chem. Biochem., 45 (1987) 19. C. Dass, J.J. Kusmierz and D.M. Desiderio, Mass Spectrometric Quantification of Endogenous/3-endorphin, Biol. Mass Spectrom., 20 (1991) 130. M. Dawson, M.D. Smith and C.M. McGee, Gas Chromatography/Negative Ion Chemical Ionization/Tandem Mass Spectrometric Quantification of Indomethacin in Plasma and Synovial Fluid, Biomed. Environ. Mass Spectrom., 19 (1990) 453. D.M. Desiderio and M. Kai, Field Desorption Mass Spectral Meaurement of Enkephalins in Canine Brain with 180 Peptide Standards, Int. J. Mass Spectrom. Ion Phys., 48 (1988) 261. D.M. Desiderio and I. Katakuse, Fast Atom Bombardment-Collision Activated Dissociation-Linked Field Scanning Mass Spectrometry of the Neuropeptide Substance P, Anal. Biochem., 129 (1983) 425. D.M. Desiderio, I. Katakuse and M. Kai, Measurement of Leucine Enkephalin in Caudate Nucleus Tissue with Fast Atom Bombardment-Collision Activated DissociationLinked Field Scanning Mass Spectrometry, Biomed. Mass Spectrom., 10 (1983) 426. B. Domon and C.E. Costello, Structure Elucidation of Glycosphingolipids and

116 Gangliosides using High-Performance Tandem Mass Spectrometry, Biochem., 27 (1988) 1534. B. Domon, J.E. Vath and C.E. Costello, Analysis of Derivatized Ceramides and Neutral Glycosphingolipids by High-Performance Tandem Mass Spectrometry, Anal. Biochem., 184 (1990) 151. C. Fenselau and P.B.Smith, High-Performance Tandem Mass Spectrometry in Metabolism Studies, Xenobio., 22 (1992) 1207. A. Ferretti, V.P. Flanagan and E.J. Maida, GC/MS/MS Quantification of 11-Dehydrothromboxane B2 in Human Urine, Prostaglandins Leukot. Essent. Fatty Acids, 46 (1992) 271. G. Fridland, Measurement of Opiod Peptides with Combinations of Reversed Phase High- Performance Liquid Chromatography, Radioimmunoassay, Radioreceptorassay and Mass Spectrometry, Life Science, 41 (1987) 809. G. Fritz, D. Fetterolf, R.A. Yost and B.D. Anderson, Oxygen Fixation into Hydroxyproline in Etiolated Maize Seedlings: Verification by Tandem Mass Spectrometry, Plant Physiol., 73 (1983) 860. S.J. Gaskell, C. Guenat, D.S. Millington, D.A. Maltby and C.R. Roe, Differentiation of Isomeric Acylcarnitines Using Tandem Mass Spectrometry, Anal. Chem., 58 (1986) 2801. P.E. Haroldsen and S.J. Gaskell, Quantitative Analysis of Platelet Activating Factor using Fast Atom Bombardment/Tandem Mass Spectrometry, Biomed. Environ. Mass Spectrom., 18 (1989) 439. P.E. Haroldsen, M.H. Reilly, H. Hughes and S.J. Gaskell, Characterization of Glutathione Conjugates by Fast Atom Bombardment/Tandem Mass Spectrometry, Biomed. Environ. Mass Spectrom., 15 (1988) 615. K.E. Ibrahim, J.M. Midgley, M.W. Crouch, M.B. Budd, R.A. Yost and C.W. Williams, Quantitative Measurement of Octopamines and Synephrines in Urine using Capillary Column Gas Chromatography Negative Ion Chemical Ionization Tandem Mass Spectrometry, Anal. Chem., 56 (1984) 1695. I. Isern-Flecha, X-Y. Jiang, R.G. Cooks, W. Pfleiderer, W-G. Chae and C. Chang, Characterization of an Alkylated Dinucleotide by Desorption Chemical Ionization and Tandem Mass Spectrometry, Biomed. Environ. Mass Spectrom., 14 (1987) 17. B.L. Kleintop, R.A. Yost and C.R. Abolin, Alternating RF/DC Isolation for Quantitation with Co-Eluting Internal Standards in Gas Chromatography/Ion Trap Mass Spectrometry, J. Am. Soc. Mass Spectrom., 3 (1992) 85. D.S. Millington, N. Kodo, D.L. Norwood and C.R. Roe, Tandem Mass Spectrometry: A New Method for Acyl Carnitine Profiling with Potential for Neonatal Screening for Inborn Errors of Metabolism, J. Inherit. Dis., 13 (1990) 321. D.S. Millington, D.A. Maltby and C.R. Roe, Rapid Detection of Argininosuccinic Aciduria and Citrullinuria by Fast Atom Bombardment and Tandem Mass Spectrometry, Clin. Chim. Acta, 155 (1986) 173. D.S. Millington, C.R. Roe and D.A. Maltby, Application of High-Resolution Fast Atom Bombardment and Constant B/E Ratio Linked Scanning to the Identification and Analysis of Acylcarnitines in Metabolic Disease, Biomed. Mass Spectrom., 11 (1984) 236. R.C. Murphy and K.L. Clay, Preparation of 180 Derivatives of Eicosaniods for GC-MS Quantitative Analysis, Meth. Enzym., 86 (1982) 547. J.O. Naim, D.M. Desiderio, J. Trimble and J.R. Hinshaw, The Identification of Serum Tuftsin by Reverse-Phase High-Performance Liquid Chromatography and Mass Spectrometry, Anal. Biochem., 164 (1987) 221. M.J. Raftery, U. Justensen, H. Jaeschke and S.J.Gaskell, Mass Spectrometric Quantifi-

117 cation of Cystein-Containing Leukotrienes in Rat Bile Using 13C-Labeled Internal Standards, Biol. Mass Spectrom., 21 (1992) 509. M.J. Raftery, G.C. Thorne, R.S. Orkiszewski and S.J. Gaskell, Preparation and Tandem Mass Spectrometric Analysis of Deuterium-Labeled Cysteine-Containing Leukotrienes, Biomed. Environ. Mass Spectrom., 29 (1990) 465. C.R. Roe, D.S. Millington, D.A. Maltby, T.P. Bohan, S.G. Kahler and R.A. Chalmers, Diagnostic and Theraputic Implications of Medium-Chain Acylcarnitines in the Medium-Chain AcyI-CoA Dehydrogenase Deficiency, Pediatr. Res., 19 (1985) 459. C.R. Roe, D.S. Millington, D.L. Norwood, N. Kodo, H. Sprecher, B.S. Mohammed, M. Nada, H. Schultz and R. McVie, 2, 4-DienoyI-Coenzyme A Reductase Deficiency: A Possible New Disorder of Fatty Acid Oxidation, J. Clin. Invest., 85 (1990) 1703. C.R. Roe, D.S. Millington, S.G. Kahler, N. Kodo and D.L. Norwood, Carnitine Homeostasis in the Organic Acidurias Fatty Acid Oxidation: Clinical Biochemical and Molecular Aspects, K. Tanaka and P.M. Coates (eds) (Alan R. Liss, New York, 1990) pp. 383-402. C.R. Roe, N. Terada and D.S. Millington, Automated Analysis for Free and Short-Chain Acylcarnitine in Plasma with a Centrifugal Analyzer, Clin. Chem., 38 (1992) 2215. M.H. Shaffer, B.E. Noyes, C.A. Slaughter, G.C. Thorne and S.J. Gaskell, The Fruitfly Drosophila Melanogaster Contains a Novel Charged Adipkinetic-Hormone-Family Peptide, Biochem. J., 269 (1990) 215. B.N. Singh, C.E. Costello and D.H. Beach, Structures of Glycophosphosphingolipids of Tritichomonas Foetus: A Novel Glycophosphosphingolipid, Arch. Biochem. Biophys., 286 (1991) 409. B.N. Singh, C.E. Costello, D.H. Beach and G.G. Holtz, DI-O-Alkylglycerol, Mono-OAlkylglycerol and Ceramide Inositol Phosphates of Leishmania Mexicana Promastigotes, Biochem. Biophys. Res. Comm., 157 (1988) 1239. F.S. Tanzer, E. Tolun, G.H. Fridland, C. Dass, J. Killmar, P.W. Tinsley and D.M. Desiderio, Methionine-Enkephalin Peptides in Human Teeth, Peptide Prot. Res., 32 (1988) 117. D. Tsikas, Analysis of Cysteinyl Leukotrienes and Leukotriene by Gas Chromatograp h y - (Tandem) Mass Spectrometry, Eicosanoids, 5 (1992) 57. J.L.K. Van Hove, W. Zhang, S.G. Kahler, C.R. Roe, T-S. Chen, N. Ternada, D.H. Chace, A.K. lafolla, J-H. Ding and D.S. Millington, Medium-Chain AcyI-CoA Dehydrogenase (MCAD) Deficiency: Diagnosis by Acylcarnitine Analysis in Blood, Am. J. Hum. Genet., 52 (1993) 958. J.Y. Wescott, K.R. Stenmark and R.C. Murphy, Analysis of Leukotriene B4 in Human Lung Lavage by HPLC and Mass Spectrometry, Prostaglandins, 31 (1986) 227. B.K. Wong, T.F. Woolf, T. Chang and L.R. Whitfield, Metabolic Disposition of Trimetrexate, A Nonclassical Dihydrofolate Reductase Inhibitor in Rat and Dog, Drug Metab. Dispos. Biol. Fate Chem., 18 (1990) 980. Z. Yamaizumi, H. Kasai, S. Nishimura, C.G. Edmonds and J.A. McCIosky, Stable Isotope Dilution Quantification of Mutagens in Cooked Foods by Combined Liquid Chromatography-Thermospray Mass Spectrometry, Mutation Res., 173 (1986) 1. J.A. Zirolli, K.L. Clay and R.C. Murphy, Tandem Mass Spectrometry of Negative Ions from Choline Phospholipid Molecular Species Related to Platelet Activating Factor, Lipids, 26 (1991 ) 1112.

119

CHAPTER 6

MASS SPECTROMETRY: ISOTOPE RATIO MASS SPECTROMETRY

THOMAS R. BROWNE ~, GEORGE K. SZABO ~ and ALFRED AJAMI 2 1Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center; 2Tracer Technologies, Inc.

Isotope ratio mass spectrometry (IRMS) determines the ratio of isotopes of a given element (e.g. H: 2H, 12C: 13C, ~4N: ~SN) present in a specimen. Typically, the total amount of each isotope also is determined. The amount of element derived from labeled drug in a specimen can be computed from the excess (over normal abundance) in labeled element isotope ratio and the total amount of element. The instruments that are traditionally associated with IRMS use multi-collector mass spectrometers that have the precision necessary to measure very small differences in isotope ratios that arise in nature. Thus, IRMS offers the potential of detecting and quantitating added label in any whole biological matrix. In pharmacologic studies, this procedure offers simplicity and flexibility similar to radioactive tracer procedures without the administrative and safely concerns of radioactive studies. Historically, whole biologcal matrix samples were combusted to elemental gases off-line, and manually collected and transfered to a dual inlet IRMS system. Two variant types of IRMS have been applied to pharmacologic research: continuous flow-isotope ratio mass spectrometry (CF-IRMS) and chemical reaction interface mass spectrometry (CRIMS). CF-IRMS uses a multi-collector mass spectrometer to measure isotope ratios of combusted gas species in a continuous flow of inert gas, while CRIMS uses traditional selected ion monitoring mass spectrometry (SIM-MS) measuring isotope ratios of small microwaveinduced plasma ion species. Neither of these techneques are classic IRMS, however, both are based on the concept of isotope ratio monitoring of tracer excess above natural abundance in the biomatrix. Both methods have been applied to mass balance/metabolite identification studies (see Chapter 11).

120 Both methods report promising preliminary results, but neither method is fully validated (see Chapter 11).

1. CONTINUOUS FLOW-ISOTOPE RATIO MASS SPECTROMETRY (CF-IRMS) 1.1. Technique

In CF-IRMS (Figure 1) organic sample is introduced into the high temperature combustion chamber of an elemental analyzer and flash combusted with a pulse of oxygen. The oxidized combustion gases (C02, N2, NOx and H20) are carried by a continuous stream of helium (hence, "continuous flow") through clean-up phases to a gas chromatography-isotope ratio mass spectrometry (GC-IRMS) device (eliminating the problematic step of transferring by hand combustion gas species to IRMS). NOx is reduced to N2 in a reduction tube (Cu); H20 is removed in an H20 trap; and C02 is removed (for N2 determina-

Samples in ------r Autosampler He -i02

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i

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121 tions) in a C02 trap. N2 and C02 are separated on a GC column and carried to an ion source where an ion beam is generated for each combustion gas. In the magnetic sector of the MS, the beam is separated into multiple beams depending on the mass-to-charge ratio of the various isotopically labeled ions. The beam for N2 is separated into three ion beams in the magnetic sector, and ion currents are measured at preset masses (m/z 28, 29, 30) in Faraday cup collectors. The intensity of ions at these m/z values are used to calculate 14N/14N, 15N/14N and 1SN/15N ratios. The mass spectrometer data system also calculates total nitrogen. In a biological sample containing a 15N tracer, an increase in masses 29 and 30 above natural abundance (atom percent) will correlate with the concentration of enriched tracer as atom percent excess (APE). Similar considerations apply for 13C (1) except that the mass spectrometer is tuned to measure m/z 44([12C02]), 45([13C 02] + [12C 170 160]) and 46([12C 180 160]), 1.2. H i s t o r y

IRMS with 15N and 13C tracers has been used extensively in the past for environmental, metabolic and nutritional studies but has been used little for pharmacokinetic studies (1-8). Hachey et al. (1) provided an excellent review of technical considerations of IRMS for nutrition and biomedical research. Benedetti and Pataky (4) measured the elimination of lSN-labeled urea in rats using an early spectroscopic analytical method. In 1974, Von Unruh et al. (9) and in 1976 Sano et al. (10) each demonstrated mass balance and metabolite identification techniques with 13C, labeled aspirin in human urine using combustion/mass spectrometric isotope ratio measurements. In 1985, Nakagawa et al. (11) performed a successful IRMS mass balance study of antipyrine in rats using 15N and 13C labeling. These studies did not lead immediately to routine animal or human applications, presumably because of the problems with early IRMS methods discussed below. Goodman and Brenna (12) described a high precision (at natural abundance) gas chromatographic combustion-isotope ratio mass spectrometric (GCC-IRMS) system capable of performing metabolite identification and mass balance for pharmaceuticals. Similar to the GC-pyrolizer-MS system used earlier by Sano et al. (10), the GCCIRMS system relies on isotope ratio measurements from combusted GC peaks. However, this is a problem in metabolite studies where thermolabile polar conjugates are present. Inadequate derivitization of nonvolatile drugs and metabolites would also be a problem with methods that used GC as the preliminary separation technique. These issues would also prove to be limitations for early GC-CRIMS methods. In 1993, Browne and coworkers (3,

122 13-16) were the first to provide evidence that stable isotope labeling and newly developed, commercially available CF-IRMS instruments could be used for whole matrix human mass balance studies and detection of labeled liquid chromatographic (LC) peaks for metabolite identification studies. However, it should be noted that LC peaks were individually collected and processed offline as discrete samples for CF-IRMS analysis. The optimal instrument would directly interface an HPLC to a continuous flow combustion isotope ratio mass analyser. Classic IRMS instrumentation was problematic because: (1) each instrument was unique and "made by hand"; (2) complete liberation of all atoms of a given molecule by manual oxidation techniques was difficult; (3) transfer by hand of N2 and CO2 gases from Dumas combustion techniques to IRMS was problematic; (4) each specimen was run manually (lack of automation); and (5) factors 1-4 made IRMS difficult and inconsistent. Recently, refined commercially-available instruments using a helium carrier gas to carry combustion products to the IRMS have become available from three sources (Europa Scientific, Ltd.; Finnegan MAT; and Micromass, Inc.). The authors purchased a Europa (Europa Scientific, Inc., Franklin, Ohio, USA) ANCL-SL (elemental analyzer) 20/20 (mass analyzer) CF-IRMS and found the instrument performed up to specifications and with very high precision as delivered (see Chapter 11 ).

1.3. Assumptions, Validation, Applications, Advantages and Disadvantages These topics are covered for pharmacologic applications of CF-IRMS in Chapter 11.

2. CHEMICAL REACTION INTERFACE MASS SPECTROMETRY (CRIMS)

2.1. Technique In CRIMS (Figure 2), the effluent molecules from a gas chromatograph (GC) or HPLC column (after solvent removal) are converted to low molecular weight ionic species, using a reactant gas in a microwave induced helium plasma with sufficient energy to break all chemical bonds (17-26). In the presence of an abundance of the reactant gas, the atoms of each analyte recombine into a common set of simple molecular species. The labeled and unlabeled species of small molecules are then detected and quantitated by selected ion monitoring (SIM) with either a magnetic sector or quadrupole mass spectrometer or

123

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an isotope ratio mass spectrometer (19). For example, in deuterated drug studies, H2 is used as a reductive reactant gas. The resulting CRIMS products are: HD (from deuterium labeling), CH4, HCN, H2S, H20 and C2H2. However, an MS with sufficient resolving power is required to measure HD at m/z 3.022 (18, 20). Strongly oxidizing reactant gases such as S02 are more useful for ~3C and ~5N isotopic detection and SIM can be performed at m/z 45 (~3C02) and m/z 31 (15N0) on a conventional quadrapole MS. Element selective detection (e.g. CL, Br, S, P, Se) is also possible with CRIMS methods for drug and metabolite molecules that contain these elements. Because of the inability of GC to analyse underivatized nonvolatile/thermolabile compounds (e.g. drug conjugates), only HPLC-CRIMS is satisfactory as a general method for mass balance/metabolite identification studies of new drugs with unknown metabolites.

2.2. History Recently, Abramson (20) provided an extensive review of the developments in CRIMS technology. CRIMS was introduced in 1982 (21) for use in conjunction with GC. In the following years a number of studies were performed using GC-CRIMS and deuterium, 13C and 15N labeling (22-24). The first attempt to combine HPLC with CRIMS utilized a moving belt interface (25). Later, a particle beam LC-MS interface (the Vestec Universal Interface) was used to join HPLC with CRIMS (26). This is the current state-of-the-art HPLC-CRIMS instrument which has been successfully applied to separation and detection

124 of deuterium, 13C and 15N labeled drugs (17-18, 26). More recently, postcolumn modifications to the HPLC interface have improved sensitivity such that LC-CRIMS had superior metabolite identification over on-line radiometric ~4C detection of a doubly labeled (~4C ~5N) anxiolytic drug (buspirone) in bile and urine (27-29). The principle need for post-column modifications of the particle beam LC-MS interface arises from the fact that HPLC gradient elution techniques are necessary to resolve polar metabolites. Unfortunately, aqueous mobile phase containing polar metabolites tends not to form uniform finely nebulized particles that readily desolvate in the universal interface. Post-column addition of an organic modifier (e.g. methanol) to the LC eluent stream reduces the variability of CRIMS response and improves instrument sensitivity for quantitation (27). This modification is critical for LC-CRIMS mass balance studies since accurate mass balance quantitation by this technique requires the measurement of the sum of all the possible labled peaks in a given sample. This is not a trivial point since each sample analyzed by LCCRIMS will have labeled peaks that cover a wide dynamic range of isotope ratio measurments. Thus, a drug that forms many minor metabolites may give total mass balance quantitation errors simply due to a lack of sensitivity of individual isotope ratio measurments for minor metabolites. As these technical details are obviated, LC-CRIMS promises to be an exciting and powerful technique for pharmacologic tracer studies.

2.3. Assumptions, Validation, Applications, Advantages and Disadvantages These topics are covered for pharmacologic applications of CRIMS in Chapter 11.

ACKNOWLEDGMENTS Supported by the United States Department of Veterans Affairs. We wish to thank Fred P. Abramson, Ph.D. for a critical review of this paper.

REFERENCES 1. D.L. Hachey, W.W. Wong and T.W. Boutton et al., Mass Spectrom. Rev., 6 (1987) 289. 2. A. Barrie, J.E. Davies and A.J. Park et al., Spectroscopy, 4 (1989) 44. 3. T.R. Browne, G.K. Szabo and A. Ajami et al., J. Clin. Pharmacol., 33 (1993) 246. 4. M.S. Benedetti and B. Pataky, Adv. Mass Spectrom. Biol. Med., 1 (1976) 73.

125 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

A.L. Burlingame, T.A. Baillie and R.R. Russell, Anal. Chem., 64 (1992) 467R. E.B. Fenn, P.J. Garlick and W.A. McNurlan et al., Clin. Sci., 61 (1981) 217. R. Guilluy, F. Billion-Fey and J.L. Brazier, J. Chromatogr., 562 (1981) 341. C.H. Suelters and J.T. Watson, Biomedical Applications of Mass Spectrometry (Wiley, Chichester, 1990), p. 1. G.E. Von Unruh, D.J. Hauber and D.A. Schoeller et al., Biomed. Mass Spectrom., 1 (1974)345. M. Sano, Y. Yotsui and H. Abe et al., Biomed. Mass Spectrom., 3 (1976) 1. A. Nakagawa, A. Kitagawn and M. Asami et al., Biomed. Mass Spectrom., 12 (1985) 502. K.J. Goodman and J.T. Brenna, Anal. Chem., 64 (1992) 1088. G.K. Szabo, T.R. Browne and A. Ajami, in 5th International Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds, J. Allen and R. Voges (eds), (Wiley, Chichester, 1995), p. 443. T.R. Browne, G.K. Szabo and A. Ajami, in 5th International Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds, J. Allen and R. Voges (eds), (Wiley, Chichester, 1995), p. 435. T.R. Browne, G.K. Szabo and A. Ajami, J. Clin. Pharmacol., 35 (1995) 935. T.R. Browne, G.K. Szabo and A. Ajami, J. Clin. Pharmacol., 35 (1995) 935. Y. Teffera, F.P. Abramson and M. McLean et al., J. Chromatogr., 620 (1993) 89. Y. Teffera and F.P. Abramson, Biomed. Mass Spectrom., 23 (1994) 776. Y. Teffera and F.P. Abramson, Abstracts, 42nd ASMS Conference on Mass Spectrometry (1994) p. 863. F.P. Abramson, Mass Spectrom. Rev., 13 (1994) 341. S.P. Markey and F.P. Abramson, Anal. Chem., 54 (1982) 2375. D.H. Chace and F.P. Abramson, in Synthesis and Applications of Istopically Labeled Compounds, T.A. Baillie and J.R. Jones (eds), (Elsevier, Amsterdam, 1989), p. 253. D.H. Chace and F.P. Abramson, Anal. Chem., 61 (1989) 2724. D.H. Chace and F.P. Abramson, J. Chromatogr., 527 (1990) 1. M. Moini and F.P. Abramson, Biomed. Mass Spectrom., 20 (1991) 308. F.P. Abramson, M. McLean and M. Vestal, in Synthesis and Applications of Istopically Labeled Compounds, E. Buncel and G.W. Kabola (eds), (Elsevier, Amsterdam, 1992), p. 113. F.Y. Hsieh, C.A. Goldthwaite and B. Nobes, Abstracts, 42nd ASMS Conference on Mass Spectrometry (1994) p. 615. C.A. Goldthwaite, F.Y. Hsieh and B. Nobes, Abstracts, 42nd ASMS Conference on Mass Spectrometry (1994) p. 618. F.Y. Hsieh, C.A. Goldthwaite and B. Nobes, Abstracts, 43rd ASMS Conference on Mass Spectrometry (1995) p. 566.

127

CHAPTER 7

ALTERNATIVES TO MASS SPECTROMETRY FOR QUANTITATION OF STABLE ISOTOPES: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH CONVENTIONAL ULTRAVIOLET DETECTION

GEORGE K. SZABO, ROBERT J. PERCHALSKI*, DAVID G. BROWNE and THOMAS R. BROWNE Neuropharmacology Laboratory, Department of Neurology, Boston University School of Medicine; Research Service, Boston Department of Veterans Affairs Medical Center; *Consultant, PO Box 12906 Gainesville, FL 32606

1. I N T R O D U C T I O N 1.1. Rationale for an Alternative

The rationale for selective quantitation of stable isotope labeled (SIL) compounds in pharmaceutical research has been discussed extensively in other chapters in this book as well as in the scientific literature at large (1-7). Historically, mass spectrometry (MS) in all its forms has been the mainstay of analytical methodology for both qualitative and quantitative studies using stable isotopes. So what is the rationale for seeking an alternative to mass spectrometry? The mass spectrometer traditionally fulfils the role of a highly selective and sensitive detector placed at the tail end of some chromatographic (separation) process. Mass-selective detection, however, involves ion formation, mass filtration and ion detection, all of which must be carefully controlled by an elaborate support system of vacuum pumps, magnets or radio-frequency controllers and a sophisticated data acquisition and storage system. Therefore, most mass spectrometric detectors are more complex and

128

expensive than any of the separation techniques and instrumentation that precede the mass analyzer. Such complexity and expense for purchase, operation and continuing maintenance has kept mass spectrometry out of the mainstream as a tool for general quantitative analysis. Although mass spectrometry, and even tandem mass spectrometry is widely used in large pharmaceutical companies, there are many investigators in small pharmaceutical firms, universities and patient care facilities who do not have access to this technology. Over a decade ago, Hoffman and Porter (8) recognized the advantage of eliminating the costly mass spectrometer for certain stable isotope applications. They developed a gas chromatographic (GC) method that resolved the antiepileptic drug (AED) valproic acid (VPA) from deuterated [2H] VPA analogues. Within six years of this application, our group had published a liquid chromatographic (LC) method that resolved two other antiepileptic drugs (phenytoin and carbamazepine) from their respective deuterated analogues (9). The critical point in this methodological approach was that mass discrimination was shifted from a detection process to a chromatographic process. Traditionally for SlL tracer studies, mass discrimination came at the end of some cleanup chromatographic process that would isolate the tracer and parent drug from potential interferences endogenous to the matrix. The purpose in GC-MS, or LC-MS was to produce an isographic peak. This peak contained tracer, parent drug and possibly an internal standard. The peak components were then fragmented to give ionic species for mass spectral identification and quantitation. An alternative to this process is to use a chromatographic isotopic separation of the tracer and its parent compound and measure the separated peaks by some simple general or selective detector against some internal standard. Depending on the intended tracer application (mass balance, drug interaction, bioequivalence, etc.), the alternative chromatograghic method has advantages of simplicity, lower cost, and reproducibility over the mass spectrometric methods. The greatest disadvantages are that deuterium is the sole source of heavy isotope, a high degree of isotope substitution is required and isotopic separation of the deuterated analogues cannot be guaranteed. The advantages and disadvantages of each method of mass discrimination and quantitation will be compared and contrasted in detail throughout this chapter. The purpose of this chapter is to review the work that has been done with chromatographic separation of stable isotope-labeled drugs for pharmacological studies.

129 1.2. Isotope Effect

The primary feature of this alternative method to mass spectrometric analysis is its reliance on a beneficial chromatographic deuterium isotope effect. Twenty years ago Blake et al. (10) wrote a comprehensive review of "Studies with Deuterated Drugs" in which adverse deuterium isotope effects were prominently featured. Van Langenhove (11) has elegantly described the consequences of adverse in vivo isotope effects for pharmacologic studies and cautions investigators on the choice and placement of stable isotopes in drug molecules. Chapter 2 in this text is devoted to a full discussion of in vivo isotope effects. A distinction should be made between the adverse in vivo kinetic (physicochemical and biochemical) deuterium isotope effects, and the chromatographic deuterium isotope effect that alternative chromatographic methods depend on for mass discrimination. An adverse in vivo isotope effect changes the rate at which a SIL compound is absorbed, metabolized, or eliminated, such that the SIL compound's behavior in the body is significantly different from that of it's unlabeled analogue. A chromatographic isotope effect results in a physicochemical difference in the rate of migration between a SIL compound and it's unlabeled analogue during the chromatographic process. The primary considerations for isotopic separation are the placement and number of deuterium atoms in a SIL molecule necessary to achieve baseline resolution. For SIL tracer studies, chromatographic conditions must be produced and maintained so that a sensitive, selective and cost effective quantitative assay is achievable. Some drug molecules lend themselves to creative deuteration allowing chromatographic isotopic separation and quantitation without mass spectrometry and without in vivo isotope effects (8, 9, 12, 14). Historically, alternative methods to mass spectrometry were not created so much by design; rather, they evolved from an SIL tracer study designed for mass spectrometric analysis. Von Unruh (13) used a tetradeutero valproic acid (VPA) isotopomer for a study of elimination kinetics in patients with epilepsy on maintenance doses of VPA. This GC-MS study was conducted on a large bore packed GC column that probably did not chromatographically resolve the isotopomers. Later, Hoffman and Porter (8) and Durden and Boulton (14) found that highly efficient capillary glass GC columns could resolve valproic acid from its deuterated analogues. Heavily deuterated isotopomers containing five or more deuterium atoms had been extensively used in GCMS assays as analytical internal standards because they were relatively inexpensive and easy to synthesize with high isotopic purity. Concern over primary and secondary isotope effects, however, often caused these compounds

130 to be passed over as tracers in SIL tracer studies in favor of more costly ~3C and ~SN analogues. Hoffman and Porter (8) were the first to demonstrate the use and potential benefits of a deuterated analogue as the tracer in a pharmacokinetic study using GC assay without MS. In our laboratory, decadeuterophenytoin ([2H~o]-PHT, Figure 1) was the mainstay internal standard for our early GC-MS work (15). Decadeuterocarbamazepine ([2Hlo]-CBZ, Figure 1) was first synthesized for us with the intended use as a GC-MS internal standard. We had noticed in the literature that other investigators had demonstrated bioequivalence of heavily deuterated isotopomers for SIL tracer studies (8, 16, 17). There were also reports of isotopic separation of deuterated compounds from their undeuterated analogues using liquid chromatography (LC) (18-22). We postulated that an LC assay with simple ultraviolet detection could be developed for these decadeuterated compounds, and were able to achieve base-line resolution of both deuterated isotopomers from their unlabeled analogues with a single isocratic LC method (5).

2. AN ALTERNATIVE METHOD

In a semi-preparative reversed phase LC method that was used for cleanup prior to GC-MS analysis, we suspected that isotopic substitution caused a decrease in the retention time of [2Hlo]-PHT relative to phenytoin (PHT) and the 13C, ~SN2-PHT analogue. Under these conditions (described below), [2H~o]PHT, ~3C, ~SN2-PHT and PHT co-eluted. If the fraction collector missed the front portion of the peak even by a few seconds, however, subsequent GCMS ion intensity readings for the deuterated internal standard were lower than when the fraction collection window started well before the front of the peak. From this observation we suspected that some isotopic separation was taking place. The mobile phase for this LC procedure was strongly organic (>50 percent) and the column packing was 10 i~m particle C18 reversed phase. Phenytoin and carbamazepine, being very nonpolar compounds, eluted late at 16.1 and 18.6 min, respectively (23). Simply lengthening the elution time of this assay by decreasing flow rate, or decreasing organic modifier, did not give base line resolution of the [2H~o] isotopomers from their parent analogues. We then tried a reversed phase C~8 column with smaller particles (5 i~m or less) and with large C~8 chain density (l~mol/m 2) to gain resolving power, and addition of a small amount of tetrahydrofuran (THF) to the mobile phase to gain more control over the carbamazepine analogues. With the addition of 0.5 percent V/V THF to a 25 percent aqueous acetonitrile mobile

131 Carbamazepine Internal Standard

Parent Drug

Isotopic Analogue

c,'=o

NH2

D

(~=O D i

NHz

NHz

Metabolites

,-, DOD

i

D

c=o

D

~=o o

NH2

I~IH2 Phenytoin

Parent Drug

Isotopic Analogue D D

H

D

Metabolites

D

o D

H

.o~

D D

~..

.O~yo ~.. Ethotoin

Parent Drug H

"~o

Isotopic Analogue D

~

"~o

N H =" ~'---N\ /D 9 O/'~- ~C/, /H O D/C, / D H /C\ D/C\D H H Figure 1. Chemical structures and isotopic labeling of drug standards and metabolites described in our studies. Deuterium [2H] substitution for protium [H] on a molecule is presented by [D].

132 CBZ

PHT-dIo

A 6

. . . . 1o TIME

2o

ao

4"o

So

MIN.

Figure 2(A). Isocratic isothermal (room temperature) chromatographic separation of carbamazepine (CBZ), phenytoin (PHT) and their respective decadeuterated isotopomers (CBZ-dlo)and (PHT-dlo).

phase, [2Hlo]-CBZ was not only resolved from CBZ and [2Hlo]-PHT from PHT but the CBZ/deutero-analogue pair eluted earlier than the PHT/deutero-anaIogue pair (see Figure 2A), the reverse of the order without THF. At ambient temperature the run time was almost 50 rain long. Increasing the column and pre-column temperature to 55~ reduced the run time to 30 rain, and improved peak shape and resolution by increasing rates of mass transfer in the stationary and mobile phases (24). Subsequent modifications to the mobile phase (Figure 2B) allowed the addition of an internal standard (10,11-dihydrocarbamazepine) which eluted between the isotopomer/analogue pairs (Figure 2B) (9). It should be noted that not all high efficiency small particle C~8-columns gave such robust resolution. The BioAnalytical Systems Phase II ODS 5 (25 cm x 4.6 mm i.d.) stainless steel column used in this assay gave the best results at the time this work was done; however, recent advances in solid support treatment and stationary phase bonding chemistry should allow even more efficient and reproducible separations. After chromatograghic optimization, a rigorous multi-sample validation study was conducted to demonstrate assay precision and accuracy suitable for SIL tracer studies (25). Investigators should choose reversed phase columns for such applications first by a column's proven ability to resolve a drug from its metabolites and possible

133 10.11

CsZ 0.S.)

a-=N o

B

i 10

2"0

30

RUN TIME IN MINUTES Figure 2(B). Isocratic, isothermal (55~ chromatographic separation of the above compounds plus the 10,11-dihydrocarbamazepine (10,11-dihydro-CBZ) structural analogue internal standard (IS) described in Szabo eta/. (9).

interferences (endogenous species and other drugs). Standard LC method development techniques can be applied to this task. If isotopic separation of deuterated isotopomers from their parent analogue is shown to be possible, a number of replicate columns and variations on chromatographic conditions should be tested before an assay is deemed suitable for tracer studies.

134 3. ISOTOPIC SEPARATION

3.1. Synthetic Considerations

Although isotopic separation is by definition an isotope effect, the rule of thumb for synthesis of a tracer compound still should be to construct the molecule to minimize in vivo isotope effects. Deuteriums should not be placed in metabolically active sites where they might be exchanged or lost altogether during biotransformation. Sometimes the synthesis itself will determine the placement of deuterons, and thus the scope of the tracer application may have to be modified to accomodate the compound. For instance, [2H~o]-CBZ was synthesized by a metal catalyzed deuterium substitution reaction, in which all protiated ring carbons, including the bridgehead carbons at the 10 and 11 positions, were deuterated. The major metabolite of carbamazepine is carbamazepine 10,11-epoxide (CBZ-E). This compound is found in blood and has anti-epileptic properties (26). [2H~o]-CBZ will form decadeutero 10,11epoxide ([2H~o]-CBZ-E, Figure 1); yet there is no way of predicting if a possible in vivo kinetic isotope effect during epoxidation of the deuterated bridgehead (10,11)-carbons is significant enough to exclude [2H~o]-CBZ in an isotope tracer study. The point here is that the substitution reaction of the entire ring system involves fewer synthetic steps than building [2H8]-CBZ carbon by carbon with protiated 10,11-carbons and deuterated benzene rings. Thus, synthesizing [2H~o]-CBZ is simpler and less costly than [2H8]-CBZ. The decadeutero phenytoin was synthesized by the incorporation of a [2H6] benzene precursor (a common NMR solvent) into the final phenytoin molecule. This relatively simple and inexpensive synthetic pathway provided a tracer with sufficient deuteration for chromatographic separation. This tracer compound has a vulnerable deuterium in the active site of the major metabolic pathway. Para-hydroxylation of the phenyl ring is the metabolic pathway that turns the decadeutero phenytoin into a nonadeutero parahydroxyphenytoin ([2Hg]-p-HPHT) metabolite (Figure 1). Caution should always be exercised when isotopic labeling occurs at metabolically active sites; however, Browne et al. (27), Mamada et al. (16) and Hoffman and Porter (8) each have demonstrated pharmacokinetic equivalence of heavily deuterated carbamazepine, phenytoin and valproic acid tracer compounds with their corresponding unlabeled analogues. Although the last two methods were GC and not LC methods, the critical feature in these methods was the elimination of mass spectrometry for tracer quantitation.

135

3.2. Chromatographic Considerations For some time, researchers have investigated the physicochemical effects of substituting deuterium for hydrogen. Results of these studies, some of which are chromatographic, indicate several effects which are linked, including: 1. carbon-deuterium bonds are shorter than carbon-hydrogen bonds (28); 2. molar volume decreases when deuterium is substituted for hydrogen (29, 30); 3. carbons bonded to deuterium have lower polarizability than carbons bonded to hydrogen (29, 31); 4. deuterated compounds have lower lipophilicity than their protiated analogues and behave accordingly in reversed phase (elute earlier) and normal phase (elute later) chromatographic systems (22, 32, 33, 34). The magnitude of the beneficial isotope effect which allows chromatographic resolution of isotopomers depends on the number and location of deuterium atoms in the molecule (35). The general resolution equation can be the best guide for optimizing the separation (36). This equation:

Rs =

(4)

(~x- 1)k/-~

[']

1 +k'

indicates that resolution between closely spaced components (Rs) is dependent on column efficiency (N, a measure of band broadening), selectivity of the mobile and stationary phase combination for the species to be separated (e, a measure of band separation), and retention as measured by the capacity factor (k', retention factor). Column efficiency (N) is dependent on the quality of the column hardware, packing material and the column packing process; however, it is also dependent on the functional groups present in the solutes (isotopomers). Assuming that hardware is optimized (minimum dead volume), selectivity (a) depends on the stationary phase and mobile phase interactions with each other and with the analyte. Successful separation of isotopomers is dependent on small differences in their affinity for the stationary phase. Therefore, the stationary phase should be chosen to give narrow symmetrical peaks for the solutes with moderate to long retention times. The time required for the separation (as measured by k') is the least important of the three parameters in this case. If selectivity is adequate, retention time

136 can be adjusted easily by changing the mobile phase or increasing column temperature to achieve the separation in a reasonable time. Chromatography is always a trade-off between resolution and speed. In order to gauge resolution, peak retention time (k' as a unit of time t) is a generally acceptable means to measure resolution. For any particular separation on the same column, the above equation can be redefined as follows (35): t~ (1/2)(twl + tw2) t2-

a s --

where relative peak separation (Rs) is calculated from the difference of retention times of adjacent peaks at tl and t2, divided by the average peak base widths of twl and tw2. This concept is graphically demonstrated in Figure 3B. Generally, the above principles will provide a system that will separate isotopomers; however, each pair of analytes will probably require independent optimization. For example, in the system described above for separation of isotopomers of phenytoin and carbamazepine, deuterated ethotoin (2HloEHN, mol. wt. 214.2, %2H = 4.7 percent) is not separated from ethotoin (EHN, mol. wt. 204.2) at k ' = 5.4. As shown in Figure 3A, the Rs value (using the second equation) was only 0.6 for both ethotoins. At the same retention time, however, deuterated carbamazepine epoxide (2H~o-CBZE, mol. wt. 262.3, %2H = 3.8 percent) is completely separated from carbamazepine epoxide (CBZE, tool. wt. 252.3). In this case with the ethotoins, it was necessary to reduce the acetonitrile concentration in the mobile phase from 16 to 6 percent to achieve baseline separation of the EHN isotopomers at k ' = 12.7. THF concentration remained at 4 percent. As shown in Figure 3B the peaks are resolved to an Rs value of 1.1. Selecting a reversed phase column can be a confusing experience. Since the introduction of bonded phase liquid chromatography in the late 1960s (37), people have recognized that all bonded phases of a particular type (i.e., C8, C~8, nitrile) are not equivalent. In fact, one of the major concerns with using bonded phase columns for analyses, particularly in regulated environments, such as pharmaceutical development and environmental monitoring, has been nonreproducibility. This non-reproducibility has not just been between nominally equivalent phases from different manufacturers, but even between lots of material from the same manufacturer. Fortunately, the cooperative efforts of manufacturers of chromatography stationary phases and columns, and academic and industrial researchers over the past 20 years, have resulted in significant improvements in lot-to-lot reproducibility of bonded phases, increased column efficiencies and much greater understand-

137

A

Rs= 0 . 6

iuu

t2

I~1 i

tl

I

1 i

Rs = 1.1

tw 1

t---~ Figure 3. Chromatograms showing relative separation (resolution Rs) of ethotoin (t2) and decadeuterated ethotoin (tl). (A) Using condition described in Szabo et al. (9), and (B) Modifying the above conditions by reducing the acetonitrile content of the mobile phase from 16 to 6 percent.

138 ing of solute interactions with the mobile and stationary phases. Much of this information is available in a special issue of the Journal of Chromatography (38). Cox (39) published a very informative review of the effects of silica structure on reversed phase separations. He concluded that traces of heavy metals in the silica cause most of the problems with reversed phase separations. Olsen and Sullivan (40) used principal components and cluster analysis to categorize 17 different C18 reversed phase columns. Columns were grouped according to separations and peak shapes obtained for selected test mixtures which characterize the stationary phase according to hydrophobic and free silanol interactions, trace metal activity and shape selectivity. This type of information can make column selection much less confusing and increase the ruggedness of HPLC methods. Carr et al. (41) present evidence that shows that retention of nonpolar solutes in reversed phase chromatography is primarily due to attractive interactions between the solute and the stationary phase rather than to repulsive interactions between the solute and mobile phase (solvophobic interactions). Tchapla et al. (42), and Dorsey and Cooper (43), have reviewed retention mechanisms for bonded phase chromatography. Although there is some controversy surrounding this subject, Tchapla et al. (42) concluded that different models for retention apply under different operating conditions, and that partition, adsorption and solvophobic theories are all valid in certain circumstances. 3.3. Detection and Quantitation

The choice of LC detection method to use once base-line isotopic separation is achieved, depends on the drug under investigation. The anti-epileptics we studied are molecules with strong chromophores that readily absorb ultraviolet (UV) light, so that any of the fixed and variable wavelength, or even photodiode array UV/VIS detectors, would be appropriate. The lower limit of quantitation (LLQ) (25) for UV detection generally ranges from the high nanogram to low microgram per milliliter of analyte concentration. Drugs that are therapeutically in the sub-~g/mL range could be quantitated by electrochemical or fluorescence detectors; however, these more selective detection methods might require use of post-column derivatization techniques to enhance drug detection sensitivity. Pre- or post-column derivatization always adds an additional level of technical complexity and a potential source of error to a quantitative assay. The point is that the LC alternative method should not become more technically labor intensive than the mass spectrometry it seeks to avoid.

139 4. CONCLUSION

The LC method we developed (5, 9) was designed for drug interaction studies. Phenytoin and carbamazepine are the two most commonly prescribed antiepileptic drugs against which a new AED entity would be compared. Drug interaction studies generally require quantitative analysis of multiple serialblood samples from multiple subjects given SIL tracers on multiple occasions. For such a volume of samples, a stable, reproducible LC method is a costeffective alternative to mass spectrometry. Bio-equivalence of the heavily deuterated tracers has been demonstrated. Moderately nonpolar and apolar deuterated drugs are well suited for iosotopic separation by chromatographic methods. The polar neurotransmitter dopamine in a heavily deuterated form has been resolved from its unlabeled analogue on a reversed phase C18 column using ion pair chromatography (35). Thus, reversed phase LC separation of polar drugs and heavily deuterated analogues seems possible as long as the hydrophobic and lipophilic interactions of column and compound are optimized. Similar applications for other drug classes are possible if investigators take the necessary steps to show pharmacokinetic equivalence of labeled and unlabled forms, and pay careful attention to the number and location of deuterium atoms on the labeled analogue.

ACKNOWLEDGEMENT

The authors wish to acknowledge the support of the United States Department of Veterans Affairs.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

T.R. Baille and A.W. Rettenmeir, J. Clin. Pharmacol., 26 (1986) 448. R.J. Perchalski, R.A. Yost and M.S. Lee, J. Clin. Pharmacol., 26 (1986) 435. G.K. Szabo and T.R. Browne, J. Clin. Pharmacol., 26 (1986) 400. R.L. Wolen, J. Clin. Pharmacol., 26 (1986) 419. T.R. Browne, D.J. Greenblatt and J.S. Harmatz et al., in Antiepileptic Drug Interactions, Pitlick (ed), Demos, New York (1989) p. 1. R.L. Wolen, in Synthesis and Applications of Isotopically Labelled Compounds, T.R. Baille and J.R. Jones (eds), Elsevier, Amsterdam (1989). T.R. Browne and G.K. Szabo, in Synthesis and Applications of Isotopically Labelled Compounds, E. Buncel and G.W. Kabalka (eds), Elsevier, Amsterdam, 1991) p. 397. D.J. Hoffman and W.R. Porter, J. Chromatogr., 276 (1983) 301. G.K. Szabo, R.J. Pylilo and T.R. Browne, J. Chromatogr., 535 (1990) 271. M.I. Blake, H.L. Crespi and J.J. Katz, J. Pharm. Sci., 64 (1975) 367.

140 11. A. Van Langenhove, J. Clin. Pharmacol., 26 (1986) 383. 12. Y. Kasuya, K. Mamada and S. Baba et al., J. Pharm. Sci., 74 (1985) 503. 13. G.E. Von Unruh, B.Ch. Jancik and F. Hoffmann, Biomed. Mass. Spectrom., 7 (1980) 164. 14. D.A. Durden and A.A. Boulton, J. Neurochem., 36 (1981) 129. 15. A. VanLangenhove, C. Costello, and K. Biller et al., Biomed. Mass. Spectrom., 7 (1980) 576. 16. K. Mamada, Y. Kasuya and S. Baba, Drug Met. Disp., 14 (1986) 509. 17. Y. Benchenkroun, B. Ribon, and J.B. Falconnet et al., J. Clin. Pharmacol., 29 (1989) 168. 18. G.P. Cartoni and I. Ferretti, J. Chromatogr., 122 (1976) 287. 19. H.D. Heck, R.L. Simon and M. Anbar, J. Chromatogr., 133 (1977) 281. 20. J.P. Thenot, T.I. Ruo and G. Paul Stec et al., in Recent Developments in Mass Spectrometry in Biochemistry and Medicine, A. Figerio and M. McCamish (eds), (Elsevier, Amsterdam, 1980) p. 373. 21. T.K. Gerding, B.F.H. Drenth and R.A. DeZeeuw, Anal. Biochem., 171 (1988) 372. 22. J.B. Falconnet, N. El Tayar and A. Bechalany et al., in Synthesis and Applications of Isotopically Labelled Compounds, T.A. Baillie and J.R. Jones (eds), (Elsevier, Amsterdam, 1989) p. 355. 23. G.K. Szabo and T.R. Browne, Clin. Chem., 28 (1982) 100. 24. R.J. Perchalski and B.J. Wilder, Anal. Chem., 51 (1979) 774. 25. G.K. Szabo, H.K. Browne and A. Ajami et al., J. Clin. Pharmacol., 34 (1994) 242. 26. J.W. Faigle and K.F. Feldman, in Antiepileptic Drugs 4th Edition, R.H. Levy and R.H. Mattson et al. (eds), (Raven Press, New York, 1995) p. 499. 27. T.R. Browne, G.K. Szabo and H. Davondi et al., Neurology, 44 (1994) 2410. 28. L.E. Sutton (eds), Tables of Interatomic Distances and Configuration in Molecules and Ions, Suppl. 1956-1959 (The Chemical Society, London, 1965). 29. L.S., Bartell and R.R. Roskos. J. Chem. Phys., 44 (1966) 457. 30. A. Leo, C. Hansch and P.Y.C. Jow. J. Med. Chem., 19 (1976) 611. 31. I.M. Kovach and D.M. Quinn, J. Am. Chem. Soc., 105 (1983) 1947. 32. N. Tanaka and E.R. Thornton, J. Am. Chem. Soc., 99 (1977) 7400. 33. N. Tanaka and E.R. Thorton, J. Am. Chem. Soc., 99 (1976) 1617. 34. N. El Tayar, H. van de Waterbeemd, M. Gryllaki, B. Testa and W.F. Trager, Int. J. Pharmaceut., 19 (1984) 271. 35. C.F. Masters, S.P. Markey and I.N. Mefford, Anal. Chem., 60 (1988) 2131. 36. L.R. Snyder and J.J. Kirkland (eds), Introduction to Modern Liquid Chromatography, 2nd edn (Wiley, New York, 1979) p. 34. 37. H.N.M. Stewart and S.G. Perry, J. Chromatogr., 37 (1968) 97. 38. Special issue, J. Chromatogr. (1993) 656. 39. G.B. Cox, J. Chromatogr., 656 (1993) 353. 40. B.A. Olsen and G.R. Sullivan, J. Chromatogr., 692 (1995) 147. 41. P.W. Carr, J. Li, A.J. Dallas, D.I. Eikens and T.C. Tan, J. Chromatogr., 656 (1993) 113. 42. A. Tchapla, S. Heron and E. Lesellier, J. Chromatogr., 656 (1993) 81. 43. J. Dorsey and W.T. Cooper, Anal. Chem., 66 (1994) 857.

141

CHAPTER 8

ALTERNATIVES TO MASS SPECTROMETRY FOR QUANTITATING STABLE ISOTOPES: APPLICATION OF NUCLEAR MAGNETIC RESONANCE IN BRAIN METABOLIC RESEARCH

K.L. BEHAR Department of Neurology, Yale University School of Medicine, New Haven, Connecticut

1. I N T R O D U C T I O N

The explosive growth in the application of NMR spectroscopy to the study of brain metabolism and function is a testament to the versatility of this noninvasive and clinically applicable method. NMR-active isotopes exist for the majority of the biologically important nuclei but only a few of these (e.g. 1H, 31p, ~3C, ~SN, ~gF) are widely used in brain studies. A steady progression of technical developments has led to studies that span from the imaging of anatomy, diffusion, and function to the spectroscopic imaging of metabolite concentrations and metabolic fluxes (1). ~H is the most sensitive and abundant of the stable NMR-active nuclei. Water is the most concentrated of brain constituents and measurement of its spatial distribution in the brain forms the basis of magnetic resonance imaging (MRI). Detection of the low molecular weight metabolites (e.g. glucose, lactate, glutamate) requires that water and peripheral tissue lipid resonances be suppressed with great efficiency (>104-10S-fold). The list of compounds that have been reported in ~H spectra of brain now exceed twenty (2, 3). Neurochemical signatures of cell-type and density and/or disease processes have been identified in the 1H NMR spectrum and combined with imaging to create intensity maps of the resonances of these compounds (1). The relatively high concentrations (>O.5mM) of phosphorylated com-

142 pounds of key importance in cellular oxidative reactions, e.g. adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (P~), have lead to the widespread use of 3~p NMR in the study of cerebral energetics in vivo (1, 4, 5). Although 31p is intrinsically less NMR sensitive than 1H (6.6 percent), the high natural abundance of the 3~p isotope in nature (100 percent) requires no specific enrichment for its detection. A unique capability of 3~p NMR is that intracellular pH can be determined simultaneously with the measurement of phosphorylated compounds (6). Phosphodiesters and monoesters detected in 3~p NMR spectra are associated with cellular membrane metabolism and these substances are altered in some disease states. A powerful application of NMR lies in the investigation of brain glucose metabolism and the quantitative determination of pathway fluxes (7, 8). The basic mechanisms regulating the transport and conversion of glucose to cellular energy can be studied using isotopic labeling techniques (e.g. ~3C, ~SN, ~gF). The use of ~3C and ~H NMR in conjunction with labeled isotopes of glucose and other substrates in vivo and in vitro have provided new information on the interactions between neurons and glia (4, 9). Measurements of the inhibitory neurotransmitter 7-aminobutyric acid (GABA) during anti-epileptic therapy in animals and humans highlights the role that NMR can play in the discovery and testing of new pharmaceutical agents (10). This chapter does not represent a comprehensive review of the literature; instead, the author has sought to show some examples of how NMR can be used to investigate cerebral metabolism and monitor both the progression of disease and treatment. Consequently, many important studies were not included and the reader is encouraged to consult the excellent reviews (1, 4 9, 11) and references within the text for detailed information concerning each application.

1.1. Some Basic Principles of NMR 1.1.1. Nuclear spin and the NMR phenomenon The basis for the NMR phenomenon arises from an intrinsic property of nuclei having odd numbers of protons or neutrons and unpaired nuclear spins. Such nuclei possess a net spin angular momentum. Because nuclei are charged particles, nuclei with spin possess a magnetic moment. In the presence of an applied static magnetic field (Bo), the nuclear moments orient themselves in discrete states given by the spin quantum number,/, w h e r e / = _+1/2 or ->1.

143 Nuclei w i t h / = 0 are nonmagnetic (e.g. ~2C) and do not give rise to an NMR signal. For a spin-l/2 nucleus, the orientation of the magnetic moment lies either with or against the field. Most of the nuclei studied in biological systems are spin-l/2 (e.g. ~H, ~3C, 3~p, ~9F) and in the presence of a magnetic field can be described by transitions between two energy levels, although spin-3/2 nuclei may be encountered (e.g. Na § Li § leading to more energy levels and more complex relaxation behavior. The proportion of spins aligned (lower energy) or opposed (higher energy) with the field can be described statistically and is given by the Boltzman distribution. At ambient temperature, there exists only an extremely small excess population of spins aligned with the Bo field (i.e. low energy state). At thermal equilibrium this small spin population difference gives rise to a "net magnetization" and it is this property, when suitably prepared, that is measured in the NMR experiment. The equilibrium net magnetization is proportional to the strength of the applied static magnetic field and the total number of nuclear spins in the sample. The intensity of an NMR signal relative to noise (i.e. detection sensitivity) is proportional to this population difference, increasing with both field strength and sample concentration. The interaction between the applied magnetic field and each nuclear moment induces the latter to precess about the direction of the applied field. Because the net magnetization is given by the vector sum of the individually precessing moments, it can also be viewed in terms of a vector precessing about the direction of the static field. The precession frequency is proportional to the applied field and is given by the Larmor equation:

v = 712]-[ x Bo

where v is the Larmor frequency, 7 is the gyromagnetic ratio and is a constant that depends on the intrinsic properties of the nucleus, and Bo is the external magnetic field strength. The frequencies encountered in the majority of clinical and high resolution NMR applications are in the MHz range (~--2-500 MHz) for magnet strengths between 1 and 12 Tesla. The highest field large bore clinical system currently available operates at 4 Tesla, although higher field systems are currently being considered. For a given field strength each NMRactive nucleus (e.g. ~H, ~3C, etc) will be resonant at a characteristic frequency as defined in the Larmor equation. A more detailed discussion of NMR theory and its application may be found in (1) and the many excellent references cited therein.

144

1.1.2. Relaxation: the concept of T1 and T2 Detection of an NMR signal involves a perturbation of the magnetization from equilibrium. A radio-frequency (rf) pulse oscillating at the resonance frequency defined by the Larmor condition is used to perturb the magnetization from equilibrium. The perturbed nuclear spins return to thermal equilibrium, through the process of relaxation with a rate defined by the spinlattice relaxation time, TI. The T1 (spin-lattice) relaxation time refers to the longitudinal (along z-axis) vector component of the magnetization (Mz) as it returns to its equilibrium value (Mo). The intensity of an NMR signal is directly proportional to Mo prior to the rf perturbing pulse. As the magnetization is tipped away from equilibrium along the z-axis and into the xy-plane, randomizing interactions between the spins and their environment causes a loss of phase coherence and magnitude of transverse magnetization, Mxy. The decay of the transverse magnetization is defined as T2 (spin-spin) relaxation. Only transverse magnetization is detected directly with the receiver coil and it is this signal that is represented by the free-induction-decay (FID). Spatial inhomogeneity in the static magnetic field also contributes to T2; differences in precession rates between spatially separated nuclear spins results in a more rapid loss of phase coherence and decay of Mxy than if the field were the same everywhere. This decay is characterized by an apparent T2 relaxation time (Tt) where Tt < T2. Because the resonance linewidth is equal to 1/(]-[ x T2), field inhomogeneity leads to shorter values of Tt and broadening of the resonance width. Overlap between closely spaced resonances is a problem regularly encountered in NMR spectroscopy in vivo. The process of "shimming" involves the adjustment of a secondary set of gradient coils to smooth out field irregularities across the sample.

1.1.3. Detection of the NMR signal The tiny oscillating magnetic field generated by the sample can be detected with a suitable antenna (i.e. loop of wire) oriented perpendicular to the direction of the static field. Transmission and reception of rf is often obtained from a single antenna coil although different coils may be used for this purpose, such as for applications involving NMR of several different nuclei from the same sample or subject. The small currents created by the oscillating field detected by the coil is amplified, digitized, and sent to a computer for further processing. Standard processing techniques involve a Fourier transformation of the digitized free induction decay to give the frequency spectrum.

145 1.1.4. Chemical shift

The resonance frequency of a given magnetic nucleus (e.g. a hydrogen atom in glucose) is determined by its electronic and chemical environment and the given magnetic field strength. The electrons within molecules shield their nuclei from the external magnetic field resulting in slight differences in their resonance frequencies. This nuclear shielding is proportional to the applied field strength. The ratio of the shielding to field strength is referred to as the "chemical shift". The chemical shift is expressed as a fraction of the applied field strength in parts-per-million (ppm) from a suitable reference and is an identifying characteristic of the NMR spectrum of a particular molecule. 1.1.5. Spin coupling

The interaction between the magnetic fields of neighboring magnetic nuclei (e.g. 1H-13C) within a molecule induces additional splitting of the energy levels for each nucleus giving rise to the phenomenon of spin-spin or J coupling. The multiplicity of resonances observed for covalently bound methylene protons of amino acids in the 1H NMR spectrum or the heteronuclear ~3C-~H splitting observed in the ~H or ~3C spectrum of a 13C labeled metabolite are examples of this type of interaction. The coupling constant, J, is given in Hz and is independent of field strength. Although a detailed discussion of spin coupling is outside the scope of this review, it is important to note that spinspin or J-coupling is the basis of many of the spectral editing techniques currently used to select and simplify the in vivo NMR spectrum (e.g. lactate and GABA) from the large number of overlapping resonances of other compounds.

2. 1H NMR IN THE NEUROCHEMICAL DETECTION OF BRAIN PATHOLOGY 2. I. N-acetylaspartate as a NeuronaI-Axonal Marker

N-acetylaspartate (NAA)is highly concentrated in animal and human brain (5-8 mM) and is the most prominent metabolite resonance in the ~H NMR spectrum. Immunohistochemical and cell culture techniques have shown that NAA is confined mainly to neurons (11, 12). While NAA is not detected in 1H spectra of astroglial cultures (13) and tumors of glial origin (14-16), significant levels have been found in some immature oligodendroglia, where it may have a role in the synthesis of myelin (13). Decreased levels of N-

146 acetylaspartate, and by inference reduced neuronal density, has been observed in the necrotic regions of cerebral infarcts (17, 18), epileptic foci (19), and in patients with acquired human immunodeficiency syndrome (20), Creutzfeldt-Jakob Disease (21), and Alzheimer's dementia (22). However, decreased NAA levels may not always be related to neuronal death, as reversible changes have been observed in white matter lesions in patients with multiple sclerosis (23, 24), which may be related to changes in synthesis and/or degradation of NAA. Although the function of NAA (and myo-inositol, see below) in the brain remains obscure, heightened interest for their use as cell-type specific markers has resulted in a renewed interest in the metabolic pathways involved in their synthesis and degradation. 2.2. Myo-inositol as a Glial Marker

Myo-inositol has been identified as a possible glial marker based on the finding that high levels are present in cultured astrocytes but not neurons (25). The role of myo-inositol in cerebral metabolism is unclear but some evidence indicates it functions as an osmolyte. Increased levels of myoinositol have been observed in ~H NMR spectra of the cerebri of patients with Alzheimer's (22) and Creutzfeldt-Jakob dementia (21), whereas reduced levels are observed in hepatic encephalopathy (26). These conditions are known to affect glial cells. As with all metabolite markers, changes in levels may reflect either altered synthesis or degradation within a given cell or a change in cell number. The coordination of studies linking clinical disease states and cellular changes with appropriate animal and in vitro models are needed to elucidate the function of myo-inositol. 2.3. Macromolecules: Lipid and Protein as Markers of Brain Disease

Low molecular weight metabolites represent only a part of the 1H NMR spectrum and techniques have been developed to reveal the broad (shorter T2), but informative, background of underlying macromolecules (27). In normal nondiseased brain tissue, the majority of macromolecule resonances arise from cytosolic proteins (27, 28) with few if any resonances identifiable as lipids. During brain injury, such as stroke, tumors, and active multiple sclerosis lesions, loss of cellular membrane integrity, myelin breakdown, and possibly macrophage infiltration can lead to marked increases in lipid signals (18, 23, 24, 29). The ability to follow metabolic changes during the inflammatory process noninvasively should aid in the monitoring of new therapies targeted at reducing the inflammation associated with brain injury.

147 3. 31p NMR MEASURES OF BRAIN ENERGY METABOLISM

3.1. High Energy Phosphate Metabolism, pH and Mg 2§ The concentrations of the high energy phosphates, phosphocreatine (PCr) and adenosine triphosphate (ATP), are substantial in brain (5 and 3 ~mol/g, respectively), and are readily detected using 31p NMR. Signal-averaging times of 1-5 min to acquire a 0.5-1 cc3 volume of animal brain and 30-60 min for a -100 cc3 volume of human brain are typical using surface coil techniques. 3~p NMR is particularly useful in the determination of intracerebral pH based on the pH dependence of the chemical shift of inorganic phosphate (6). Free intracellular [Mg 2§ can be estimated from the chemical shift of ATP (30). Based on the equilibrium reaction maintained by creatine kinase (see below), both [ADP] and [AMP] concentrations can be calculated from the measured changes in [PCr], [ATP] and [H § in the 31p NMR spectrum (31). Changes in PCr, ATP, P~ and pHi in the 3~p NMR spectrum are sensitive indicators of tissue oxygenation. Cerebral hypoxia and ischemia leads to rapid depletion of high energy phosphates, elevation of P~, and cellular acidosis in animals (32) and in patients with stroke (33). Phosphomonoesters (PME) and phosphodiesters (PDE) in 3~p NMR spectra, which are linked to cellular phospholipid synthesis and catabolism, are altered in some disease states such as AIDS (20) and Alzheimer's dementia (34). As for other NMR-active nuclei detected in vivo, phosphorylated substances must be relatively mobile on the NMR time-scale to give narrow peaks in the 3~p NMR spectrum; phosphorylated proteins, which are low in concentration and restricted in motion, cannot generally be observed in vivo.

3.2. Creatine Kinase Flux Measured In Vivo Using Magnetization Transfer Cellular levels of phosphocreatine and ATP in brain tissue are enzymatically linked through the creatine kinase (CK) reaction. It has been possible under certain conditions to extract information on high energy phosphate turnover by measurement of the unidirectional fluxes for phosphoryl group transfers catalyzed by CK using magnetization transfer techniques (35). In the saturation transfer method, a low power rf field is applied to saturate either of the phosphate resonances of PCr or the ?,-phosphate of ATP, which is the phosphate group undergoing chemical exchange with creatine. Transfer of this "magnetization label" to creatine by the CK reaction results in a reduction in the intensity of PCr. By increasing the length of time between the end of the

148 saturating rf and the acquisition of the 31p spectrum in successive experiments, a time course of the recovery of PCr to its equilibrium state (or ATP7 if PCr is saturated) is obtained and used to derive the pseudo-first-order unidirectional rate constants for the forward reaction (PCr~ ATP) and the reverse reaction (ATP-~ PCR). In principal, both forward and reverse rate constants and fluxes can be determined. However, the reverse rate constant is more difficult to measure accurately (36) so that many investigators report only the forward rate constant. Results of a saturation transfer study of rat brain showed that phosphoryl group exchange between PCr and ATP is several-fold faster (>5 times) than the rate of ATP utilization (37). Evidence that CK is operating at, or near, equilibrium has been reported both in rabbit brain in vivo (36) and in superfused brain slices (38). The fast rates of exchange between PCr and ATP relative to the rate of ATP hydrolysis probably accounts for the lack of ATP changes during acute hypoxia or seizures (31, 39). In human grey matter the CK forward flux is 2-fold greater than in white matter (40), suggesting that CK activity is greater in neuron-rich areas. An activity dependent relationship in both the forward rate constant and flux of CK has been reported during thiopental anesthesia and seizures in rats (41). Changes in CK kinetics are also seen during development where a 4-fold increase is observed in the CK forward reaction rate over a narrow time period between 12-17 days after birth. This time period parallels the increase in the activity of the mitochondrial CK iso-enzyme when the latter is expressed as a percentage of total brain CK activity (42). The timing for the increase both in CK flux and mitochondrial CK expression is also coincident with development-dependent changes observed in the time course of PCr and ATP depletion during prolonged hypoxia. These studies suggest that ATP utilization and synthesis are closely associated with the maturational increase in CK flux.

4. NMR MEASURES OF CARBOHYDRATE METABOLISM

Glucose is the major fuel of the mature brain accounting for >90 percent of oxygen consumed in the well nourished state. During development, substrates other than glucose contribute appreciably; lactate and ketones (/3hydroxybutyrate and acetoacetate) may be oxidized to a greater extent than glucose, a process that appears to be related both to neuronal and glial development and the concentrations of these fuels in the blood. The availability of ~3C labeled isotopes of glucose, lactate and /3-hydroxybutyrate

149 provides the opportunity to explore many facets of cerebral energy metabolism. NMR spectroscopy permits measurement not only of the properties of key steps in the metabolism of glucose, such as the affinity and capacity of glucose transport, but the rates of the major pathways of energy metabolism. The determination of the TCA cycle flux allows the determination of several other fluxes, which include: glucose utilization, a-ketoglutarate/glutamate exchange, glutamine synthesis, and GABA synthesis. These pathways are of fundamental importance in brain energy metabolism, are altered during disease, and are uniquely suited to measurement with NMR methods.

4.1. Glucose Transport The concentration of intracellular glucose in the brain (G;) is determined by the kinetic parameters of the glucose transport proteins that reside in the microvasculature of the blood-brain-barrier (Kt, Tmax), the blood glucose concentration (Go), and the rate of glucose metabolism by the brain (CMRg~u). Glucose levels have been measured in rat (43) and human brain using 13C NMR (44, 45) and ~H NMR methods (46-48). At steady state, when blood and brain levels of glucose are constant, the unidirectional inflow and outflow velocities can be described in terms of the Michaelis-Menten formulation according to the following equation:

/max"~_

dG* = ('Go x Tmax~_ ( G ; x dt \ i G o +~)t) J (G~-+ Kt) /

CMRg,u = 0.

When this equation is fitted to sets of paired measurements of blood and brain glucose and iterated with the parameters Kt and the ratio of Tmax-tOCMRg~u, the values of Kt and Tmax of glucose transport are obtained. Both rat and human brain glucose transport kinetic parameters have been determined in this way using 13C NMR detection of [1-13C]-Iabeled glucose (43, 44). The observation that Tmax/CMRg~u > 1 in both rat and human brain in vivo shows that under normal conditions, transport is not limiting the utilization of glucose. Brain diseases associated with decreased transporter density and Tmax, e.g. Alzheimer's Disease, would be expected to narrow this margin. Another approach is to measure the change in the glucose signal in the brain following a rapid step-up in blood glucose concentration. The glucose infused may be either labeled or unlabeled, the choice determining the parti-

150 cular detection technique. ~H NMR detection of glucose in difference spectra of human occipital cortex (46) allows spectra to be acquired in as little as 3 min (48, 50). The rapid time resolution possible with ~H NMR allows dynamic changes in individual subjects to be assessed from the rise of the brain glucose signal (48). Intersubject variability can also be assessed directly. Because the transport kinetic parameters can be determined for individual subjects, the step-up method should be of particular value in clinical investigations of suspected glucose transport alterations and measurements of glucose utilization in paired stimulus-response functional studies. In the absence of changes in the transport parameters, changes in glucose levels during cortical activation can be related to changes in glucose utilization. Glucose levels have been observed to decrease in visual cortex during photic stimulation in humans which is consistent with an increased rate of glycolysis (7, 8, 49, 50).

4.2. Glucose Utilization 4.2.1. Glycolysis and lactic acid

The end product of the anaerobic utilization of glucose in mammalian brain is lactic acid. Under basal aerobic conditions, brain lactate levels measured in vivo using 1H NMR editing techniques are about 1 mM. Although hypoxia and ischemia can lead to high concentrations of lactate in vivo (17, 18, 32, 51), increased levels can be produced when the rate of glycolysis exceeds oxidation and lactate removal by the blood (e.g. seizures or hypocapnia), a subject that has fueled recent controversy in studies of functional activation (7, 8). Under hypoxic or ischemic conditions, glucose utilization through the glycolytic pathway can be determined from the change in the lactate concentration verses time in a series of sequentially acquired 1H NMR spectra. Lactate can be measured either without isotopic labeling (e.g. difference spectroscopy (51) or editing (52)) or after the enrichment of the precursor glucose pool (53) and both approaches have their specific advantages. Because the heteronuclear ~3C-edited ~H difference technique (53) permits both labeled and unlabeled species to be measured from a single set of acquired spectra, the fractional enrichment of lactate-C3 reflects the sum of the pathways contributing carbon atoms (both ~3C and ~2C) to the C3 position of lactate. During ischemia, the brain is essentially a closed system such that inflow of glucose and outflow of products is halted and oxidative pathways are inhibited. Under conditions of constant glucose labeling in the blood, the contribution of unlabeled endogenous glycogen to pyruvate (and lactate) production during ische-

151 mia resulted in the dilution of labeled lactate from which the concentration of glycogen could be estimated (53). Because glycogen is known to be localized in glia, its hydrolysis is a measure of glial metabolism; in principal similar measurements could be used to determine the contribution of glial glycogen metabolism during seizures. ~3C-labeling of the large lactate pool observed in human stroke (54) has shown that the lactate observed in ~H NMR spectra is metabolically active and in communication with blood glucose. Lactic acid may also be produced actively from leukocytes within weeks of cerebral infarction (18) and may be suspected when macrophage-associated lipid resonances are present. The ability to monitor these changes noninvasively provides new opportunities to assess the efficacy of different therapeutic approaches.

4.2.2. Hexose monophosphate shunt The hexose monophosphate shunt (HMP) or alternatively, the pentose phosphate cycle, diverts glucose carbon from the glycolytic pathway for the production of pentoses for nucleic acid synthesis and NADPH for synthesis of lipids and maintenance of reduced glutathione. In the adult brain, HMP activity accounts for only 3-5 percent of glucose metabolism but is increased during oxidative stress as shown in studies of rat brain using 13C NMR (55, 56). Discrimination between differently labeled glucose isotopes in the HMP pathway occurs at the level of 6-phosphogluconate dehydrogenase, which catalyzes the decarboxylation of C1 of 6-phosphogluconate produced from glucose-6-phosphate. For glucose labeled at C1, flux through the HMP pathway will result in the loss of label as 13C02 and in a lower enrichment of lactate C3. The measurement of HMP flux has been accomplished by monitoring label incorporation into lactic acid after the addition of equal mixtures of [1~3C] and [6-~3C]glucose to transformed glial cells in culture (57); an approach that corrects for the significant recycling of trioses that can lead to overestimation of HMP flux when using [1-~3C]glucose alone. HMP flux can also be determined from the relative distribution of ~3C in the C4 and C5 of glutamate at steady state using [2-13C]glucose (58).

4.2.3. Fluoro-deoxyglucose and alternate pathways of glucose metabolism Fluoro-deoxyglucose (FDG) competes with glucose for transport into brain tissue and is readily phosphorylated by hexokinase to fluoro-deoxyglucose6-phosphate (FDG-6-P). When FDG is administered in the blood in tracer

152 quantities, the fluorinated tag is trapped in tissue predominately as FDG-6-P, since the latter is not a substrate of phosphohexose isomerase (glycolytic pathway) and is only slowly metabolized by enzymes of the HMP pathway. However, under conditions of high loading doses (>400 mg/kg) of FDG, the product FDG-6-P may rise substantially in brain (>1 ~mol/g) as shown in rats using both 31p NMR (59) or ~gF NMR (60, 61). FDG-6-P can be metabolized further, albeit slowly, by the enzymes of the HMP and aldose reductase sorbitol (ARS) or polyol pathways. Administration of the [2-~9F] and [3-~9F] analogues of DG to rats leads to the appearance in brain tissue of the corresponding [2-~9F] and [3-19F] analogues of fluoro-deoxy-6-phosphogluconate, fluoro-deoxyfructose, and fluoro-deoxysorbital, metabolites of the HMP and ARS pathways (60, 61). Pre-treatment of rats with sorbinil, an inhibitor of aldose reductase, at pharmacologic doses reduced the flux of FDG through the ARS pathway (61, 62). ~gF NMR employing high loading doses of 2- and 3-fluorinated analogues of FDG in conjunction with analysis of fluorinated metabolites in extracts may be useful probes to investigate the regulation of the HMP and ARS pathways in the brain.

4.3. TCA Cycle Flux and Oxygen Consumption (CMR02) The metabolism of glucose in the brain can be traced in great detail through the use of 13C-labeled isotopes (7-9). The position of the isotopic label in the glucose molecule determines the specific labeling patterns observed in the various metabolites of glucose (Figure 1). For example, if the glucose molecule is labeled at C1, then pyruvate produced from glucose by the glycolytic pathway will be labeled at C3. Rapid chemical exchange between pyruvate and lactate catalyzed by lactate dehydrogenase will result in the labeling of lactate at C3. Oxidation of pyruvate in the TCA cycle leads to labeling of eketoglutarate C4, and through rapid isotopic exchange with glutamate, labelling of glutamate C4. The rate of isotopic exchange between a-ketoglutarate and glutamate is >60 times faster than the TCA cycle flux in rat and human brain (63, 64, 91). The fast exchange rate together with the high concentration of glutamate in brain tissue (about 10-12mM) makes glutamate a highly efficient ~3C-label trap. Glutamine and GABA synthesized from glutamate will be labeled initially at C4 and C2, respectively. Movement of the ~3C label around the TCA cycle results in the labeling of other carbon positions in glutamate and its products. The rate at which these other positions are labeled (e.g. C3) relative to C4 is a function of the rate of a-ketoglutarate/glutamate exchange and the rate of TCA cycling. The isotopic labeling of glutamate can

153

LABELING

OF BRAIN METABOLITES

FROM

13C G L U C O S E

CCCCCC Glucose

ccc ~

' ,I, ~cc ccc

Lactate

i,3,yco,~,, I

J/ pyruvate

o

CCCC o

cccc ~

ccccc ~__ @

J

ket~lu~r~e

Glutamate

1

succlnate oo 9

eoo

CCCCC

CCCC

Glutamlne

GABA

1

1

Figure 1. Labeling of brain metabolites from [1-13C]glucose. Glutamate efficiently traps 13C, first at C4 (filled circle) and then at other carbon positions due to movement through the TCA cycle (open circles). Carbon numbering is left-to-right as indicated for each molecule.

be detected either with 1H NMR using 13C-editing (65) or direct 13C NMR (91). When the labeled glucose is raised rapidly in blood and held constant, typically 50-60 percent enrichment, glutamate is labeled rapidly at C4 and more slowly at C3 as shown in human brain (Figure 2). Analysis of the time courses of the ~3C enriched spectral peaks using mathematical modelling permits both a-ketoglutarate/glutamate exchange and TCA cycle flux to be determined (63, 64). The glutamate measured with NMR may be associated with the "large glutamate pool" of neurons (65). The TCA cycle rate is coupled to oxygen consumption and can be used as an indirect but sensitive measure of CMRO2 (7). Measurement of the carbon-carbon J-couplings of ~3C-labeled metabolites in conjunction with isotopomer analysis (66-68) can give specific information about metabolism occurring within neurons and astrocytes (see below).

154

Time course of glutamate and other amino acids labelled from [1J3C]glucose in ~3C NMR spectra of human occipital cortex GluC4I 60 min "~

~ GlnC4 GIuC3

5

(ppm)

25

30 min

GluCA

....

c

;4'

'

0 min J0

i

i,

1

30 (ppm)

Figure 2. Time course of 13C labeling of glutamate, glutamine and aspartate in 13C NMR spectra of human occipital cortex during an intravenous infusion of [1-~3C]glucose. Each spectrum was acquired in 4-min blocks. (Reproduced from Gruetter et al., 1994, Ref. 91.)

5. METABOLIC COMPARTMENTATION" NMR MEASUREMENTS OF NEURONAL AND GLIAL METABOLISM Functional nervous tissue represents a bewildering degree of complexity where compartmentation exists at the anatomic, cellular and subcellular level. A major challenge of contemporary NMR spectroscopy as applied to the brain

155 is the development of methods and strategies to assess the contribution of major cell populations (e.g. neurons and glia) to the signals that are measured. Methodological developments include techniques to identify, select, and quantitate resonance intensities and isotopic enrichment (e.g. homonuclear and heteronuclear editing (10, 52, 53, 65)) while isotopic labeling strategies take advantage of known metabolic pathways and cell-specific enzymatic reactions to provide detailed information about neuronal and glial metabolism (67-69). The metabolism of glutamate and GABA associated with nerve terminals has been linked to a substrate cycle between neurons and astrocytes involving glutamate, GABA, and glutamine, generally termed the "glutamine cycle" (70). The efficient functioning of the glutamine cycle is made possible by the physical segregation of certain enzymes between neurons and glia. For example, glutamine synthetase and pyruvate carboxylase are astroglial enzymes. GABA synthesis catalyzed by glutamic acid decarboxylase is confined to neurons. Glutaminase catalyzes the production of glutamate from glutamine and is enriched in neurons. NMR measurements of the fluxes associated with these enzymatic reactions permit investigation of neuron and glia specific metabolic pathways. The synthesis of GABA in neurons and glutamine in astroglia are important examples. These reactions involve rearrangements of both carbon and nitrogen groups and provide opportunities for NMR investigations employing ~3C and 15N isotopes in vivo and in vitro as described below. 5. I. Information Contained in Isotopomer Distributions

A powerful method to determine the contributions of competing metabolic pathways occurring in vivo is based on the analysis of homonuclear 13C-13C couplings in 13C NMR spectra (66). Analysis of the mutiplet structure of ~3C NMR spectra of metabolites isolated from extracts of whole tissue, brain slices, or cell cultures following the use of doubly or universal labeled isotopes (e.g. [1,2-~3C]glucose, [U-~3C glucose], [1,2-~3C]acetate, [U-13C]glutamate) has been used to assess metabolism of neurons and glia in vitro and in vivo. For the case of two adjacent ~3C atoms in a molecule, spin coupling between the nuclei results in the splitting of each resonance into a doublet with a characteristic coupling constant, J (Hz). Singlet resonances arise from the 1.1 percent natural abundance of ~3C and reflect the contribution of unlabeled pathways to the carbon atom at that position in the molecule. Since the probability of observing a doublet in the ~3C spectrum from natural abundance ~3C is low, only 0.0001 percent, detection of a doublet indicates that the doubly

156 labeled carbon skeleton is incorporated intact; other pathways incorporating unlabeled carbon atoms will lead to isotopic dilution of adjacent carbons and the appearance of singlets. Therefore, the proportion of doublets to singlets at steady state represents the ratio of fluxes from labeled and unlabeled pathways. Differences in the isotopomer distributions between the TCA cyclelinked amino acids, glutamate, glutamine, and GABA is consistent with physically separate TCA cycles and the "compartmentation" of their metabolism (67-69). The distribution of ~3C labeling among the five carbons atoms of glutamate gives specific information on the flows of carbon into the cycle. Amino acids synthesized from glutamate, e.g. GABA and glutamine, will reflect the isotopic distribution of the glutamate and TCA cycle involved in their synthesis. Analysis of the isotopomer distributions by appropriate steady state mathematical models (66, 71, 72) has allowed the relative contributions of these pathways in neurons and glia to be assessed (68, 72). 5.2. Differentiation of Neuronal and Glial Metabolism from Isotopic Labeling

The distribution of isotopic labeling of glutamate, glutamine, and GABA in NMR spectra obtained from extracts of animal brain tissue in vivo (67, 68, 73) and in vitro (69, 74) following application of 13C-labeled substrates has been interpreted in terms of neuronal and glial compartmentation and the "glutamine cycle". Particularly advantageous for isotopic studies is the observation that neurons and glia can be differentiated by their preference for different substrates. Whereas neurons are almost solely dependent on glucose, glial cells are capable of metabolizing acetate carbon (9). The metabolism of [113C] or [1,2-~3C] acetate in rats leads to different isotopomer distributions of glutamate and glutamine in ~3C spectra of brain extracts (67). In superfused brain slice preparations glutamine is more highly labeled than glutamate from [2-~3C]acetate but not [1-~3C]glucose (69). Glutamine labeling from acetate occurs readily in cultured glial cells and co-cultures of glia and neurons but not in neuronal cultures ( 7 5 ) - a finding that is consistent with the known localization of glutamine synthesis in glia. Because glutamate serves as the direct precursor of glutamine, unequal 13C isotopic distributions between glutamate and glutamine must arise from physically separate glutamate pools. Together these findings are consistent with separate TCA cycles and separate pools of glutamate in neurons and glia. In vitro findings have provided an important framework for the interpretation of labeling patterns observed in vivo and will continue to be important to the development and refinement of realistic multicompartment metabolic models for the analysis of in vivo data in animal and human brain (63, 64, 66, 71, 72).

157 5.3. Glutamine Synthesis

Glutamine synthesis from [1-13C]glucose has been measured from the time course of glutamine labeling in13C NMR spectra obtained from extracts of rat brain (73), rat brain in vivo (76), and human occipital cortex (91). Metabolic modeling analysis of the glutamine and glutamate enrichment time courses in conscious human brain and rat brain in vivo have indicated that glutamine synthesis is more rapid than generally thought and may reflect, in part, the rapid exchange of labeled glutamate and GABA between neurons and glia (64, 76). This finding is consistent with a study of brain slices (77), where glutamine labeling from [13C]glucose was found only following KCI depolarization, suggesting that glial glutamine synthesis from glucose is enhanced in response to increased activity. The concentration of blood and brain ammonia has a major role in the regulation of brain glutamine and the glutamine cycle. Brain glutamine levels are increased in hyperammonemia as shown in experimental animal models (78-80) and human patients with hepatic encephalophy (26). Elevated glutamine has also been observed in some epilepsy patients on anti-epileptic medications (81) as discussed below. The time course of [5-15N] glutamine labeling in 15N NMR spectra following administration of ~SN-labeled ammonium chloride has permitted the rate of glutamine synthesis to be measured in the hyperammonemic rat brain in vivo (80). The disappearance of 15N-labeled glutamine in 15N-NMR spectra following the administration of an inhibitor of glutamine synthesis has been used to estimate the rate of phosphate-dependent glutaminase (82). The reaction catalyzed by glutaminase is believed to be crucial for the neuronal replenishment of transmitter glutamate and GABA. 5.4. Anaplerotic Flux and the Contribution of Gila to Glucose Metabolism

The major anaplerotic enzyme in brain tissue, pyruvate carboxylase (PC), is highly enriched in glial cells and is the main pathway capable of replenishing the carbon skeletons of TCA cycle intermediates lost from glia during the dynamic process of neurotransmitter cycling and ammonia detoxification. The proportion of glucose metabolized through PC represents about 10 percent of total glucose metabolism (83). Isotopic labeling of brain glutamate and glutamine from glucose or acetate can be used to measure PC activity. Pyruvate formed from glucose through the glycolytic pathway may undergo direct decarboxylation and acetyI-CoA formation via pyruvate dehydrogenase (PDH) or carboxylation to oxaloacetate via pyruvate carboxylase (PC). Because

158 c~-ketoglutarate C4 (and glutamate C4 by exchange) originates from the methyl group of acetyI-CoA, labeling at that position and that of glutamine C4 will be derived only from C3-1abeled pyruvate. In contrast, carboxylation of pyruvate labeled at C3 will lead to an additional flow of label into oxaloacetate C2 and/or C3 (depending on the state of equilibration with fumarase) leading to more label at glutamate C2 (and glutamine C2) than would be expected based on flux through PDH alone. Values of 38 percent of total glial metabolism (72) and 60 percent (84) of PDH flux have been calculated for the contribution of the PC pathway using this approach. If the flux through PC accounts for 10 percent of total glucose metabolism, then glia could account for ~--27 percent of brain glucose metabolism (84). 5.5. Detection of Neurotransmitters In Vivo: The Unique Case of GABA

The concentration of substances that can be detected in vivo is limited generally to a few hundred micromolar which exceeds by 7-fold the concentrations of neurotransmitters such as acetylcholine, dopamine, or serotonin. However, the amino acids glutamate and GABA, which exist at millimolar levels, are readily detected and quantitated with specialized techniques. For example, GABA (0.9-1.0 mM) can be measured in 1H spectra of single brain volumes of about 8 mL in <10 min of signal averaging when using J-editing and difference spectroscopy (10). The GABA and glutamate measured in 1H NMR spectra is predominately intracellular because extracellular levels are low. However, the cellular location (neuronal versus glial) cannot be distinguished based on the measurement of concentration alone. Therefore, interpretation of glutamate and GABA spectra in terms of neurotransmitter metabolism poses a challenging problem. As discussed below, GABA levels measured by ~H NMR in human cortex correlate positively with CSF levels and negatively with seizure frequency following GABA-T inhibition (85) suggesting that changes in cortical GABA levels measured in vivo reflect parallel changes in GABA concentration at the synaptic level. Rates of synthesis can often be localized with great specificity. For example, the measurement of the rate of GABA synthesis will reflect only a specific population of cells (e.g. GABAergic neurons). 5.5.1. Effects of an anti-epileptic drug on GABA metabolism

GABA is the major inhibitory transmitter and central to the etiology of epilepsy. GABA levels can be increased in brain through inhibition of the enzyme which degrades it, GABA-transaminase, an enzyme present both in neurons

159 and glia. Highly specific, irreversible inhibitors of GABA-T have been introduced (e.g. ~/-vinyl GABA and gabaculine) and these agents lead to marked increases in GABA levels which can be readily measured in animal and human brain using J-editing techniques (10). Studies of the effects of the GABA-T inhibitor vigabatrin on GABA levels of epilepsy patients have shown that GABA levels rise with vigabatrin dose up to 3 g/day but rise no further with increasing dose (85). Progressive decreases in GABA has been reported in some patients receiving a constant high dose of vigabatrin over a period of several months (85). In a study of rat brain, the cortical rate of GABA synthesis from [1-~3C]glucose was reduced 70 percent 24 h after vigabatrin administration (86) and suggests that the plateau of GABA levels observed in patients receiving vigabatrin is due to reduced GABA synthesis. Both observations are consistent with the down-regulation of one of the two major isoforms of brain GAD by increased GABA levels (87).

6. FATTY ACID METABOLISM

Fatty acids are metabolized more slowly than glucose in brain compared to other tissues such as heart and liver and few in vivo NMR studies of cerebral lipids using isotopic labeling in vivo have appeared. Many potential applications exist, however, particularly during brain development when membrane lipid turnover is more rapid. Cunnane et al. (88) reported that a mixture of C16 and C18 uniformly 13C-labeled polyunsaturated fatty acids administered intragastrically in neonatal rats were incorporated into longer chain C20 and C22 phospholipids (e.g. arachidonic acid and docosahexaenoic acid). De novo synthesis of cholesterol and monounsaturated fatty acids from the labeled precursors was also observed. Although direct observation of brain lipids using 13C in vivo is of limited usefulness due to broadened lines and loss of multiplet resolution, a great deal of structural information is readily available in both ~3C and ~H NMR spectra after chloroform-methanol extraction (89). A ~SC isotopomer-based NMR method has been developed for investigation of fatty acid metabolism in heart. Gavva et al. (90) used mixtures of multiply-labeled octanoate ([2,4,6,8]~3C and [1,2,3,4113C octanoate) to investigate mitochondrial/3-oxidation of fatty acids in perfused heart under different conditions of work load, ischemia and anoxia. Based on the isotopomer distribution of glutamate C4 in the ~3C NMR spectra, this measurement, which does not require steady state conditions, could be of potential use in studies of developmental abnormalities of cerebral lipid metabolism.

160 7. APPLICATION OF NMR IN DRUG PHARMACOKINETICS

7.1. ~H NMR of Drugs in Biofluids Studies of biofluids (plasma, urine and CSF) can yield much information on the catabolites of administered drugs aiding in the assessment of their rates of accumulation, excretion, and metabolism and mechanism of potential toxicity. Another advantage is that both the drug and its catabolites are measured simultaneously along with many other metabolites important in cellular metabolism and function. 1H NMR detection of a drug or drug metabolite in vitro requires a concentration of ->501~M; a limit that depends on the number of protons on the groups being detected (e.g.--CH, INCH2,--CH3, etc.), multiplicity due to spin-coupling, and the presence of other overlapping resonances from nondrug molecules. The drug may be detected in the biofluid with little or no sample preparation; for nonvolatile substances, simple lyophilization and dissolution into D20 will often suffice. In some cases, quantitation and selectivity may be improved by prior chromatography using solid phase extraction (92) or HPLC (93; and references therein). Several drugs have been detected in plasma and urine, including oxypentifylline, ibuprofen (93), acetaminophen (92, 94), naproxen (92), penicillins (95) and metronidazole (96). For example, numerous conjugates of paracetamol (e.g. cysteinyl, Nacetylcysteinyl, glucuronide and sulfur conjugated paracetamols) appear in urine within a few hours of a subject ingesting the compound (97). Increased levels of these conjugates and specific changes in their ratios have been reported in cases of paracetamol (acetaminophen) overdose and may be related to the hepatotoxicity of this drug (98). New metabolites of drug metabolism have been described, such as the appearance of diketopiperazine in the ~H spectrum of urine after administration of ampicillin (95). Specific labeling of drugs with ~3C and ~gF (see below) can provide much additional information about drug metabolism. In addition, studies of the metabolism of drugs in cell suspensions, such as that of acetaminophen in hepatocytes can lead to detailed information about cell and organ specific metabolism of the drug (94). Similar techniques should be as readily adaptable to in vivo and in vitro investigations of drug metabolism in the CNS.

7.2. ~gF NMR Detection of Fluorinated Drugs and Ion Sensitive Ligands The 19F nucleus possesses a high NMR sensitivity relative to 1H (83 percent) and is the predominant isotope of fluorine with a natural abundance of 100 percent. Fluorine is not a natural constituent of biological tissue and presents

161 no background spectrum. Therefore, 19F NMR is ideally suited for pharmacokinetic investigations of drugs and their metabolites. 7.2.1. Neuroleptics

Several of the neuroleptics used in the treatment of psychiatric disorders contain atoms of fluorine and can be measured using 19F NMR. 19F NMR detection of fluphenazine (99, 100), trifluoperazine (100, 101), fluvoxamine (102), and fluoxetine + norfluoxetine (103, 104) have been reported in the brains of animals and human patients following administration of these compounds. Renshaw et al. (103) reported in a small number of patients administered fluoxetine, that the parent compound and its active metabolite norfluoxetine, which is not resolved from fluoxetine in the ~9F spectrum, accumulates more slowly and to a greater extent in brain (brain-to-plasma ratio, 2.6), long after steady state levels were reached in the plasma. Studies of autopsy brain samples of patients treated with fluoxetin confirmed the presence-of norfluoxetin (104). The tri-fluorinated serotonin selective reuptake inhibitor fluvoxamine (102) required days to weeks for the drug to attain steady state levels in the brains of patients under treatment. The T1 spin-lattice relaxation times reported for these lipophilic drugs in human brain 19F NMR spectra appear to be highly variable, possibly reflecting the known binding of these compounds to membranes and proteins in plasma and tissue. Reported differences in the chemical shifts of these resonances between human brain spectra and solutions of the pure compound have been ascribed to binding of the drug to its receptor; however, sample temperatures were not reported. Because 19F shifts are very temperature sensitive, ~9F spectra of pure solutions of the drug should be obtained at 37 ~ in order to match physiological conditions. Some of the problems encountered in the detection of these compounds appear to be related to poor field homogeneity, low signal-to-noise ratios and/or lack of spatial localization. Uncertainties in spectral resonance assignments, quantitation, and relaxation times in ~9F NMR spectra of brain could be readily assessed in animals administered the drug in question. 7.2.2. Anesthetics

Accumulation of the fluorinated inhalation anesthetics halothane, methoxyflurane, and isoflurane was first reported in rabbit brain using ~9F NMR (105). Significant levels (20 percent of maximum signal) of these fluorinated compounds were detected in their brains up to 4 days following termination of

162 the anesthetic, a result indicating that the half-life for their clearance was longer than originally thought. Subsequent studies of halothane and other related anesthetics (e.g. enflurane) have focused on the kinetics of accumulation and clearance and the formation of metabolites from the parent compound. A study of halothane distribution in rabbit brain (106) indicated that elimination is biexponential; the appearance of multiple halothane peaks was interpreted as representing the distribution of halothane between distinct chemical environments within the brain. A long-lived water soluble metabolite of halothane detected in the in vivo ~gF spectrum was tentatively assigned to trifluoroacetate, a compound that could have specific effects on glial metabolism. The studies show clearly that the elimination kinetics of inhalation anesthetics such as halothane from the brain are not perfusion-limited as previously believed.

7.2.3. Chemotherapeutic drugs ~gF NMR is an important tool in the pharmacokinetics evaluation of the anticancer fluorinated pyrimidines, which include 5-fluorouracil, 5-fluorouridine and 5-fluoro-deoxyuridine (107). The rates of activation and clearance of the fluoropyrimidines to the fluorinated nucleosides and nucleotides are important both in the chemotherapeutic effectiveness and toxicity of these agents. The major catabolic pathway of 5-fluorouracil (5-FU) to ~-fluoro-/3-alanine (FBAL) has been shown in the livers of both mice (107) and humans (108) in vivo and in the plasma and urine of cancer patients treated with 5-FU (109). Trapping of 5-FU into the tumors may be an important factor in therapeutic outcome (110). The conversion of 5-FU to the toxic fluoro-nucleosides, deoxynucleosides and deoxynucleotides (single unresolved peak in 19F NMR spectrum in vivo) in the anabolic pathway are observed in tumors but not in liver (107).

7.2.4. ~gF Indicators of ion concentrations ~gF NMR spectroscopy has been used in conjunction with ion sensitive fluorinated ligands for the measurement of Ca2§ (fluoro-BAPTA (111, 112)), Na § (FCryp-1 (113)), pH (fluoromethyl alanines (114)), and oxygen tension (perfluorocarbons (115, 116)). A review of the many applications of 19F indicators in vivo may be found in (117). For the polar calcium and pH indicators, intracellular loading is achieved by use of their membrane permeant acetoxymethyl ester derivatives. Nonspecific esterases cleave the ester linkages and effectively trap the indicator within the cell. ~gF NMR measurements of intra-

163 cellular Ca2+ in superfused brain slices using the Ca2+ indicator fluoro-BAPTA showed increased [Ca;2 + ] under conditions known to raise [Ca,-2+] levels (112, 118). 19F NMR measurements of [Ca;2 + ] using fluoro-BAPTA combined with 31p NMR of high energy phosphates has shown that a disturbance of energy metabolism in response to the excitotoxic amino acid NMDA occurs independently of the NMDA-mediated rise in [Ca,.2+] (119). Deutsch and Taylor (114, 120) have described a number of fluorinated amino acid and aniline derivatives for use as intracellular pH indicators. The di- and tri-fluoromethyl alanines have pKa's in both the acid and neutral pH range (114) and provide potentially more accurate values of pH/than 3~p NMR under conditions of moderate to extreme acidosis (e.g. ischemia) where the P; chemical shift is relatively insensitive to changes in pH. ~gF NMR measurements of intracerebral pH in rat brain following intraventricular loading of the fluoromethyl alanines (121) yielded pH values slightly more acid than pH values obtained with 3~p NMR suggesting the possibility of their selective accumulation into a compartment with lower pH (neurons?).

7.3. Detection and Quantitation of Lithium

Lithium is used in the treatment of bipolar affective disorders. Detailed information on the biodistribution of lithium in the brain and body tissues in vivo has been limited by the absence of appropriate measurement techniques. The major isotope of lithium, 7Li, has a relatively high intrinsic NMR sensitivity (27 percent of 1H) and a high natural abundance (92.6 percent). Measurements of lithium levels in 7Li NMR spectra of animal and human brain (122-125) and pharmacokinetics following single intraperitoneal doses (126) have been reported in rats. Lithium accumulation in the brain progresses more slowly than in serum or muscle with a time course of hours to days; elimination from the brain is also slower with a half-time of 48 h (124). Consistent with the quadrupolar relaxation mechanism of this spin-3/2 nucleus and its binding to macromolecules in the tissue, both T1 and T2 appear biexponential and can be described by more than a single rate constant (123). The different relaxation times have been used to calculate a correlation time of 3.6 x 10 -8 s between free and bound lithium in the immature dog brain (123). The relatively long T~ relaxation time of 7Li in brain tissue (~3-7 s) has limited the practical application of 7Li spectroscopic imaging to relatively low spatial resolution.

164

ACKNOWLEDGEMENT The author wishes to acknowledge the support of grant HD32573 from the National Institutes of Health.

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169

CHAPTER 9

ALTERNATIVE TO MASS SPECTROMETRY FOR QUANTITATING STABLE ISOTOPES: ATOMIC EMISSION DETECTION

J.L. BRAZIER Faculty of Pharmacy, Universit& de Montreal, CP 6128 Succursale Centre-Ville, Montreal, H3C 3JC, Quebec, Canada

1. I N T R O D U C T I O N

Atomic emission detection is becoming an increasingly popular and powerful methodology for gas chromatographic analysis. This mode of detection allows multielement measurements and combines the temporal selectivity and high resolution of capillary gas chromatography with the specificity of the spectral resolution given by atomic emission. Gas chromatography-atomic emission detection (GC-AED) is performed by passing eluates from GC directly into a plasma induced by microwave where elements are atomized, excited and then returned to their ground state. This results in emission of photons with a wavelength characteristic of the emitting atom. Such a system allows the qualitative and quantitative determinations of a wide range of isotopes and elements and it may be feasible to attain elemental and/or isotopic composition via the calculation of inter-element ratios. Interest in methodologies using stable isotopically labeled (SIL) molecules continuously increases with the development of analytical methods like gas chromatography coupled to mass spectrometry, isotope ratio mass spectrometry or atomic emission spectroscopy. SIL molecules can be used as ideal internal standard for quantitative analysis (see Chapter 12). They are used much more as nonradioactive tracers in various fields of biology such as biochemistry, clinical pharmacology and clinical pharmacokinetics (see Chapters 15-21). They are also powerful tools for medical diagnosis (see Chapter 20). Among the main stable isotopes, 13C is widely used because it

170 does not give rise to significant biological isotope effects, while deuterium can induce kinetic isotope effects or metabolic switching (see Chapter 2). lSN can be used in the same manner as 13C for the labeling of organic molecules with nitrogen containing chemical groups.

2. STABLE ISOTOPES AND ATOMIC EMISSION

The past decades have seen a keen and growing interest in the applications of stable isotopically labeled molecules to chemical and biological problems. Due to a quantitative and specific labeling, SIL molecules can be used as perfect tracers in the studies of mechanisms, metabolic pathways and many other fields in pharmacology, biochemistry, diagnosis methodologies and functional investigations. The absence of radioactivity of stable isotopes has opened very wide areas of applications for investigations in humans because of the safety of these tracers. From the analytical point of view, the main driving forces for this interest are: - the application of magnetic nuclear resonance techniques to many complex chemical or biological molecules, facilitated by the judicious use of stable isotope labeling on various molecular sites. - the development of gas chromatographic mass spectrometric techniques, either in the fields of organic mass spectrometry or isotopic mass spectrometry. More recently, the development of continuous flow gas chromatography-isotope ratio mass spectrometry (CF-GC-IRMS) has given a new dimension to stable isotopic labeling in many biological applications. The areas covered by these applications go from authentication of natural compounds to noninvasive methodologies for medical diagnosis (1) and forensic sciences (2) (see Chapters 8-20). - the availability of deuterated compounds, together with the increasing availability of ~3C, ~SN, ~80, and even depleted ~2C and ~4N molecules in high isotopic purity. In the early years of SIL work, the price of analytical instrumentation and isotopes prevented the rapid development and wide use of SIL molecules, despite their great interest, The development of new generations of analytical instrumentation and electronics and new analytical techniques, together with the publication of numerous and ever-increasing number of papers on the various applications

171 of stables isotopes, progressively lowered the costs of these methodologies and opened much wider fields for their applications. Despite the many uses applicable to SIL, certain inherent limitations still prevail. One is the isotopic effect. From the physical and physicochemical point of view, isotopic effects create differences in physical and physicochemical properties which can be used in various areas especially for isotope detection. But these isotope effects have to be avoided when using SIL molecules as tracers. The marked biological effects of deuterium labeling are not observed when ~3C is substituted for ~2C, or ~SN for ~4N. The source of this differential effect is likely to be the much larger kinetic isotope effect associated with deuterium as compared to the stable isotopes of carbon and nitrogen. It is obvious that the heavy isotopes of carbon and nitrogen may be expected to have qualitatively effects similar to those induced by deuterium, but the magnitude of these effects is generally small enough to be within the range of the normal biological control systems (see Chapter 2 for a detailed review of isotope effect). Consequently, this chapter treats the detection of molecules labeled with 2H, ~3C and ~SN, and used as markers, or tracers, in biological investigations and trials. In the domain of atomic spectroscopy, let us remember that the first direct observation of deuterium was made in 1931 by Urey et al. (3), who observed weak satellites of four of the Balmer lines of hydrogen which were shifted to a shorter wavelength by an amount ranging from 1.9~ for He at 6536~ to 1.12 ~ for H$ at 4102 ~. Within experimental error, the shifts were in exact agreement with the prediction of quantum mechanics for the effect of mass " 2 " nucleus on the reduced mass of the atom. For multielectron atoms, isotope effects are manifest not only in the changes in hyperfine structure rising from nucleus spin changes, but also in small shifts in the energy of electrons which may be attributed to changes in the nuclear dimensions. Using such isotopic shifts, spectroscopic methods have been used for the analysis and determination of the isotopic composition of hydrogen by Veinbert and Zaidel (4), carbon by Zaidel and Ostrovskaya (5), and nitrogen by Zaidel and Ostrovskaya (6). The development of theoretical and analytical atomic spectroscopy has assumed an increasingly important place in element detection and, more recently, in isotope detection. The development and current status of atomic emission spectroscopy (AES), and the concept and implementation of chromatographic detection, led to the coupling between a chromatographic separation and atomic emission spectroscopy or detection (GC-AES, GC-AED).

172 These analytical devices allow for an element selective detection. Moreover, the objective of element selective chromatographic detection is to obtain quantitative and qualitative information on eluates, generally in the presence of interfering background matrix, by virtue of their elemental composition. Element selective detectors have been developed for gas chromatography: (1) Alkali Flame Thermoionic Detector (AFID, NPD) for nitrogen and phosphorus; (2) Flame Photometric Detectors (FPD) for sulfur and phosphorus; and (3) Hall detector for halogens, nitrogen and sulfur. Despite their wide use, none of these detectors is able to detect several elements simultaneously and the number of elements that can be monitored is restricted. So the development of detectors using atomic spectroscopy interfaced with chromatographic separation is a very important improvement in analytical chemistry because atomic spectroscopy may be recognized as the most fundamental analytical technique for elemental determination.

3. PHYSICAL BASIS OF ISOTOPE ATOMIC EMISSION DETECTION

3.1. Carbon Isotopes In 1956, Ferguson and Broida (7) reported stable carbon isotope analysis by optical spectroscopy. The reported work concerned the use of the C2 radiation from a flame for the measurement of relative concentrations of carbon isotopes 12C and 13C. Acetylene samples with various ~3C contents were burned and the emission spectra of the flames were recorded. The relative intensity of the 1,0 bandheads of the 3]-[-3R system due to the isotopic C2 radicals ~2C-~2C, ~2C-13C, and ~3C-~3C in the 4735-4755 ~ region were measured. Plots of the observed ~2C-~3C/12C-~2C bandhead intensity vs. ~3C acetylene contents were studied. With samples containing less than 15 atom percent carbon 13, the ~3C-~2C and 12C-~3C intensities were very nearly proportional to the expected isotopic C2 concentrations calculated by assuming a random distribution of the isotopes in C2. It was estimated that the measured intensity ratio alone could be used directly to determine the 13C abundance to the nearest 1 percent in this range. When oxygen is introduced in the discharge tube, intense CO bands appear in the spectrum. Among the CO bands, the 12CO band at 4123~ is the most convenient one for spectral analysis when the ~3C content is low, since the head of the corresponding band of the ~3CO molecule is displaced 8.2 toward the red from the ~2CO band and is free from its superimposed rotational structure. The problem of the differential detection of both ~2C and

173

0.8nm

4 ~ A

.L

'1;0

3411

342

344

346

nm

Figure 1. (0--> 3) and (1--> 4) emission bands of the fourth positive system of 12CO and 13CO.

13C from 13C-labeled molecules is much more difficult because the molecular bands corresponding to ~3CO and ~2C, respectively, are quite superimposed, and particular recipes are necessary to estimate the specific proportion of each carbon isotope. Figure 1 shows the 0-3 and 1-4 bands of the fourth positive system of CO. Figure 2 shows the three-dimensional display of the snapshot of 12C and ~3C bands recorded during the elution of the chromatographic peak of caffeine labeled with 13C on the methyl groups, in the wavelength range (338-346nm). It can be seen that the emission spectra are overlapping, but the respective signal of both carbon isotopes can be automatically extracted from the whole analytical response using the recipe developed by Quimby et al. (8). These results have been obtained on a GC AED system (HP5921), the spectrum being recorded by a diode array detector. The Quimby's recipe is the software algorithm used to make an element selective chromatogram. It is conceived to detect the raw atomic emission from the element and the interferent. A particular recipe was adjusted to reject compounds with the ~3C natural abundance (1.1 percent). With such a recipe, a selectivity of 2,500 was achieved for ~3C-labeled compounds over unlabeled compounds, otherwise the selectivity for spiked compounds could not exceed 100.

174

~2C0 ~SCO

~2C0

16.2

16:71'1"1 340

min

342

t 344

f 346

Fim

Figure 2. GC-AED: three-dimensional display of the snapshots of 12COand 13COemission bands recorded during the elution of a chromatographic peak corresponding to [~3C3] caffeine between Rt = 16.2 and 16.7 min (A = 338-346 nm).

Remember that selectivity corresponds to the multiplying factor of the 12C amount, necessary to produce the same analytical response as that obtained for the measured element. The band spectra of CO near 171 nanometers can be used to detect 13C. The spectra in second order (A13C = 341.712 nm and A~2C = 342.574 nm) are preferred. The CO molecular bands are emitted inside the plasma cavity using 02 and H2 as reactant gas. The wavelength difference of the first-order bands (A13C 170.86 nm and A~2C = 171.28 nm) is ~A = 0.43 nm, and is too narrow for a selective detection of both isotopes. As CO has to be produced inside the plasma, the flow rates of the plasma gas and reactant gases have to be optimized to obtain the maximum residence time of the element inside the plasma for a best yield of the reaction, and to obtain the highest sensitivity of ~3C detection. Such an optimization carried out for the detection of ~3C using an experimental design has been published by Leclerc et al. (9).

3.2. Nitrogen Isotopes As for carbon isotopes, an alternative method of determining 14N/15N ratios has been developed by molecular emission spectroscopy, for bands in the

175 electronic vibrational spectrum of the nitrogen molecule which show an isotope shift (10, 11). The advantage of this optical method is that very small samples can be measured. As little as 0.2 ~g of total nitrogen was sufficient for Goleb and Middleboe (12) to obtain 14N/15N ratios. In the many optical emission spectroscopies described in the literature for the 14N/1SN ratio determination, isotopically shifted bandheads of a single transition corresponding to the electronic transition system C (3]-[u) ~-> B (3]-[g) of the 2-~ 0 transition have been used. In this system, the I"N-I"N bandhead has a wavelength of 297.7 nm, the 14N-1SN bandhead 293.3 nm and the lSNlSN bandhead 298.9 nm. The possible use of the 3--, 0 bandhead is weak and can suffer interferences from coincident OH bands in the 281-283 nm region. The large isotope shifts of the (3-0) and (4-1) bandheads produce a pattern of overlapping bands. However, Burridge and Hewitt (13) demonstrated that the simultaneous use of bandheads from two transitions (3--> 0) and (4-> 0) can provide an adequate basis for the optical emission spectroscopy determination of 14N/1SN ratios. A14N2 (3.0) = 281.98 nm, A14N 15N(4.1 ) = 282.11 nm A14N-1SN (3.0)= 282.71 nm and AlSN2 (4.1)= 282.78 nm The detection of 14N and lSN can be carried out using the emission of the diatomic molecule CN in the 420 nm region. Figure 3 shows the 3D emission spectrum obtained from caffeine labeled with lSN at the 1 and 3 positions by gas chromatography coupled to the atomic emission detector HP 5921 equipped with a diode array detector. The spectrum shows two bands corresponding to the B2]-[-A2]-[ system. The Arnax are 420.1677 nm for 15N and 421.4646 nm for 14N, respectively. The wavelength difference 8,~ is only 1.2969 nm. As for carbon isotopes, a careful optimization of the plasma gas (He) and reactant gases (CH4, 02, N2) flow rates has to be carried out for an optimal formation of CN inside the plasma and excellent sensitivity and limit of detection. Recently, a new plasma discharge source for assaying nitrogen isotope abundance has been designed by Hoult and Preston (14).

3.3. Isotopes of Oxygen and Hydrogen The detection of oxygen isotopes 180 and 160 is difficult. It can be performed from the emission of the CO band using ethane, or methane, as reagent gases. If the 180 chromatograms are too long or recorded too often, the diode array can be damaged because the emission line of nitrogen near 175 nm is

176

C15N

7

8. min

423 nm

Figure 3. GC-AED: three-dimensional display of the snapshots of C14N and C15N recorded during the elution of a chromatographic peak corresponding to 1.3-[15N2], 2-[13C] caffeine.

so intense that the photodiode array becomes blind rapidly. The CO emission band detected at 171 nm for 180 determination corresponds to that used for the 13C determination as 13CO. Oxygen can also be detected when reacted with hydrogen as an OH radical, the Amax of this band is located near 307 nm. At last hydrogen isotopes can also be measured using atomic emission detection. The emission lines recorded are 656,039 nm for 2H and 656,302 nm for 1H, respectively. It can be seen that for increasing the resolution molecular bands are generally used for isotope determination using atomic emission, especially when it is coupled to gas chromatography. Table 1 gives the main characteristics of these determinations. Table 2

TABLE 1. Molecular Bands Used for Isotope AE Detection with ~maxand Reagent Gases Used for Producing the Chemical Emitting Species Inside the Plasma

Isotope

Molecular band

Wavelength (nm)

Reagentgas

12C, 13C, 14C

CO OH CN CO

171 307 419 171

Oxygen Oxygen Ethane, methane Ethane, methane

2H, 1H 14N, ~SN 160, 180

177 TABLE 2. Selectivity vs. Carbon for the Determination of Stable Isotopes by GC-AED

Isotope

Selectivity vs. carbon

13C 2H* 1SN 180

2400 2200 2500 2900

*The selectivity of 2H VS. 1H at 656.1 nm is 300.

TABLE 3. Atomic Weight and Terrestrial Natural Abundance of Various Stable Isotopes

Element

Atomic weight

Terrestrial natural isotopic abundance (percent)

1H 2H 12C 13C 14N 15N 160 170 180

1.0078 2.0141 12.000 13.003 14.003 15.004 14.994 16.999 17.999

99.985 0.015 98.85 1.11 99.63 0.36 99.759 0.037 0.204

shows the values of selectivity vs. carbon for the main stable isotopes determined by GC-AED using the GC AED HP 5921. This table shows that the selectivity of stable isotope detection by atomic emission is excellent, but not sufficient to measure variations in the natural abundance range of stable isotopes found in different biological materials (Table 3).

4. C H R O M A T O G R A P H Y COUPLED WITH A T O M I C SPECTROSCOPY DETECTION

Different types of atomic spectroscopy have been interfaced with chromatographic systems. Among these are atomic absorption (AAS), flame emission (FES), atomic fluorescence (AFS) and atomic emission (AES) spectroscopies.

178 The capability of AES for simultaneous multielement measurement, while maintaining a wide dynamic measurement range and good selectivities and sensitivities over background elements, has led to atomic plasmas becoming widely used during the last decade (15). The main advantages of chromatography AES are: - monitoring the elemental composition of eluates directly with high elemental sensitivity. - monitoring of particular molecular functionality by means of interelement ratio measurement or by using specific derivatization reagents containing elemental labels. toleration of nonideal chromatographic separation. Elemental detection is very specific and its inherent selectivity enables quantitation of compounds with poor chromatographic resolution when working on complex matrixes like biological samples. - simultaneous detection of various elements, computation of interelement ratios, determination of empirical and molecular formula from a chromatographic peak. - detection and quantitation of some isotopes, especially those of elements constituting organic molecules: ~3C, 2H, ~5N and ~80. -

4.1. Plasma Sources The main atomic plasma emission detectors that have been developed for GC coupling can be summarized as follows: ICP: Inductibility Coupled Plasma In such a plasma the discharge results from interaction of a radiofrequency fields with argon flowing through a quartz tube set within a copper coil. The ICP discharge is well suited for liquid chromatographic detection since it is usually configured for a liquid inlet stream (spray chamber, nebulizer). DCP: Direct-Current Plasma Direct-current excitation sources for atomic emission involve a low voltage (10-500), high current (1-35 A) discharge. The direct-current plasma jet stabilized by flowing inert gas (argon) can be coupled with liquid chromatography. The argon based ICP and DCP are interfaced with HPLC or supercritical fluid chromatography for specific elemental determinations.

179 MIP: Microwave-Induced Plasma MIP is the most used plasma source for gas chromatographic (GC) applications. A helium plasma is maintained within a resonant cavity which allows focusing the power from a microwave source (2.46 GHz) into a discharge cell or discharge tube (quartz). Although the plasma temperature is lower in helium MIP than in ICP or DCP, the chemistry of helium discharges produces high spectral emission intensities. The first presentation of the use of a microwave-induced plasma as a detector for GC was performed in 1965 by McCormack et al. (16) using an argon plasma working at atmospheric pressure. Bache and Lisk (17) studied a plasma of helium at low pressure, the device was later improved by Van Delhen et al. (18). There was a very large increase in the applications of GC coupled to microwave plasma (19) after the development of the Beenakker cavity (20, 21). More recently, Yieru et al. (22) showed that the chemical structure, as well as the microwave energy, could influence the analytical response. SWP: Surface Wave Plasma SWP is an alternative to the electromagnetic resonant cavity for the transmission of microwave energy to the plasma SWP using a "Surfatron" power launching device (23). GC-MIP Coupling In a GC-AED system, the separation of sample components takes place on a capillary column within a standard gas chromatograph oven. The column is extended out of the oven through a heated interface and directly connected to the detector. Thus, the entire column effluent enters the microwave-induced plasma. Make-up and reagent gases are automatically added according to the element to be recorded in order to generate chosen chemical species inside the plasma. The cavity can be a Beenakker type resonant cavity which enables the microwave energy to be focused in the centre of a quartz discharge tube. The capillary column ends just before the beginning of the plasma. The discharge tube has to be cooled by a water circulation in order to avoid or minimize erosion and interactions of sample components with the silica tube walls. The atomic emission exits the discharge tube and is transmitted into the spectrometer. The exit chamber and window are flushed by pure helium. In the spectrometer, the light is diffracted by a holographic grating which projects it onto a flat focal plane. An adjustable photodiode array covering 50 nm can move along this focal plane to provide a good resolution of the monitored emission lines and allows one to obtain elemental chromatograms and atomic spectra. The signal recorded by each of the 211 photodiodes can

180 be treated for background and interference subtraction, especially for isotope determination (natural isotope abundance). This type of analytical instrument and detector allows one to acquire two kinds of analytical information.

4.2. Elemental Chromatograms By recording all along the chromatographic run the various emission lines corresponding to particular compounds, it is possible to obtain the chromatograms corresponding to each of the elements or isotopes monitored. Figure 4 shows the elemental chromatogram of a mixture of xanthines containing caffeine labeled with deuterium. The elements monitored were carbon at 193.03 nm, hydrogen at 656.30 nm and deuterium at 656.04 nm. The chromatogram of deuterium clearly points out the presence of deuterium-labeled caffeine with a retention time of 6.5 min. The qualitative response on the presence of such or such element can be completed by the quantitative measurement of each elemental response. So it is possible to quantify every element and to calculate interelement ratios which are very useful for structure or empirical formula determinations. These values can afford additional information to mass spectrometric analysis, especially in the presence of labeling stable isotopes.

3000

C i93.03 nmJ

2000

t JD656"~nmJ'/

1000.

o-

.....

,~

~

Ill

7

~

~

1"0 min

I

Figure 4. Elemental chromatogram of a mixture of xanthines containing deuterated caffeine. Elements monitored: C at 193.03 nm, 1H at 656.30 nm and 2H at 656.04 nm.

181 4.3. Snapshot

Snapshots are three-dimensional displays (A, retention time, intensity) recovered within a wavelength range during the elution of a chromatographic peek. These 3D spectra which are specific for an element or isotope certify the presence of this particular atom in the analyzed molecule (Figures 2 and 3).

5. LIMIT OF DETECTION OF ISOTOPES DETECTION 5.1. Limit of Detection of 7SC at 341.712nm

The limit of detection of isotopes can be expressed according to several methods. We will express the limit of detection according to the IUPAC method (24), Oppenheimer's method (25) and that of Quimby and Sullivan (26). IUPAC M e t h o d The limit of detection is defined as the concentration (CL) obtained from the smallest measurement (XL) which can be detected with a sufficient confidence level during a given analytical procedure. First, the standard deviation of the noise measurement Sb is determined from a blank sample (Sb) in the interval of retention times where the studied compound will be eluted. According to the International Union of Pure and Applied Chemistry (IUPAC), the following equation can be used: XL = XB + KSB

where K is a numeric factor chosen according to the expected confidence level. If K = 3, the confidence level is 99.86 percent, there is a 0.14 percent risk in obtaining a signal which is only a fluctuation of the noise. The CL concentration is a function of XL according to, CL = (KSB)Im

CL = (3SB)/m

This method, used for the calculation of the limit of detection, generally gives values which are artificially low. It is why IUPAC recommended the use of K = 3 (CLUE=31). Indicative comparisons between analytical methods and/or instruments can be carried out using CL values calculated according to the IUPAC method.

182 Oppenheimer" s M e t h o d Oppenheimer defines the limit of detection X~ using the regression lines and their confidence intervals. These limits are expressed in terms of concentrations with an e and/3 risk. This method is restricting but it is statistically the more relevant and very close to the analytical factuality. The limit of detection Y1 for a signal X~, form a regression line y = aX = b is given by, Y~ = b + (t(~_,) + t(1-f3))Sr[1 + 1/n + {E(x)2/S(X)})] ~

where b = estimated intercept; t(l_,) and t(l_~)= the values of the student's test for = a =/3 = 0.05; n = number of replicates; Sr = residual standard deviation; E(X) 2 = X mean square; and S(X) standard deviation of X values. Calculation of the Residual Standard Deviation The residual standard deviation is calculated using the following equation: Sr

=

{,~-~(Yest

-

Ymes)2/( n -

2)} ~

where Yest = estimated Yvalue using the regression line; Ymes = measured Y value; and n = number of data. M e t h o d of Quimby and Sullivan According to Quimby and Sullivan, the limit of detection for a given element is the amount of the element necessary to give a peak twice the height of the noise (X2) divided by the width at middle height of the peak (Wo.5) expressed in seconds: LD = (2 * X2)lWo.s

This way of expressing results (pg/s) is difficult to compare with the limit of detection expressed as analytical concentrations; nevertheless, it can be use to compare the sensitivities of various analytical methods or instruments. After applying a four-factor experimental design corresponding to the various flow rates of plasma gas and reagents gas, Leclerc et al. (9) determined the limit of detection of ~3C from an anti-inflamatory drug, Fenoprofen, transformed into its ~3CH3 methyl ester. Under their experimental conditions, and after injection of 1 i~1 of the analytical sample, the limits of detection were: 1 pg/i~l according to the IUPAC method, 12 pg/l~l according to Oppeinheimer and 0.1 pg/s according to Quimby and Sullivan. The detec-

183 TABLE 4. Limits of Detection Measured from Trideuteromethylcaffeine by GC-AED (C, N, 1H, 2H) and Expressed According to Quimby, Oppenheimer and IUPAC Methods Method

C

N

1H

2H

Quimby (pg/s)

1.07

8.30

0.40

6.50

IUPAC (pg) (pg/s)

1.04 0.20

13.80 4.60

1.70 0.30

6.50 1.10

52.00 8.70

287.00 47.80

13.00 2.10

66.00 11.00

Oppenheimer (pg) (pg/s)

tion of 12CO and 13CO was performed by recording the emission lines at 341.712 and 342.574 nm, respectively.

5.2. Limit of Detection of Deuterium The same methods of determination of the limit of detection were carried out on deuterium using the molecule of caffeine labeled with 9 deuterium atoms on its three methyl groups, on positions: N1, N3 and N7, as analyte. According to the three methods previously described, the results are presented in Table 4 for carbon measured at 193.031 nm, nitrogen at 174.261 nm, 1H at 656.302 nm and 2H at 656.039 nm (27).

5.3. Limit of Detection of Nitrogen 75N The limit of detection of lSN was determined after optimization of four factors affecting sensitivity: helium flow rate (plasma gas), methane pressure, as well as oxygen and hydrogen pressures (reagent gas). Emission lines of ~SN and I"N were detected at 420.17 and 421.46 nm, respectively. The results for the limit of detection are shown in Table 5. The molecule used for these optimization and limit of detection determinations was caffeine labeled with two atoms of lSN at positions 1 and 3. According to Quimby and Sullivan, the minimum detectable limits for deuerium and 180 are 16.0 and 140 pg/s, respectively.

184 TABLE 5. Limit of Detection of lSN Form [~5N2(1-3)]Caffeine

Method

lSN

IUPAC (pg) (pg/s)

4.6 1.9

Quimby (pg) (pg/s)

5.52 2.3

Oppenheimer (pg) (pg/s)

100 36

6. LINEARITY

Analytical parameters, such as selectivity and limit of detection or minimum detectable level, are good when using GC coupled with atomic emission detection. Linearity and linear dynamic range are also very good. The values of the extent of the linear dynamic range for several elements are shown in Table 6. During a linearity study of deuterium performed by Bannier et al. (27), the possible variation of the response according to the number of deuterium atoms substituting for hydrogen atoms in a caffeine molecules has been studied for the same overall deuterium concentration. Three caffeine isotopomers were used [7C2H3] caffeine (7d3 caf), 1,3 [C2H312 caffeine (1,3 d6 caf) and

TABLE 6. Atomic Emission Line, Wavelength of Emission; Minimum Detectable Level and Linear Dynamic Range of Several Common Elements Measured by GC-AED

Element

Wavelength (nm)

Minimum detectable level (pg/s)

Linear dynamic range (K)

C 1H

193.1 486.1 656.1 777.2 174.2 180.7

0.2 1.0 2.0 50.0 15.0 1.0

10 9 10 3 10 10

2H O N S

185 12000

12C

10000

9

J

9

8000

6000

4000 -"

.

.

.

.

.

. _ _

. _ _ . _ _

. _ _

2000

0 0

5

10

15

20

25

NANOGRAMS OF 12C AS CAFFEINE Figure 5. Mixture of 13C labeled (constant amount = 6.9ng of 13C) and varying amounts of unlabeled caffeine measured by GC AED using ~2C and 13C signals.

1,3,7 [C2H313 caffeine (d9 caf). An effect of the number of deuterium atoms per molecule would make the slope of the regression lines, calculated between the deuterium signal and the deuterium amount, exhibit a change for each isotopomer. The parallelism of the lines was checked and showed that there is no influence of the number of deuterium atoms per molecule of caffeine on the deuterium quantitative determination. For the same deuterium concentration, the detection is the same whatever the number of deuterium atoms in the isotopomer. Figure 5 shows the responses of 12C and lSC in a mixture containing a constant amount of caffeine labeled with 13C (corresponding to 6.9 ng of ~3C) and increasing amounts of unlabeled caffeine (up to 25 ng). This figure clearly shows that AED responses are independent of the other isotope when monitoring two isotopes of the same element. The same results are shown on Figure 6 with a mixture containing caffeine labeled with lSN (constant amount of 7.8 ng ~SN) and increasing amounts of the unlabeled molecule. This result leads to the same conclusion than that obtained with carbon isotopes. Dual isotope measurements by gas chromatography coupled with atomic

186 5000 4500

14N

4000 3500 3000 2500 2000 -'-'-'"11

. . . .

1500

B"

O/

""

-1 . . . . 7,8

,, -

-

-

--,-

NANOGRAMS

-

OF

--~

15[~

. . . .

r

1000 500

0

5

10

NANOGRAMS

15

20

OF

14N A S

25

30

35

CAFFEINE

Figure 6. Mixture of lSN labeled (constant amount=7.8ng of lSN) and varying amounts of unlabeled caffeine measured by GC-AED using ~4N and ~SN signals.

emission detection may enhance results for quantitative analysis. Adding a known amount of an isotopically labeled form of a target analyte in each sample can compensate for irreproducibilities or uncertainties associated with sample pretreatment, as well as fluctuations in AED parameters (28).

7. SOME APPLICATIONS OF GC-AED ISOTOPE DETECTION

7.1. Determination of the Number of Isotopes Incorporated into a Molecule (27) The various isotopomers used in the reported study were: 1-mono deuteromethyl caffeine, 3-mono deuteromethyl caffeine, 7-mono deuteromethyl caffeine: (84 ng/l~l) and 1.3-dideuteromethyl caffeine, 1,7-dideuteromethyl caffeine, 3,7 dideuteromethyl caffeine (82 ng/~l). The analytical signals corresponding to 1H, 2H and 12C were recorded and the areas corresponding to elution peaks were measured. In order to check the influence of the number of the deuterium atoms and of the location of these atoms on the analytical response, the 2H/~2C ratios were calculated as follows:

187 2H/12C = (area of the 2H peak/number of 2H atoms)/(area of the ~2C

peak/number of ~2C atoms) There are three deuterium atoms in the monodeuteromethyl isotopomers of caffeine and six in the various dideuteromethyl isotopomers. All the caffeine molecules contain eight carbon atoms. The 2H/12C values calculated from isotopomers containing three atoms of deuterium (d3 caffeine) were respectively: 0.011 (1-d3 caffeine), 0.010 (7-d3 caffeine), 0.011 (3-d3 caffeine). The same ratios calculated from the d6 isotopomers gave the following values: 0.012 (1.3 d6 caffeine), 0.013 (1.7 d6 caffeine) and 0.012 (3.7 d6 caffeine). The mean value of the 2H/12C ratio from all the d3 isotopomers (0.011 _+ 0.00062) was not significantly different (p < 0.01) from that obtained from the d6 isotopomers (0.012 _+ 0.00048). In order to calculate the number of deuterium atoms present in every other isotopomer, the factor of proportionality between the responses of deuterium and carbon, KCDw a s calculated from 1-d3 caffeine taken as standard

Kco = [(Number of 2H atoms) x (area of C)]/[(Number of C atoms) x (area of 2H)] The number of deuterium atoms in each isotopomer was calculated as follows: Number of 2H atoms = KCD • number of C x (area of 2H/area of C) where Kco is the factor of proportionality between C and 2H; Number of C is the number of carbon atoms in the standard (8 in 1-d3 caffeine); Area of C and 2H are the areas of the corresponding peaks of the measured isotopomer. The results are shown in Table 7. It can be seen from these results that there is an unequivocal determination of the number of deuterium atoms in the various deuterated isotopomers. Moreover, these results show that there is no influence of the location of the labeling on the determination of the number of labeling isotopes.

7.2. Studies of Metabolic Pathways Pharmaceutical and biomedical analysis needs much more efficient separative techniques like capillary gas chromatography coupled with specific detectors. The interest of coupling such specific modes of detection with highly resolu-

188 TABLE 7. Number of Deuterium Atoms Measured in Each Caffeine Isotopomer

Caffeine isotopomer

1-d3

KCD

87.47

7-d3

3-d3

1,3-d6

1,7-d6

3,7-d6

Number of 2H calculated

3.00

2.72

2.85

6.00

6.16

6.05

Number of 2H theoretical

3

3

3

6

6

6

tive techniques increases with the development of various kinds of chromatographic systems coupled with organic mass spectrometry (MS), isotope ratio mass spectrometry (IRMS) and atomic emission detection (AED). Such a detector can be used for the determination of drug metabolites after administration of a parent drug labeled with the stable isotope 13C. The use of stable isotopically labeled (SIL) drugs for the studies of metabolic pathways in humans increases from year to year. These SIL molecules act as safe and nonradioactive tracers. They can be used according to the "ion cluster" technique where a mixture of unlabeled and SIL parent drug is administered to a subject. Thus, all the metabolites which are formed from this mixture are also labeled with the same isotopic content as the parent drug. They can be detected easily from the total ion current by the presence of ion clusters corresponding to unlabeled and labeled ions. Also the SIL parent drug can be administered alone and its labeled metabolites specifically detected by atomic emission detection. For example, the study of the urinary metabolites of caffeine (1,3,7-trimethylxanthine) labeled with three atoms of 13C on the three methyl groups located at the N1, N3 and N7 positions was reported by Boukraa et al. (29). Caffeine can be used as a metabolic probe for exploring oxidative metabolic pathways (cyt P450 IIA2) and the numerous factors that can potentially alter these enzymatic activities. Demethylation mediated by microsomial mono-oxygenases and oxidative reactions on the C8 position, lead to the production of di- and monomethylxanthines (MX), tri-, di- and monomethyl uric acids (MU) as well as ring-opened uracil related metabolites. With such an extensive metabolism, more than 85 percent of the dose of caffeine administered to a human is eliminated as urinary metabolites. If caffeine is labeled with ~3C on the three methyl groups, all the methylated

189 metabolites which are eliminated are also labeled and can be detected from the biological fluids by the specific signal of 13C. For this trial a GC AED System (Hewlett Packard HP 5921) was used. This system consists of a HP 5890 Series II standard gas chromatograph equipped with a HP 7673A auto-sampler and a HP 5895 A Chemstation. The choice of the plasma gas (Helium 99.9999 d. percent) and reagent gas (H2 and 02 under a pressure of 1 bar) and their flow rates were adjusted in order to optimize to the yield of atomic emission of the selected elements. The emission lines monitored were: 348.424nm for nitrogen, 342.574nm for 12C and 341.712 nm for ~3C. The overall flow rate in the detector was 32 ml/min. Caffeine metabolites were extracted from urine and transformed into their pentylated derivatives on the residual free NH groups before injection into the chromatographic system. Figure 7 shows the chromatograms obtained by gas chromatographyatomic emission detection from an aqueous solution containing caffeine and its metabolites at a concentration of 20 i~g/ml for methylxanthines (MX) and 30 i~g/ml for methyluric acids (MU). An aliquot of this solution was submitted to the whole analytical procedure described above. Figures 7a, 7b and 7c, respectively, show the elemental chromatograms monitored at 342.574 nm for ~2C (a), 341.712 nm for ~3C (b) and 348.424 nm for N. As these wavelengths are inside the 50-nm interval covered by the photodiode array, the three emission lines were recorded simultaneously during the same chromatographic run. When these three emission lines are used for detection, the compounds of interest which contain both stable isotopes of carbon (~2C and 13C) and N can be detected perfectly well. It can be observed that the chromatograms corresponding to the detection of ~2C and ~3C are almost identical because these compounds display an isotopic abundance which is the natural ~3C abundance (#1.1 percent). These results show that the AED is able to detect ~3C from organic compounds with an isotope enrichment corresponding to the terrestrial natural abundance of this isotope (1.01-1.15 percent). A blank urine from a subject who was not a caffeine consumer was spiked with a solution containing caffeine labeled with 13C and twelve unlabeled metabolites as well as IBMX (internal standard) in order to obtain a final concentration of 20 ~g/ml for MX and 30 i~g/ml for MU. An aliquot sample was processed according the described procedure. Figure 8 shows the elemental chromatograms from the extracted urine where the following elements or isotopes are recorded: ~2C (a), ~3C (b) and N (c). As every compound present in urine contains both carbon isotopes, the

190 i

12C 9 342.574 n m 1200-.

3 3'

8

4

IOO0"

r~

67

9

2

800-

~ 400-. 2000:

11

I

600-..

"

13

X,o ! "

I-8

"

"

-2.0-

"

-

- 52

-

- 2 ~

"

~

"

~

t "

-~-

- - ~

"

3 ~

-

min

i

i30"

,

341.712 n m

33' 8 4

~0-

9

26o-

2

I

.

"

1

11

13

o

.....

18

"

- 2"0

. . . .

52-

-

-2~1

"

"

"

2'6"."

" 2~

30

.....

-

"

-

32

3"4 . . . . . . . .

. . . .

min

N ' 348,424 nm ~ ' 5001

5O0~00:

3001

I00" O"

.

.

18

.

,

.

.

.

.

20

.

.

.

.

.

.

22

. 2"4"

"

"

~b

.....

28" ,

"

"3"0-

"

-3~2"

"

"

,~4 . . . .

m i n ,

Figure 7. GC-AED" elemental chromatograms of 12CO at 342.574nm (7a), 13CO at 341.712 nm (7b), N at 348.424 nm (7c). Caffeine (1) and its metabolites (2) 1.3 MX, (3) 1.7 MX, (3')3.7 MX, (4)IBMX, (5) TMU, (6)3 MX, (7) 1 MX, (8) 7 MX, (9) 1.3 MU, (10) 1.7 MU, (11)3.7 MU, (12) 1 MU, (13)3 MU, (14) 7 MU.

191

12C : 343 n m

3 000

13C : 342 n m

2 000. i

1 000'

lO

15

"

2b

2~5 min. ;

Figure 8. Elemental chromatograms for 12C (a), 13C (b) and N (c) from an urine extract containing 3-[~3C] caffeine and its metabolites.

corresponding elemental chromatograms are quite nonspecific and unable to detect the presence of caffeine and its various metabolites. Numerous molecules excreted by urine also contain nitrogen and the N chromatogram is no more specific for caffeine detection. The very large peak on the chromatogram corresponds to hippuric acid whose concentration in urine is very high. A specific detection has been performed by GC-AED. The 13C chromatogram in Figure 8b corresponds to the analytical response given by the natural abundance of this carbon isotope. All the compounds which appear on this chromatogram have the same 13C enrichment (#1.1 percent) caffeine excepted which is artificially enriched on the three methyl groups. This ~3C elemental chromatogram can be processed in order to subtract the natural abundance of ~SC. The signal used for this subtraction is taken from a portion of the chromatogram free of peaks corresponding to labeled molecules (1013 min). The use of this "Suppress" function results in the total removal of the natural ~3C response. Hence, only the compounds enriched with ~3C over the natural abundance are specifically detected. The same method was used to find the various metabolites of caffeine from the urine of a subject who absorbed an oral dose of 50 mg of caffeine labeled with three atoms of ~3C. The ~3C enrichment of each methyl group was 99 percent, so the total amount of ~3C administered as caffeine was 9.8mg. Figure 9a shows the isotopic 13C chromatogram from the urine collected before caffeine intake spiked with the internal standard ~3C isobutyl methyl xanthine and processed to subtract the natural ~3C contribution. No significant

192

I

1.4E4-" 1.2E4-

13

4

CO : 341.712 nm

1000080006000-

5 6

4000.

_~

I [iii i i I lb

b

...... ........o "

--

.... ---

~Omin

Figure 9. (a) 13C chromatogram of a blank urine spiked with [13C] isobutylmethylxanthine (internal standard) obtained after subtraction of the ~3C natural abundance ("Suppress" command). (b) ~3Cchromatogram of a urine extract after ~3C natural abundance subtraction. Urine collected after 3-[~3CH3] caffeine administration. (1) 3-[13C] TMX; (2) 3-[~3C]theophylline; (3) 3-[13C]theobromine; (4) 3-[~3C]trimethyluric acid; (5) 3-[~3CH3] xanthine; (6) 3-[~3CH3] dimethyluric acid.

peak can be observed on the chromatogram because all the molecules extracted from urine and eluted from the chromatographic column have the same 13C content which is the natural one. Figure 10 shows the subtracted 13C chromatograms of urine samples collected at various times after labeled caffeine intake. The only peaks which can be observed on this subtracted chromatogram are those of caffeine and its urinary metabolites, because these molecules are enriched in ~3C over the natural abundance and are not erased by the natural ~3C subtraction. Figure 11 shows the chromatogram of an urine extract corresponding to a subject who absorbed orally 100 mg of 3-~3CH3 labeled caffeine. After subtracting the natural abundance, only the metabolites containing a ~3C atom on the 3N position can be detected. This example clearly shows that atomic emission detection, coupled with gas chromatography, is a powerful tool for the screening of compounds from complex matrices and mixtures using the selective detection of an element

193

3C0 9 3 4 1 . 7 1 2 n m 2

4

1 400 1 200

I

3

5 7

8

1112

I 000 i""-

d

800 600' 400" 200" 5

10 "

15

20

25

min

Figure 10. 13Cchromatogram of urine extracts after 13C natural abundance subtraction. Urine collected after 1,3,7-[13 (CH3)3] caffeine absorption. (a) to-2H; (b) t2-6H; (c) t612H; (d) t~2-24H. (1) 1,3,7-13 C]3 TMX; (2) 7 [~3C] IBMX; (3) 1,3-~3C]2 MX; (4) 1,7-~3C]2 MX and 3,7[13C]2 MX; (5) 3-~3C] MX; (6) 1-~3C]MX; (7) 7-~3C] MX; (8) 1,7-~3C]2MU; (9) 1,3-[13C]2 MU; (10) 3,7-[~3C]2 MU; (11) 3-[~3C] MU; (12) 1-[~3C] MU; (13) 7-[~3C] MU.

or isotope. Despite the very small wavelength difference between the emission lines of 12C0~ and 13C0~ radicals (0.85 nm for the second order), the algorithm proposed by Quimby allows the extraction of both isotopic signals and the perfect recording of the specific ~3C chromatogram. These secondorder emission lines are preferred to the first-order one (at 171.3 and 170.8 nm, respectively) which are more intense but too close one from each other to allow a good extraction of the ~3C response at the ~3C natural abundance level. The processing of the ~3C chromatogram, and the judicious use of the "Suppress" function, allows the subtraction of the natural contribution of ~3C and a specific detection of the only molecules whose ~3C enrichment is over the natural one. Hence, gas chromatography coupled with atomic emission detection is a very appropriate tool for metabolic studies where the parent drug is labeled with stable isotopes (like 13C). If the labeled site is chosen correctly so as not to be removed during the metabolic process, the label remains in all the metabolites formed from the parent drug. A nonspecific extraction of the biologic fluid to be studied, followed by a chromato-

194

~3C0" 341.712 nm

40.

20.

2 ,.,, i13

10

4 5

15

6

20 min

Figure 11. 13C chromatogram of a urine extract after 13C natural abundance subtraction. Urine collected after 3-[~3CH3] caffeine administration. (1) 3-[~3C]TMX; (2) 3-[13C]theophylline; (3) 3-[13C]theobromine; (4) 3-[13C]trimethyluric acid; (5) 3-[~3CH3] xanthine; (6) 3-[~3CH3] dimethyluric acid.

graphic analysis with 13C atomic emission detection with the subtraction of the 13C natural abundance, allows one to easily point out the various ~3C enriched compounds corresponding to the drug metabolites from the whole chromatogram. Hence, a specific profile of metabolites can be obtained. Another alternative to detect metabolite is GC-MS and the ion-cluster technique with coadministration of a mixture of both unlabeled and labeled parent drugs. When using such a method, ion-clusters have to be detected for each unknown metabolite. An algorithm can be used to systematically seek these cluster all along the total ion current. This detection can be very difficult if the compounds are at a very low level and their corresponding ion-cluster within the noise intensity. An alternative is to use the selected ion monitoring mode (as shown above).The disadvantage of this method is the necessity to know the characteristic fragment-ions of the metabolites and generally neither the metabolites nor their characteristic ions are known before the study of metabolic pathways. Consequently, the major advantages of GC-AED for the screening of the metabolites of labeled drugs relies on the possibility to: (i) monitor various elements or isotopes, among them the tracer isotope ~3C, during the same chromatographic run,

195 (ii) use and compare the elemental responses for interelement ratios calculations, (iii) use the subtraction of the ~3C natural abundance from the whole ~3C chromatogram in order to easily point out only the labeled compounds.

7.3. Bioavailability Studies A study was designed in our laboratory (LEACM - unpublished results) by Besacier and Croin to compare the performance of GC-MS and GC- AED in bioavailability simulations. The study design was that an i.v. dose of unlabeled caffeine was administered simultaneously with an oral dose of either 3(13CH3) caffeine or 1,3,7 (13CH3)3 caffeine. Plasma samples were spiked with these caffeine isotopomers in order to simulate pharmacokinetic curves corresponding to a theoretical bioavailability of the oral dose of 70 percent vs. the i.v. dose. Plasma samples were then assayed by GC-MS (HP 5972) and GC-AED (HP 5921) using ~3CH3 isobutylmethylxanthine (IBMX) as internal standard. When 3(~3CH3) caffeine is used with caffeine and their concentrations measured by GC-MS, there is an important overlapping of the molecular ionclusters of both isotopomers which are used for the quantification. When the mixture of the extracted unlabeled caffeine and labeled caffeine is analyzed by AED, the two isotopomers are coeluted from the chromatographic column and it is not possible to detect the proportion of the analate due to the labeled isotopomer. The response corresponding to the ~3C signal is the sum of the ~3C natural abundance of the unlabeled molecule and of the 13C content of the labeled one. The problem is the same with the ~2C response. Two methods can be used to selectively obtain the signal of ~2C from the unlabeled molecule and of ~3C from the labeled one. The first is the use of the recipe allowing one to subtract the ~3C natural abundance, the second is the resolution of a series of equations derived from standard curves. The use of the 13C subtraction has been discussed above, and so we will develop the mathematical method. S(~2C) and S(~3C) are the areas of unlabeled and labeled molecules, respectively, measured from the ~2C and ~3C chromatograms of the mixture. Se(12C) and Se(~3C) are the areas corresponding to the internal standard measured on the same chromatograms. [M] and [M*] are the respective concentrations of unlabeled and labeled molecules and [Se] the internal standard concentration. We can develop the

196 system of four equations: S(12C)/Se(~2C) = (A1 x [M]/[Se] + B1)+ (A2 x [M*]/[Se] + B2)

(1)

S(120)/Se(13C)- (A3 x [M]/[Se] + B3)+ (A4 x [M*]/[Se] + B4)

(2)

S(13C)/Se(120) = (A5 x [M]/[Se] + B5)+ (A6 x [M*]/[Se] + B6)

(3)

S(13C)/Se(13C) = (A7 x [M]/[Se] + B7)+ (A8 x [M*]/[Se] + B8)

(4)

The A1, B1, A3, B3, A5, B5, A7 and B7 coefficients are determined from the standard curves of the unlabeled molecules. The A2, B2, A4, B4, A6, B6, A8 and B8 coefficients are determined from the standard curves of the labeled molecule. Combining Eqs. (1) to (4), [M] and [M*] can then be calculated according four different ways. For example, combining Eqs. (1) and (3): [M*]/[Se] = [(S~3C)/Se(~2C)- B5 - B 6 - A5) x (S(~2C)/Se(~2C) - B1 - B 2 ) / A 1 ] / ( - A 6 - A2 x A5/A1) and [M]/[Se] = [(S(~2C)/Se(12C)- B1 - B2 - A2) x ([M*]/[Se])]/A1 In the bioavailability study simulation the following standard curves were calculated for caffeine concentrations ranging from 0 to 50 ng/l~l: S(~2C)/Se(~2C) = S(~2C)/Se(~3C) = S(~3C)/Se(~2C) = S(~3C)/Se(~3C) =

f([Caf]/[IBMX]) f([Caf]/[IBMX]) f([Caf]/[IBMX]) f([Caf]/[IBMX])

Table 8 shows the parameters of the standard curves corresponding to unlabeled and tri- ~3C-labeled caffeine molecules. The concentrations were calculated from the following derived equations: S(12C)/Se(12C) = (A1 x [M]/[Se] + B1)+ (A2 x [M*]/[Se] + B2)

(1)

S(12C)/Se(12C) = (A5 x [M]/[Se] + B5)+ (A6 x [M*]/[Se] + B6)

(4)

The correlations between measured concentrations and target values were

197 TABLE 8. Regression Lines Parameters of a Mixture of Labeled and Unlabeled Caffeine Determined by GC-AED

S(12C)/Se(12C)

Unlabeled caffeine

Labeled caffeine

Slope Intercept Coef of regression S(12C)/Se(13C)

0.84 _+0.02 0.06 _+0.03 99.49%

0.549 _+0.009 0.03 _+0.01 99.77%

Slope Intercept Coef of regression S(13C)/Se(12C)

3.56 +_0.09 0.2 -+ 0.1 99.52%

2.27 _+0.04 0.13 -+ 0.017 99.73%

Slope Intercept Coef of regression S(13C)/Se(13C)

0.115 _+0.002 0.007 _+0.003 99.78%

0.409 _+0.008 0.02 _+0.01 99.73%

Slope Intercept Coef of regression

0.486 _ 0.008 0.027 _+0.01 99.80%

1.69 _+0.03 0.09 _ 0.05 99.71%

excellent on the i.v. and oral pharmacokinetic curves. r = 0.9997 for the ~3C labeled caffeine concentrations (oral) r = 0.9971 for the unlabeled caffeine concentration (i.v.) The derived kinetic parameters were in good agreement. Two simulations were performed for the absolute bioavailability study. Unlabeled caffeine was administered by i.v. route and either 3-13CH3 caffeine or 1,3,7 (13CH3) caffeine were simultaneously administered by oral route. Table 9 shows the target pharmacokinetic parameters and the measured values derived from both labeled caffeine plasma levels determined by 13C atomic emission. For relative bioavailability, the two 13C-labeled caffeine isotopomers were administered orally in a two-phase experimental design. Each isotopomer was administered with unlabeled caffeine. As in Table 9, Table 10 shows the pharmacokinetic parameters derived from plasma curves. It can be seen from this example that molecules labeled with ~3C can be used for biopharmaceut-

198 TABLE 9. Comparison of Pharmacokinetic Parameters Obtained from Plasma Level of 313CH3 Caffeine, and 1,3,7 ~3(CH3)3 Caffeine Administered Orally, with the Target Values of the Model. tm.x = Time for Plasma Peak, Cm.x = Maximum Plasma Level, AUC = Area Concentration-time Curves, F = Factor of Bioavailability

Target values

tmax (h -1)

2.54

2.00

2.50

Cmax (mg/I)

3.13

3.14

3.11

34.46

30.18

32.10

AUC(0-inf)(mg.h.1-1) Difference (%) F (%)

~

(~= 12.4

70

3-~3CH3 caffei ne

1,3,7 (13CH3)3 caffeine

Parameter

~" (~= 6.84 61.26

~/ 69.35

TABLE 10. Comparison of Pharmacokinetic Parameters Obtained from Plasma Level of 3(13CH3) Caffeine, and 1,3,7 13(CH3)3 Caffeine Administered Orally, With the Target Values of the Model. tm.x = Time for Plasma Peak, Cm.x = Maximum Plasma Level, AUC = Area Concentration-time Curves, F = Factor of Bioavailability.

Parameter

3-(13CH3)

Target values

caffeine

1,3,7 (13CH3)3 caffeine

tmax (h)

2.54

2.50

2.50

Crnax (mg/I)

3.13

3.18

2.89

AUC(0-inf)(mg.h.1-1)

34.46

35.22

32.80

Difference (%)

~ '~

F (%)

70

(~= 2.19

~/ ~ = 4.83 71.53

~/ 66.62

ical studies using an experimental design with co-administration of unlabeled and labeled molecules and that AED is quite functional for the determination of both isotopomers. And that even with only one labeling isotope the experimental results are in very good agreement with the target values calculated by the theoretical pharmacokinetic model.

199 8. GC-AED COMPARED TO OTHER ANALYTICAL INSTRUMENTS FOR ISOTOPE MEASUREMENT

When stable isotopes are used several analytical methods are now available to measure their concentration, or to measure isotopes ratios between labeled and unlabeled compounds. Numerous mass spectrometric methods have been utilized in their detection. The most common of these methods has been the twin ion or ion-cluster method which allows both detection of metabolites using the ion cluster and structural identification by their mass spectrum. This technique is structure-dependent, and labeled compounds or metabolites in low amounts may not be detected due to interference with overlapping mass spectra of other compounds. Structure-independent detection may be an advantage when the structure of the investigated compounds are not known. Several structure-independent detectors have been developed. Among them, three kinds of analytical instruments can be mentioned for isotope determination:

- the first correspond to chromatography atomic emission spectroscopy coupling and especially GC-AED. - the second is CRIMS (chemical reaction interface mass spectrometry). Markey and Abramson (30) developed the chemical reaction interface. It is a microwave powered device that completely decomposes a complex molecule to its elements in the presence of helium. The addition of reagent gas forms stable products such as C02, S02 and H20 which reflect the elemental composition of the original analyte and are detected by a conventional quadrupole mass spectrometer. CRIMS is able to selectively detect compounds labeled with 13C, lSN and 2H from a biological matrix (31). HPLC can also be interfaced with such CRIMS (32). Detection limits are in the low to high ng/ml concentration range, and quantitative precision is in the 3-6 percent range (33). See Chapters 8 and 13 for more details on CRIMS. - t h e third is CF-GC-IRMS (continuous flow gas chromatography mass spectrometry). This isotope ratio mass spectrometer is able to measure isotopes ratios of carbon and nitrogen from very small samples with high precision and accuracy. For ~3C/~2C ratio measurements, organic carbon has to be transformed into C02, and for ~SN/~4N ratio measurement organic nitrogen into N2. Special devices have been developed for the determination of ~3C/~2C ratio from C02 contained in gaseous samples produced by micro-C02 generators.

200 Deruaz et al. (34) compared the limit of isotope enrichment detectable by GC-MS, GC AED and GC IRMS using ~3C-labeled progesterone as analyte. Progesterone (4 p r e g n e n e - 3.20 dione) was labeled with two atoms of ~3C at positions 3 and 4. It was chromatographed as its 2-enol pentafluoropropionic ester. The limit of isotope enrichment was the smallest value of molar enrichment significantly different from the response given by the unlabeled compound. For 10 ng of progesterone injected into the GC MS system (HP 5970A), the limit of isotope enrichment was 0.6 percent (over the natural ~3C abundance). The ~3C enrichment which can be determined routinely for progesterone by GC MS is about 2 percent (measurement on the molecular and base peak at m/2 = 462). When using GC AED, and the same chromatographic conditions, the limit of isotope enrichment was 1.8 percent for 10 ng of progesterone injected (HP 5921A). GC IRMS is specifically dedicated for the precise and accurate determination of small isotope enrichment. The smallest amount of CO2 necessary for a carbon isotope ratio is 10 nmol. That amount corresponds to 524 ng of unlabeled progesterone. The limit of isotope enrichment obtained by GC IRMS was 4.810 -3 percent for 2 nmol of progesterone injected (628 ng) (VG ISOCHROM II). See Chapters 6 and 21 for more details on CF-GC-IRMS. It can be observed from these results that GC AED and GC MS can measure carbon isotope enrichments in the same range of values (---2 percent) with small samples. GC IRMS is a complementary method which allows the determination of very small enrichments but needs larger amounts of analytical samples. In thesame way, the comparison between a quadrupole mass spectrometer (RIAL QM 130-CS) and an emission spectrometer (JASCO NIA 1) was reported on the determination of ~SN abundance in low enrichment nitrogenous materials (0.36-0.99 percent) by Cervelli et al. (35).

9. CONCLUSION

Current AED instrumental development allows one to attain high levels of sensitivity and specificity in elemental detection. With chemical, instrumental and algorithmic techniques, it is possible to lessen the interferences which limited the applications of AES coupled to chromatography. Hence, technique is now found to be practical and broadly useful in the analytical laboratory and in the areas of analysis of complex mixtures like biological samples, in biochemistry and pharmacology. These analytical performances allow use of a new kind of safe tracers,

201 stables isotopes, which can be easily detected and measured using gas c h r o m a t o g r a p h y coupled to atomic emission spectroscopy.

ACKNOWLEDGEMENTS The author thanks Dr D. Deruaz w h o has made productive this area of research in the LEACM, and M. Buisson for her skilful typographic assistance.

REFERENCES 1. R. Guilluy, F. Billion-Rey, C. Pachiaudi, S. Normand, J.P. Riou, E.J. Jumeau and J.L. Brazier, Anal. Chim. Acta, 259 (1992) 193. 2. J.L. Brazier, in Forensic Application of Mass Spectometry, in J. Yinon (ed) CRC Series Modern Mass Spectrometry (CRC Press, Boca Raton, FL, 1995), p. 259. 3. H.C. Urey, F. G. Brickwedde and G.H. Murphy, Phys. Rev. (1931) 722. 4. G.V. Veinbert, A.N. Zaidel and A. Petrov, Optr. I Spektr., 2 (1950) 972. 5. A.N. Zaidel and G.V. Ostrovskaya, Optics and Spectroscopy, 9 (1960) 78. 6. A.N. Zaidel and G.V. Ostrovskaya, Optr. I Spektr., 1 (1960) 137. 7. R.E. Ferguson and H.P. Broida, Anal. Chem., 28 (1956) 1436. 8. B.D. Quimby, P.C. Dryden and J.J. Sullivan, Anal. Chem., 62 (1990) 2509. 9. F. Leclerc, D. Deruaz, A. Bannier and J.L. Brazier, Anal. Lett., 27 (1994) 1325. 10. M. Hoch and H.R. Weisser, Helv. Chim. Acta, 23 (1950) 2128. 11. H.P. Broida and M.W. Chapman, Anal. Chem., 30 (1958) 2049. 12. J.A. Goleb and V. Middleboe, Anal. Chim. Acta, 43 (1968) 221. 13. J.C. Burridge and I.J. Hewitt, Anal. Chim. Acta, 153 (1983) 347. 14. D.I. Hoult and C.M. Preston, Rev. Sci. Instrum., 63 (1992) 1927. 15. P.C. Uden, Atomic Spectral Chromatographic Detection in Element Specific Detection by Atomic Emission Spectroscopy, in P.C. Uden (ed), ACS Symposium series 479 (1992), p. 1. 16. J.A. McCormack, S.C. Tong and W.D. Cooke, Anal. Chem., 37 (1965) 1470. 17. D.H Chose and F.P. Abramson, Anal. Chem., 61 (1989) 2724. 18. J.P. Van Delhen, P.A. De Lezenne Coulander and L. De Galan, Spectrochim. Acta, 33B (1978) 545. 19. D. Deruaz and J.M. Mermet, Analusis, 14 (1986) 107. 20. C.I.M. Beenakker, Spectrochim. Acta, 31B (1976) 483. 21. C.I.M. Beenakker, Spectrochim. Acta, 32B (1977) 173. 22. H. Yieru, O. Qingyu and Y. Weile, J. Chromatogr. Sci., 28 (1990) 584. 23. J. Hubert, M. Moisan and A. Ricard, Spectrochim. Acta, 34B (1979) 1. 24. G.L. Long and J.D. Winefordner, Anal. Chem., 55 (1983) 712. 25. R L. Oppenheimer, T. Capizzi, R. Weppelman and H. Mehta, Anal. Chem., 55 (1983) 638. 26. B.D. Quimby and J.J. Sullivan, Anal. Chem., 62 (1990) 1027. 27. A. Bannier, D. Deruaz, C.Weber and J.L. Brazier, Anal. Lett., 25 (1992) 1073. 28. T.C. Laurence and R.L. Terry, J. Chromatogr., 586 (1991) 309. 29. M.S. Boukraa, D. Deruaz, A. Bannier, M. Desage and J.L. Brazier, J. Pharm. Biomed. Anal., 12 (1994) 185.

202 30. S.P. Markey and F.P. Abramson, Anal Chem., 54 (1982) 2375. 31. D.H. Chace and F.P. Abramson, Anal. Chem., 61 (1989) 2724. 32. F.P. Abramson, M. McLean and M. Ustal, in Synthesis and Applications of Isotopically Labeled Compounds, E. Buncel and G.C. Kabalka (eds) (1991), p. 133. 33. D.H. Chace and F.P. Abramson, Biomed. Environ. Mass Spectrom., 19 (1990) 117. 34. D. Deruaz, B. Deruaz, A. Bannier, M. Desage and J.L. Brazier, Analusis, 22 (1994) 241. 35. S. Cervelli, F. Di Govanni and S. Ferrari, Rapid Comm. Mass Spectrom., 5 (1991) 48.

203

CHAPTER 10

THE USE OF ISOTOPES FOR PROBING LIGAND-PROTEIN INTERACTIONS AND LIGAND STRUCTURE: THE RHODOPSIN G-PROTEIN COUPLED MEMBRANE RECEPTOR PARADIGM

PETER J.E. VERDEGEM, JOHAN LUGTENBURG and HUUB J.M. DE GROOT Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands

1. INTRODUCTION

Rhodopsins are the light-collecting and transducting molecules in visual perception for three phyla of animals. Vertebrate, mollusks and anthropods. The most studied is bovine rhodopsin, which is the 40kDa G-protein coupled photoreceptor in the rod outer segments of the bovine retina that initiates the visual signal transduction cascade (1). It is an intrinsic membrane protein with 348 amino acids, which is folded into seven hydrophobic e-helices flanked by hydrophilic loops (Figure 1) (2, 3). The light-absorbing ligand in the active site of rhodopsin is called the chromophore. It is an 11-cis-retinylidene group covalently bound with a Schiff base link to lysine-296 of the polypeptide chain. The chromophore in rhodopsin is positively charged since the Schiff base is protonated. The charge is stabilized by a negative counterion in the protein. Glu 113 is thought to represent the primary counterion in rhodopsin, although it is likely part of a hydrogen-bonded network involving one or more water molecules, similar to the complex counterion in the somewhat related bacteriorhodopsin protein (4, 5). Capture of a photon by rhodopsin initiates the photochemical isomerization of the 11-cis chromophore to a distorted alI-E configuration within ~--200fs, leading to the primary photointermediate bathorhodopsin (6). The groundstate energy of bathorhodopsin is 145 kJ/mol higher than for rhodopsin and

204

205

Rhodopsin light Bathorhodopsin

l l l l

Lumirhodopsin

Metarhodopsin I

Metarhodopsin II

opsin + a/l-E-retinal Figure 2. The photosequence of rhodopsin.

--~60 percent of the photon energy of ---2 eV is temporarily stored in the primary photoproduct. Subsequent dark-reactions lead to the other intermediates in the sequence, lumirhodopsin, metarhodopsin I and II, and finally opsin and free all-E-retinal (Figure 2). Thus, a cis-trans photoisomerization is sufficient to convert the chromophore from an excellent antagonist into an optimal agonist for the receptor. The apoprotein is selective with respect to the binding of the 11-cis isomer. After the photosequence has ended, enzymatic isomerization of the all-Eretinal to the 11-cis form is necessary before it can bind again with the opsin to form the light active rhodopsin. The metarhodopsin I to II transition involves the discharge of the Schiff base through deprotonation and leads to the activation of the G-protein transducin and visual signal transduction (7).

Figure 1. The structure of the rhodopsin G-protein coupled visual photoreceptor, with the seven trans membrane e-helices and the 11-cis-retinylidene chromophore. Color picture courtesy of Prof. S.O. Smith.

206 In this chapter some recent applications of enrichment with stable 13C isotopes and Magic Angle Spinning (MAS) NMR studies of rhodopsin are presented. It will be demonstrated that comprehensive information about the electronic and spatial structure of the ligand can be obtained when bound to the membrane with isotope labeling and MAS NMR. First, we present a short overview of the labeling and MAS NMR studies aiming at a characterization of ligand-protein interactions for rhodopsin via the determination of chemical shifts. These studies have been performed in collaboration with several other research groups. To complement the NMR shift investigations, we discuss experimental data for the spatial structure of the ligand in the binding site in rhodopsin. To illustrate the capabilities of MAS NMR for studying photoproducts, shift data on the ligand-protein interactions in bathorhodopsin, structural data for the pre-discharge metarhodopsin I, and finally, shift data for the discharged metarhodopsin II photointermediate, will be reviewed. This includes a brief presentation of some of the newest structural results that were obtained recently in the last year in our own laboratories and will be published extensively elsewhere. The rhodopsin can be considered a paradigm for other G-protein coupled receptors. With hundreds of potential drug targets within companies worldwide, the importance of tools for the study of ligand structure and ligand-protein interactions can hardly be overestimated.

2. EXPERIMENTAL METHODS

MAS NMR, in conjunction with selective isotope enrichment, is the method of choice for NMR investigations of membrane protein receptors when in the membrane in their natural environment. With technologies for membrane protein expression continuously improving, it can be anticipated that many membrane receptors will become available in sufficient quantity for NMR spectroscopy in the near future. In this review, the major focus will be on the characterization of ligand-protein interactions at the atomic level, and how to examine specific details of the ligand structure with pairs of isotopes, incorporated by total synthesis at strategic positions in the ligand.

2.1. Magic Angle Spinning NMR MAS NMR is a technique for obtaining high-resolution NMR data from solids. In a MAS NMR experiment, the chemical shift anisotropy broadening of the NMR response in the solid state is suppressed by macroscopic sample rotation around an axis at the magic angle /3,7,= 54o44' with respect to the

207 applied magnetic field. A detailed treatment of the MAS averaging of the NMR response of a solid can be found elsewhere (8). Briefly, during MAS individual molecules are subject to physical sample rotation and the chemical shift of every molecule varies periodically in time. Since many different chemical shift trajectories will be possible, the macroscopic nuclear magnetization collapses in a very short time, but refocuses after every completed rotor cycle. In the frequency domain MAS generates an infinite number of sidebands at integral multiples of the spinning speed O~r,with respect to the isotropic shift (r~, which is the average chemical shift experienced by every molecule in the rotating sample.

2.2. The Rotational Resonance MAS NMR Technique Rotational resonance is a high resolution solid state NMR technique that 1 allows the measurement of internuclear distances between / = ~ nuclei through the interference of the MAS with the homonuclear dipole interactions within the pair of spins (9, 10). Rotational resonance occurs when (.Or matches the difference in resonance frequency A~O~s= ~o~- ~Os of the two spins. In an established approach to measure distances between a pair of ~3C atoms, one of the spins is selectively inverted and rotor-driven exchange of magnetization is followed in time by collecting a series of 1D datasets (11). At the n = 1 rotational resonance condition, the line shapes change and a broadening, or in favorable cases, a splitting of the line shape A(o~ can be observed. Recently, the relationship between A~o~ and the scaled dipolar interaction b~s/27r~/8 was investigated and calibrated experimentally with a series of four doubly-labeled retinal model compounds (12). Here,

b,s = t -j['~tOt ,)/2~ r3s

(1)

is the dipolar coupling constant. Analysis of the A(o~, by taking the second derivative of the rotational resonance spectrum and subsequent fitting of the shape with a pair of second derivative Lorentzians, provides a way to measure internuclear distances accurately (12). A linear relationship

bls/27r~/8 = al(A~ol/2~T) + ao

(2)

was found with a~ = 1.15 and ao a small offset depending on the total line

208 width (12). Using Eqs. (1) and (2), dipolar couplings b~s and internuclear distances r~s can be calculated from the experimentally determined A~o~.

3. MAS NMR INVESTIGATIONS OF RHODOPSIN

3.1. Ligand-Protein Interactions in Bovine Rhodopsin

The NMR response can be used to probe, with atomic resolution, the chemical environment of ligands bound to membrane receptors. The technique complements other spectroscopic approaches, in particular resonance Raman spectroscopy with isotope labeling to assign vibrations. By labeling with isotopes in the ligand, an NMR assay of the ligand-protein interactions can be constructed, for instance, by comparing the chemical shifts for the retinylidene in the protein with the corresponding shifts for a protonated Schiff base model in solution. This is illustrated in Figure 3, which shows the 11-cisretinylidene protonated Schiff base, like the antagonist-type ligand in rhodopsin. To construct a shift assay for the chromophore in situ, retinals labeled along the polyene chain were first synthesized (13) and reconstituted with bovine opsin (14). Subsequently, NMR experiments were performed to determine the ~3C shifts of the isotope labels in the protein and the signals for protonated Schiff bases in solution (15). The shift differences are reliable indicators of ligand-protein interactions. In Figure 3 they are encoded with colors, going from blue via green to yellow and red for increasing interaction strength. The result is a genuine assay of ligand-protein interactions with atomic resolution, almost like a "snapshot" of the ligand when bound to the receptor, in its natural lipid environment. Chromophore-protein interactions are observed for almost the entire length of the conjugated chain. The strongest interactions, indicated by the red and yellow spots, are detected for the cis region of the chromophore. This is of interest, since it is exactly the section of the chromophore involved in the isomerization to the agonist form of the ligand. It can be concluded that for rhodopsin the shift assay works particularly well, since the region of the chromophore that is most critical for the function, is immediately transpiring from the representation of the NMR data. Using the shift differences and by correlating the NMR data with other experimental results from e.g. site-directed mutagenesis studies, Han et al. (16) were able to identify the location of the counterion to the Schiff base. Using this counterion as an anchor, they were also able to fit the chromophore into the electron diffraction map that was recently obtained for rhodopsin (2),

....... ....

k,

i

x

\

O

r

t-

t.~

(D

O

0 0

0 o

(I) t-

r-"r_

,-

.Q

c-

0

O

e.-

*-,

"o

o

e-

0 o *" o o - L,,

c-

O

~ZE "~

e"

L r ~- e " e-- , - .--

~"

0-0 e"

.__..- ~

0

L

0

0

e"

--

o.~-o

L_ ~

o-o~

0 f--

Eg-o 0

~

~-o

O'>'-

e-~

m

0

. F . ~ -~

N

~ l=-' L 0 0 " ~

Z ~- o ~_ ~

~-~o

209

210 thereby providing the first comprehensive low-resolution structure for a Gprotein coupled membrane receptor shown in Figure 1 (3). In principle, similar assays can also be made based on other NMR characteristics. For instance, the examination of the variations in relaxation properties can help to detect steric nonbonding ligand-protein and intra-ligand interactions. The measurements presented in Figure 3 were performed using selectively labeled compounds, which is a laborious procedure, since every labeled position needs a separate organic synthesis. However, recent advancements in 2D MAS NMR dipolar correlation spectroscopy allows the use of uniformly labeled ligands to obtain a complete picture in one set of labeling and reconstitution experiments, with improved accuracy (17, 18). Such experiments will benefit greatly from novel ultra-high field MAS NMR equipment currently under development with the NMR industries.

3.2. Probing the Spatial Ligand Structure in Bovine Rhodopsin With NMR and isotope labeling we can examine the structure of the isomerization region by measuring critical intra-ligand internuclear distances. In Figure 4A, the chemical structure of the 11-cis-retinylidene chromophore in rhodopsin is shown. It is thought that nonbonding interactions between the C10-H and the C20-H3 provoke an out-of-plane deformation in the is0merization region of the chromophore which is of prime importance for the speed and efficiency of the isomerization. The presence of an out-of-plane deformation can be verified with 1D MAS NMR rotational resonance spectroscopy (19). The particular focus will be on the r~o,2o, between the vinylic C10 and the C20 methyl group, which were labeled for this purpose. The doubly labeled retinal was reconstituted into freshly prepared bovine opsin maintained in its natural lipid environment. In Figure 5, the off rotational resonance MAS spectrum of the reconstituted [10,20-~3C2]-rhodopsin is shown. The narrow label resonances are indicated with a filled circle for the vinylic signal with (~; = 127.1 ppm, and with a square for the methyl response with ~; = 15.9 ppm. To determine the n = 1 rotational resonance condition &~s from the NMR response, both label resonances were analyzed with Lorentzians and, from the center frequencies ~O~oand (02o, the rotational resonance condition was calculated as A~O~s= 12.592 kHz. The vinylic part of the rotational resonance spectrum of [10,20-~3C2]-rhodop sin, collected with OJr= A~O~S,is depicted in Figure 6A, showing in the upper trace the C10 signal superimposed on the broad resonance from the unsaturated lipids in the natural membrane. At rotational resonance, a small splitting of the label response due to rotational resonance dipolar recoupling can be

211 16

17

, , o, i l . :

A 2

~

12

13

3 4

18

,5 L%NtH LLys296 2O | FI

Lys296

Figure 4. Chemical structures of 11-cis retinylidene (A) and alI-E retinylidene in rhodopsin. In scheme A the IUPAC numbering for the entire chromophore group is indicated, while in scheme B the two positions that were labeled with isotopes to measure the out-of-plane distortion in the rhodopsin and the metarhodopsin-I intermediate are emphasized with their IUPAC numbers.

observed. To analyze the data, we first apply a 40 Hz apodization to suppress background noise, and fit the second derivative spectrum with a pair of second derivative Lorentzians, yielding A~o1/2~r=75_+4Hz. The second derivative of the signal and the computer analysis are shown in the lower traces in Figure 6. In the second derivative, the splitting is enhanced which facilitates the analysis. Calculation of the scaled dipolar coupling constant using Eq. (2) gives Ib,sl/2~/8 - 98 _+ 4 Hz, corresponding with an internuclear distance rlo,2o=0.302-+0.01 nm, significantly longer than the rlo,2o = 0.295 nm expected for a predominantly planar chromophore. The rlo,2o distance measurement provides direct and unambiguous experimental evidence that the chromophore is indeed the 11-cis form and that a considerable outof-plane distortion is present in the isomerization region of the chromophore of rhodopsin, probably due to nonbonding interactions between C10-H and C13-methyl. Simple molecular modeling, using the crystal structure of 11-cis12-s-cis-retinal (20) and our internuclear distances, provides an estimate of the angle between the C7-C10 and C13-C15 planes of the chromophore of

212

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250

200

150

l O0

50

0

Chemical shift (ppm)

Figure 5. 100.6 MHz 13C-CP/MAS spectrum of [10,20-13C2]-rhodopsin collected with ~r/2~T = 7.000 kHz. The markers indicate the centerbands of the C10 (O) and C20 (11) label resonances.

---42~ in agreement with ab-initio molecular dynamics results (21). The distance between C11 and C20 has also been determined, r11,2o = 0.294 nm. This measurement confirms the previous conclusions with respect to the out-ofplane deformation of the chromophore (19). Using a limited set of precise distance constraints, Bifone et al. (22) were able to model the structure of the isomerization region. In particular, they found a distribution of the ---42~ torsion over the Clo-C~-C~2-C~3 moiety, which has been confirmed recently with a novel MAS NMR method for measuring torsional angles (23).

3.3. Probing the Electronic and Spatial Structure of Intermediates An accurate determination of the electronic and spatial structure of the ligand after activation will be essential also for understanding the molecular mechanisms behind the initial stages of visual signal transduction. The retinylidene ligand in rhodopsin and its photoproducts are brightly coloured, which makes them accessible to an extensive array of optical techniques. Recent ultrafast time-resolved optical absorption experiments, and resonance Raman experiments, have provided conclusive evidence that both the speed and the effi-

213

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Chemical shift (ppm) Figure 6. Vinylic region of 100.6 MHz 13C n = 1 rotational resonance CP/MAS spectra of [10,20-13C2]-rhodopsin and -metarhodopsin-I. The lower traces represent the second derivatives of the NMR response with the computer, analyses indicated by the dashed lines.

214 ciency of the isomerization strongly depend on a precise tuning of the structure in the central part of the ligand, well in line with the MAS NMR shift assay discussed in the previous section (24, 25). In addition, the isomerization is ultrafast and is completed in less than ---200 fs (26). Even a small ligand cannot undergo major structural changes on such a short timescale. The observation that the photochemical isomerization is ultrafast thus implies that the bathorhodopsin photoproduct constitutes a formal alI-E chromophore in globally the binding pocket optimized to accommodate the 11-cis form. This inference is supported by the ab initio molecular dynamics simulations, which provided compelling evidence that the conformation of the ligand in the rhodopsin inactive ground state, together with its stabilization by the counterion or complex, largely determines the structure, the amount of strain, and the charge delocalization of the chromophore in the active bathorhodopsin state (27). An accurate determination of the electronic structure of the chromophore in bathorhodopsin along the polyene chain, and in the vicinity of C11C12 double bond, is then also essential to understand the mechanisms of isomerization and energy storage in the primary photointermediate, which can be considered as the agonist form of the ligand. The photointermediates in the rhodopsin photosequence can be trapped at low temperature and accessed with MAS NMR once isotopes have been incorporated. To achieve this, either reconstituted rhodopsin or the 9-cis isorhodopsin receptor is activated by illumination with an intense light source, with the sample packed in the NMR rotor and cooled to liquid nitrogen temperature. In this procedure, the bathorhodopsin primary photointermediate is formed with good efficiency. Subsequently, the NMR rotor can be inserted in a pre-cooled probe. By raising the temperature in steps, the various photointermediates can be trapped and their electronic and spatial structure examined with MAS NMR. Again the NMR complements the vibrational at studies where structural information about the chromophone can be obtained from resonance Raman spectroscopy and isotope labeling to assign the normal modes. MAS NMR spectra were obtained for the trapped bathorhodopsin photointermediate, starting from isorhodopsin samples reconstituted with retinals labeled at positions 8, 10, 11, 12, 13, 14 or 15 (28). A shift assay of the ligandprotein interactions in the isomerization region of the primary photointermediate was constructed by comparison with the shifts from alI-E retinal protonated Schiff bases. The largest difference amounts to 6.2 ppm and is observed for the position 13 in the bathorhodopsin photoproduct. Small differences in chemical shift between bathorhodopsin and the alI-E protonated Schiff base chloride salt are also observed at positions 10, 11 and 12. The effects are almost equal in magnitude to the shifts observed in rhodopsin (Figure 3).

215 Consequently, the energy stored in the primary photoproduct does not give rise to any substantial change of the average electron density at the labeled positions. The data indicate that the electronic and structural properties of the protein environment are similar to those in rhodopsin and isorhodopsin. In particular, the counterion, which is thought to be located near C13, appears not to change its position significantly with respect to the chromophore upon isomerization. The NMR provides strong evidence that the light energy is rapidly converted into strain, and is not primarily stored in the form of charge separation between chromophore and protein. Starting from the rhodopsin structure and our NMR charge and distance constraints, the bathorhodopsin structure and energy storage function were modelled with ab-initio molecular dynamics methods (27). It appears that the energy storage is mediated by a charge defect or soliton, which is already present in the ground state (22). After excitation the molecule is bistable, in the sense that the defect can easily move between the two extremes of the polyene chain. Fully in line with the MAS NMR shift data for the bathorhodopsin intermediate, it is the interaction with the counterion that locks the defect at the Schiff base end of the chromophore. This yields a distribution of electronic change similar to rhodopsin and gives rise to a strained molecule capable of accumulating the energy for triggering the subsequent steps in the sequence. In this respect, it is also of interest that discharge of the Schiff base probably requires the motion of another solitonic defect at the same energy scale, e.g. a proton in a hydrogen bonded network, from the Schiff base nitrogen into the protein environment. It is fair to state that these novel functional descriptions have emerged from the interpretation of the data from NMR and isotope labeling studies (5, 22). Very recently, we have been able to obtain genuine structural data for various intermediates in the rhodopsin sequence. This is illustrated in Figure 6, which shows the vinylic region of a dataset collected after illumination and accumulation of the [10,20-~3C2] metarhodopsin I intermediate together with its second derivative trace. The response of the [10,20-~3C2] yields two contributions to the second derivative spectrum, with cr/= 130.6 ppm from the metarhodopsin I intermediate and with cr; = 127.1 ppm from the rhodopsin that could not be converted in the illumination procedure. Although the data for Figure 6 were collected at the n = I rotational resonance condition for the [10,20-~3C2] in the metarhodopsin I, there is no resolved splitting of the second derivative signal like in Figure 6 for the rhodopsin. This shows that the C10C20 distance is longer in metarhodopsin I than for rhodopsin, which is due to the photoisomerization to the alI-E form (Figure 4B). The distance in the photointermediate can be estimated by measuring the excess broadening of

216 the n = 1 rotational resonance in the second derivative response. From Figure 6 an rlo,2o > 0.4 nm can be estimated, which indicates that the chromophore is almost planar in the metarhodopsin I intermediate. This would imply a considerable structural change of the ligand of ~--0.5 nm already before the discharge of the chromophore and the activation of transducin by the deprotonated metarhodopsin II form occurs. The actual discharge of the ligand has been investigated with ~3C MAS NMR and retinal labeled at C14 or C15. To achieve this, MAS !3C NMR spectra were collected from 13C-labeled rhodopsin reconstituted into 1,2dipalmitoleoylphosphaditylcholine bilayers to increase the amount of meta II trapped at low temperature (29). Both the C13 and the C15 shifts are characteristic of an unprotonated Schiff base, providing unambiguous evidence that the ligand is discharged in the meta I to meta II transition.

4: CONCLUSIONS

NMR in conjunction with isotope enrichment can provide assays of ligandprotein interactions to atomic resolution for ligands when bound to the receptor. In the future, such NMR assays will be useful for the identification and characterization of pharmacophores, conformational and configurational changes of the ligand induced by binding, charging or discharging, nonbonding interactions and novel functional mechanisms for triggering signal transduction. 1-Dimensional rotational resonance and other, more sophisticated MAS NMR techniques, in conjunction with specific isotope enrichment, are powerful methods for obtaining accurate structural information of biological systems. It is illustrated how the ligand conformation in the rhodopsin Gprotein coupled receptor can be examined with MAS NMR. Our newest data measure the out-of-plane deformation in the isomerization region, which is essential for the molecular mechanism of function, since it determines the speed and efficiency of the first step in the visual signal transduction process (25). After capture of a photon and isomerization to an alI-E conformation in bathorhodopsin, the chromophore adapts a relaxed alI-E structure already in the pre-discharge metarhodopsin.

217

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

L. Tang, T.G. Ebrey and S. Subramaniam, Isr. J. Chem., 35 (1995) 193. J. Baldwin, EMBO J., 12 (1993) 1693. M. Han and S.O. Smith, Biochemistry, 34 (1995) 1425. H.J.M. De Groot, G.S. Harbison, J. Herzfeld and R.G. Griffin, Biochemistry, 28 (1989) 3346. H.J.M. De Groot, S.O. Smith, J. Courtin, E.M.M. van den Berg, C. Winkel, J. Lugtenburg, R.G. Griffin and J. Herzfeld, Biochem., 29 (1990) 6873. R.W. Schoenlein, L.A. Peteanu, R.A. Mathies and C.V. Shank, Science, 254 (1991) 412. K.P. Hofmann, O.P. Ernst, S. J~ger, Isr. J. Chem., 35 (1995) 339. M. Mehring, Principles of High Resolution NMR in Solids (Springer-Verlag, Berlin, 1983). M.H. Levitt, D.P. Raleigh, F. Creuzet and R.G. Griffin, J. Chem. Phys., 92 (1990) 6347. F. Creuzet, A. McDermott, R. Gebhard, K. van der Hoef, M.B. Spijker-Assink, J. Herzfeld, J. Lugtenburg, M.H. Levitt and R.G. Griffin, Science, 251 (1991) 783. D.P. Raleigh, M.H. Levitt and R.G. Griffin, Chem. Phys. Lett, 146 (1988) 71. P.J.E. Verdegem, M. Helmle, J. Lugtenburg and H.J.M. De Groot, J. Amer. Chem. Soc., 119 (1997) 169. M. Groesbeek and J. Lugtenburg, Photochem. Photobiol., 56 (1992) 903. W.J. De Grip, F.J.M. Daemen and S.L. Bonting, Methods in Enzymol., 67 (1980) 301. S.O. Smith, I. Palings, M.E. Miley, J. Courtin, H.J.M. De Groot, J. Lugtenburg, R.A. Mathies and R.G. Griffin, Biochem., 30 (1991) 1991. M. Han, B.S. De Decker and S.O. Smith, Biophys. J., 65 (1993) 899. T.S. Balaban, A.R. Holzwarth, K. Schaffner, G.-J. Boender and H.J.M. De Groot, Biochem., 34 (1995) 15259. T. Egorova-Zachernyuk, B. Van Rossum, G.-J. Boender, E. Franken, J. Ashurst, J. Raap, P. Gast, A. Hoff, H. Oschkinat and H. De Groot, Biochem., 36 (1997) 7513. P.J.E. Verdegem, P.H.M. Bovee-Geurts, W.J. De Grip, J. Lugtenburg and H.J.M. De Groot, submitted. R.D. Gilardi, I.L. Karle and J. Karle, Acta Cryst., B28 (1972) 2605. A. Bifone, H.J.M. De Groot and F. Buda, Chem. Phys. Lett., 248 (1996) 165. A. Bifone, H.J.M. De Groot and F. Buda, J. Phys. Chem., 101B (1997) 2954. X. Feng, P.J.E. Verdegem, Y.K. Lee, D. Sandstr6m, M. Eden, P.H.M. Bovee-Geurts, W.J. De Grip, J. Lugtenburg, H.J.M. De Groot and M. H. Levitt, J. Amer. Chem., in press. Q. Wang, G.G. Kochendoerfer, R.W. Schoenlein, P.J.E. Verdegem, J. Lugtenburg, R.A. Mathies and C.V. Shank, J. Phys. Chem., 100 (1996) 17388. G.G. Kochendoerfer, P.J.E. Verdegem, I. van der Hoef, J. Lugtenburg and R.A. Mathies, Biochem., 35 (1996) 16230. Q. Wang, R.W. Schoenlein, L.A. Peteanu, R.A. Mathies and C.V. Shank, Science, 266 (1996) 422. F. Buda, H.J.M. De Groot and A. Bifone, Phys Rev. Lett., 77 (1996) 4474. S.O. Smith, J. Courtin, H.J.M. De Groot, R. Gebhard and J. Lugtenburg, Biochem., 30 ( 1991 ) 7409. S.O. Smith, H.J.M. De Groot, R. Gebhard and J. Lugtenburg, Photochem. Photobiol., 56 (1992) 1035.

219

CHAPTER 11

MASS BALANCE

THOMAS R. BROWNE, 1 GEORGE K. SZABO ~ and ALFRED AJAMI 2 1Departments of Neurology and Pharmacology, Boston University School of Medicine, Neurology Service, Boston Department of Veterans Affairs Medical Center; 2MassTrace, Woburn, MA

1. INTRODUCTION

Regulatory agencies in many countries require a human mass balance/metabolite identification (MB/MI) study as part of the testing of a new drug. Historically, MB/MI studies have been performed by: (1) administering radioactive (14C) labeled drug; (2) measurement of radioactivity in urine and feces (to measure mass balance); (3) chromatography of urine to divide dissolved material into peaks; (4) detection and quantitation of peaks containing drug or metabolite by measurement of radioactivity; and (5) identification of structure of drug or metabolite in "hot" chromatography peaks by various mass spectrometry techniques. Preliminary data on the pharmacokinetics of the drug can be obtained from radioactivity versus time relationships in serum, blood and urine. The analytic aspects of this methodology are simple and have served pharmaceutical research well for many years. Recently, alternatives to traditional radioactive labeling techniques for performing MB/MI studies have been sought for several reasons: (1) regulations for storage and disposal of radioactive specimens have become more restrictive; (2) institutional review boards are increasingly reluctant to approve any work exposing humans to radioactivity; (3) it has been nearly impossible to perform radioactive tracer studies in children, even though drug metabolism in children often differs significantly from adults; (4) synthesis of radioactive analogues of some compounds can be difficult; and (5) the sponsor may be assuming long-term liability risk if the subjects or employees later develop cancer or other diseases. These considerations may increase the cost and delay performance of MB/MI studies. Human MB/MI data should be obtained as early in drug development as possible to determine the presence and

220 extent of potentially active or toxic metabolites and to guide future pharmacokinetic studies (e.g. collection of patient urine samples for drug analysis may, or may not, be useful, depending on results of MB/MI studies). This chapter reports on work to develop simple general methods for performing an MB/MI study on any new drug using stable isotope labeling (SIL) and detection as an alternative to radioactive labeling and detection. Two SIL techniques show promise of achieving this objective of a simple general MB/MI methodology: (1) continuous flow-isotope ratio mass spectrometry and (2) high-performance liquid chromatography combined with chemical reaction interface mass spectrometry. There are many examples of combining SIL and MS for identification of specific metabolites of specific drugs which are covered in Chapters 3-5, and 12.

2. CONTINUOUS FLOW-ISOTOPE RATIO MASS SPECTROMETRY (CF-IRMS) 2.1. Technique and History These topics are covered in Chapter 6.

2.2. Assumptions in Using CF-IRMS for MB/MI Studies Use of CF-IRMS methods for performing human MB/MI studies assumes: (1) simple, reliable CF-IRMS instruments are commercially available; and (2) the commercially-available CF-IRMS instruments possess the necessary sensitivity, precision and accuracy to determine label in urine, feces, serum and blood and to detect and quantify labeled drug and metabolites in HPLC peaks collected from urine, feces and serum.

2.2.1. Reliable instrumentation Early IRMS instrumentation was problematic because: (1) each instrument was unique and "made by hand"; (2) complete liberation of all atoms of a given molecule by oxidation was difficult; (3) transfer by hand of N2 and C02 gases from elemental analyzer to IRMS was problematic; (4) each specimen was run by hand (lack of automation); and (5) factors 1-4 made IRMS difficult and inconsistent (1, 2). Recently, refined commercially-available instruments using a helium carrier gas to carry combustion products to the IRMS have become available from tl~ree sources (Europa Scientific, Ltd., Finnegan MAT and VG Instruments). The authors purchased a Europa (Europa Scientific, Inc.,

221 Franklin, Ohio, USA) ANCL-SL (elemental analyzer) 20/20 (mass analyzer) CFIRMS and found the instrument performed up to specifications and with very high precision as delivered (see below).

2.2.2. Adequate sensitivity: Theoretical computations It is possible to compute the lowest quantity of drug quantifiable with a precision (coefficient of variation, CV) of 5 percent or less for a given drug using the maximal resolution and minimal total sample size of a CF-IRMS instrument, the carbon or nitrogen content of a biological specimen, and the molecular weight and 15N or 13C content of the tracer drug. The equation for this computation is as follows: LQ = MR,- x M(c,n) x Tmw x N

(1)

where MR/is a mass spectrometer's instrument resolution taken as the mole ratio of 15N/14N focused on masses 29/28 or 13C/~2Cfocused on masses 45/44 at natural abundance which can be measured with 5 percent or better precision (data taken from manufacturer's or literature values); M(c,n)is the moles of natural abundance isotopolog(s) per unit volume (time) in the biological matrix that is to be spiked with 15N or 13C tracer (data taken from published elemental composition of various biological matrices); Tmw is the molecular weight of a tracer drug (assumed to be 200 g/mol in this paper); N is the number of labeled atoms per mole of tracer drug. The results of Eq. (1) applied to a typical CF-IRMS instrument are shown in Table 1. Note the following: (1) procedures which reduce background carbon or nitrogen (deproteinization, extraction, chromatography) increase sensitivity; (2) sensitivity for a given molecule increases directly with the number of atoms labeled with an additional neutron; and (3) the theoretical sensitivity of CF-IRMS appears sufficient to perform MB/MI studies on drugs of medium or low (but not high) potency using one 15N or two 13C labels.

2.2.3. Adequate sensitivity, precision and accuracy: Empirical studies In 1993, we presented preliminary evidence that an early commercial CFIRMS instrument (Europa Roboprep CN/Tracer Mass) may possess sufficient sensitivity, precision and accuracy to quantitate some drugs with one 13C or two 15N labels (2, 3). Stable isotope labeling in therapeutic and subtherapeutic quantities of 15N2 13C-phenobarbital were quantitated in urine and in HPLC peaks from urine. Standard curves were reproducible and linear (r2> 0.985)

222 TABLE 1. Lowest Quantity of Drug Quantifiable with a Precision (CV) of 5% or less Using CF-IRMS 1

Desired value

lSN1 label

~3C~ label

~3C2 label

A. Total label Blood (whole)

0.1 ~g/mL

42.2 i~g/mL

>42.2

>42.2

Blood (deproteinized)

0.1 ~g/mL

0.006 ~g/mL

Serum (whole) 0.1

0.1 i~g/mL

1.4 i~g/mL

Serum (deproteinized)

0.1 i~g/mL

0.006 i~g/mL

0.006 ~g/mL

0.003~g/mL

Urine (whole)

1 i~g/mL

1.0 i~g/mL

1.4 i~g/mL

0.7 ~g/mL

Feces (whole)

1 mg/24 hr

0.3 mg/24 hr

2.3 mg/24 hr

1.2 mg/24 hr

Feces (extracted)

1 mg/24 hr

0.05 mg/24 hr

0.7 mg/24 hr

0.4 mg/24 hr

0.006 i~g/mL

0.003~g/mL

0.006 ~g/mL >1.4 i~g/mL

0.003~g/mL >1.4 ~g/mL

B. Labeled drug or metabolite in an HPLC peak drug Serum or urine

0.02 ~g/mL

0.006 ~g/mL

1Europa ANCL-SL 20/20 instrument.

over the range of 3-100 i~g/ml for whole urine (15N2 or 13C labeling) and 0.1-8.0 ~g/ml for HPLC peaks derived from urine (lSN2 labeling). The lower limit of quantitation values for urine drug concentration were 0.46-2.62 ~g/ml in whole urine and 0.10-0.701~g/ml in HPLC peaks. Validation samples quantitated with these standard curves yielded close to expected values. We have been working since then on further empirical verification of CFIRMS analytic determinations of stable isotope-labeled drug concentration in biological matrices using a newer CF-IRMS instrument (Europa ANCL-SL 20/20). We calculated the new instrument should be more sensitive than the older instrument (4). Several interim reports of our (not yet completed) work to confirm these calculations are available. We first studied ~SN~ labeled drug in human urine (5, 6). Standard curves of atom percent excess of ~SN (above natural abundance) times total nitrogen values versus drug (~SN-acetaminophen) concentration were regressed over a concentration range (in whole urine) of 1.0 to 200.0 ~g/ml (Figure 1). Weighted (1/X 2) and unweighted least squares linear regression analysis techniques gave coefficient of determination (r 2) and lower limit of quantitation (LLQ)

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224 TABLE 2.

Quantitation Characteristics of lSN-acetaminophen Urine 2

Urine (diluted)

Urine (urease treated)

LLQ1 ~g/ml

Run A Run B

1.6 1.0

0.9 0.7

1.5 2.2

WT 1 LLQ ~g/ml

Run A Run B

0.3 0.1

0.2 0.3

0.9 0.3

0.3680

0.3687

0.3776

0.3%

0.3%

0.1%

Mean atom% (Blank 2) C.V. n = 6 Mean total Nitrogen (w/g) C.V. n = 32

260.6 3.8%

214.4 2.9%

39.9 2.9%

1LLQ based on weighted (1/X2) least square linear regression. =Blank urines at natural abundance. From Szabo et al. (5) with permission.

values of 0.9998 to 1.000 and 0.24 to 2.2 i~g/ml, respectively. Coefficients of variation for atom percent at natural abundance ranged from 0.1 to 0.3 percent for 15N and 0.8 to 3.8 percent for total nitrogen. Spiked validation urine samples containing drug concentration values determined using standard curves showed close agreement of observed and expected concentrations for values greater than 1.51~g/ml. Similar determinations were performed in whole urine and urine diluted with water or treated with urease to reduce background nitrogen. One milliliter whole urine samples were treated with either 100 I~L of urease (10 mg/mL, 6200 units of activity/mL) or diluted with an equivalent 100 i~L of distilled water. The best results were obtained with diluted whole urine (Tables 2 and 3). Urease treatment may introduce errors because of isotope effects. When whole urine, which contains high concentrations of urea, is treated with urease two products are formed: (1) ammonia (NH3) which is volatile and can be removed under vacuum; and (2) amm o n i u m carbonate ((NH4) CO3) which crystalizes out of solution in the neutral to slightly acidic conditions optimal for urease activity. Urease treated blank urines (isotopically unenriched), measured higher atoms percent of ~5N than equivalent untreated urines. In the urease treated samples 15N at natural

225 TABLE 3. Accuracy Validation of 15N-acetaminophen (observed concentration ixg/ml) Expected concentration (l~g/ml)

Urine

Urine (diluted)

Urine (urease treated)

0.75 3 30 90

0.3 1.9 28.7 87.2

1.1 3.0 30.2 90.4

2.0 3.9 30.8 87.6

1See Table 2. From Szabo et al. (5) with permission.

abundance could be concentrating in the ammonium carbonate product, while 14N more readily forms the volatile ammonia product. The isotope effect could be due to 15N's preferential ammonium carbonate crystal formation. This explanation needs to be confirmed. As shown in Table 2, urease effectively reduces a sample's total background nitrogen load, however, the increased ~5N background levels (due to the isotope effect) limits the assay sensitivity. In a second set of experiments (7), SIL drug (~5N ~3C2 acetaminophen) was measured in urine by the dual selective detection of ~SN label in N2 gas and subsequent 13C label in CO2 gas. Twenty-five microliters of urine was added directly into tin combustion capsules and evaporated and analyzed directly. Drug spiked standards and validation control points in whole urine were assayed. Dual measurements of atoms percent of ~SN and total nitrogen as well as atoms percent ~3C and total carbon were obtained simultaneously from a single combusted sample. Standard curves of atom percent excess (APE) of ~5N (above natural abundance) times total nitrogen (APEXTN) and ~3C times total carbon (APEXTC) values, versus drug concentration were regressed over a concentration range of 0.5 to 200 i~g/ml. Our results were are follows (Tables 4 and 5). Weighted (1/X 2) and unweighted least square linear regression analysis techniques gave coefficients of determination [r 2] values of 0.9773 to 0.9995. Weighted regressions gave greater confidence at concentration values at low (near natural abundance) atom percent measures. Coefficients of variation for atom percent at natural abundance ranged from 0.01 to 0.02 percent for ~5N and ~3C with CVs of 4.8 to 5.0 percent for total nitrogen and total carbon. Observed and expected values for spiked urine samples showed close agreement for concentration >1 ~g/ml. APEXTN

226 TABLE 4. Quantitation Characteristics of ~SN, ~3C2-acetaminophen Urine whole matrix ~3C derived values

Urine whole matrix ~5N derived values r2 Weighted 1 r~

0.9994 0.9986

0.9995 0.9773

Mean atom% (Blank 2)

0.3667

1.1116

C.V. n = 4

0.01%

0.02%

Mean total Nitrogen (l~g) or carbon (~g) (Blank 2) C.V. n = 4

313.8

233.6

4.8%

5.0%

l r2 based on weighted (1/X 2) least square linear regression. 2Blank urines at natural abundance.

TABLE 5. Accuracy Validation of lSN, 13C2-acetaminophen (observed concentration i~g/ml) Expected concentration (~g/ml)

Urine whole matrix ~5N derived values

Urine whole matrix ~3C derived values

Unweighted

Weighted 1

Unweighted

Weighted

0.75 3.0 30.0 90.0

0.75 2.3 28.4 92.6

1.0 2.6 28.6 92.7

2.0 3.4 27.2 92.4

1.4 2.9 28.5 98.4

1Values based on weighted (1/X 2) least square linear regression.

values versus APEXTC values were also regressed and gave a correlation coefficient [r] of 1.0000 and an r 2 of 0.9999 (Figure 2). In a third set of experiments (8), SIL drug (~3C6 levodopa) was measured in serum. Twelve and a half microliters of whole matrix serum, or twentyfive microliters of diluted serum (1:1 with H20) was added directly into tin combustion capsules and evaporated and analyzed directly. Drug spiked

227 i3C 2

VS

is N

CORRELATION

70 60

-

7

i,

6

-

5

.

4

3

50

, i

-

CD H-

X

2

1

40

0

0

1

2

3

4

W 0_

30 20 10

0 0

10

20 3O APEXTN

40

50

Figure 2. Unweighted least square regression plot demonstrating correlation of 15N 13C2-acetaminophen CF-IRMS measures of atoms percent excess 15N times total measured N (APEXTN) versus atoms percent excess ~3C times total measured C (APEXTC). The insert shows the lower instrument measures (corresponding to lower ~SN ~3C2-acetaminophen concentrations) and the dotted lines represent the 95 percent confidence interval.

standards and validation control points in whole matrix serum and diluted serum (to reduce carbon load) were assayed. Measurements of atoms percent of ~3C and total carbon were obtained. Standard curves of atom percent excess (APE) of ~3C (above natural abundance) times total carbon values versus drug concentration were regressed over a concentration range of 0.01

228 TABLE 6. Quantitation Characteristics of 13Cs-acetaminophen

Serum whole matrix

Serum whole matrix diluted

0.9998 0.9926

0.9999 0.9977

Mean atom% (Blank2)

1.1030

1.1145

C.V. n = 4

0.05%

0.02%

r2

Weighted 1

r 2

Mean total Nitrogen (l~g) of carbon (i~g) 535.1 (Blank2) C.V. n = 4

0.6%

520.4 2.0%

lr2 based on weighted (1/X 2) least square linear regression. 2Blank serums at natural abundance.

to 100 ~g/ml. Our results were as follows (Tables 6 and 7). Weighted (1/X 2) and unweighted least square linear regression analysis techniques gave coefficients of determination [r 2] values of 0.9926 and 0.9998, respectively, for undiluted whole matrix serum samples from a range of 2.5 to 100 ~g/ml. Diluted serum samples were linear from 0.25 to 100 i~g/ml with respective r = values of 0.9977 and 0.9999. Coefficents of variation for atom percent at natural abundance ranged from 0.02 to 0.5 percent for 13C and 0.6 to 2.0 percent for total carbon. Observed and expected values for spiked serum standard curve samples showed close agreement for concentrations > 1.0 i~g/ml. The above results are in agreement with predictions of Table 1 and suggest current CF-IRMS instruments possess sufficient sensitivity, precision and accuracy to perform MB/MI studies of many drugs. More confirmatory work is necessary.

2.3. Advantages of the CF-IRMS Method The CF-IRMS method eliminates the problem of special facilities for radioactive specimen storage and disposal, radioactive drug synthesis, special human review procedures, and sponsor liability for exposure of subjects and

229

TABLE 7. Accuracy Validation of 13Cs-levodopa (observed concentration i~g/ml) Expected concentration (l~g/m)l

Serum whole matrix

Serum whole matrix diluted

Unweighted

Weighted 1

Unweighted

Weighted

0.01 0.25 0.5 1.0 2.5 5.0 10.0 20.0 40.0 70.0 100.0

OR2 OR2 OR2 OR2 3.3 5.1 9.3 19.7 39.9 NC3 100.1

OR2 OR2 OR2 OR2 2.6 4.6 9.0 20.0 41.5 NC3 105.4

OR2 0.38 0.58 1.2 2.6 5.0 9.7 19.6 40.0 70.5 99.8

OR2 0.26 0.46 1.1 2.5 5.0 9.7 19.7 40.3 71.2 100.9

1Values based on weighted (1/X 2) least square linear regression. 2OR = Out of range of standard curve. 3NC = Not included in assay runs.

research personnel to radioactivity. This should speed up the performance of MB/MI studies and make MB/MI data available earlier in drug development. The relative cost of an MB/MI study done with stable isotopes versus one done with radioactive methods will vary depending on costs for subjects, special facilities, drug synthesis, and analytic work. In general, the cost for an MB/MI study done with CF-IRMS methods should be comparable to, or lower than, the cost for an MB/MI study done with radioactive methods. An added advantage of the CF-IRMS method is that the specimens can be stored indefinitely without loss of label or special precautions and later analyzed for drug serum concentration versus time relationships using any convenient method. Thus, the subject observations and plasma and specimens obtained in the MB/MI study can also be used to generate a single dose volunteer safety/pharmacokinetic study. This reduces subjects and the time necessary for the single dose volunteer studies required for FDA Phase 1. An economic analysis of SIL methods is contained in Chapter 24. Counting of radioactive label in biological matrices has many biomedical applications in addition to MB/MI studies. It can be predicted that quantitation

230 of stable label in biological matrices by CF-IRMS also will find many biomedical applications.

2.4. Disadvantages of the CF-IRMS Methods CF-IRMS methods have five disadvantages. First, the lower limit of quantitation of CF-IRMS methods (Table 1) may not be adequate to quantitate potent drugs whose concentrations in biologic matrices are in the nanogram/ml range (although use of multiple labels on a molecule improves sensitivity). Second, special synthesis of drug with 13C or lSN label(s) is required. Third, CF-IRMS instruments are relatively scarce at the present time. However, CF-IRMS are available from three commercial suppliers, and CF-IRMS services are available through contract laboratories. Fourth, each HPLC fraction analyzed requires special handling and input to the CF-IRMS. This makes analysis of multiple HPLC fractions labor intensive and time consuming. HPLC-CRIMS may obviate this problem (see below). Fifth, it has not yet been proven that the CF-IRMS method will provide data on new drugs of a quality acceptable to regulatory agencies.

3. CHEMICAL REACTION INTERFACE MASS SPECTROMETRY (CRIMS)

3.1. Technique and History These topics are covered in Chapter 6.

3.2. Assumptions The use of HPLC-CRIMS depends upon several assumptions: (1) reliable and proven equipment is available; (2) the HPLC solvent(s) must carry all of the drug products in a sample (i.e. no drug product is lost in the solvent front or remains behind on the column); (3) the drug product is completely broken down by the microwave-induced plasma; and (4) all of the atoms being monitored react with the reactant gas and are transported to the MS. Note also that all solvents and buffers as well as reactant gas must be volatile. There is preliminary evidence from the owners of the CRIMS technology that these assumptions are true based upon studies using known drugs and metabolites and using solvent systems designed for them (9-12). Extensive and independent validation of HPLC-CRIMS on older drugs has not been

231 performed, and study of new drugs with unknown metabolites by HPLCCRIMS has yet to be reported. 3.3. HPLC-CRIMS Aplications to Mass Balance Studies

By summing the label counted in each HPLC peak, it is possible to estimate the total label present in a sample of urine or other biological material. This technique has been successfully demonstrated in vivo for cortisol (9) and acetaminophen (10). 3.4. HPLC-CRIMS Applications to Metabolite Identification Studies

Rapid, on line, continuous measurement of stable isotope label in HPLC peaks makes CRIMS and extremely powerful technique for detecting labeled peaks in HPLC effluents derived from samples containing unknown metabolites. This technique has been successfully employed to detect the urinary metabolites of cortisol (9) and acetaminophen (10). 3.5. HPLC-CRIMS: Advantages

For both mass balance and metabolite identification studies, HPLC-CRIMS has the following advantages: (1) absence of radiation and associated problems (see above); (2) absence of effects of analyte structure on methodology; (3) no specimen preparation; (4) combination of mass balance and metabolite identification data from one analytic specimen; and (5) quantitative results (912). For metabolite identification, HPLC-CRIMS offers the following additional advantages: (1) rapid and simple detection of all metabolites in a specimen; and (2) preliminary identification data can be obtained using enzymes (to cleave conjugates) and atom specific monitoring to detect rare atoms derived principally from drug (e.g. S, CI, 14C) (9-12). 3.6. Disadvantages of HPLC-CRIMS

Disadvantages of HPLC-CRIMS for MB/MI studies include: (1) difficulty proving assumption of complete recovery listed above; (2) requirement of mass spectrometer, interface, CRIMS and appropriate peripherals; and (3) absence of proof to date that method will provide data on new drugs of a quality acceptable to regulatory agencies.

232

4. STATE OF THE ART Both CF-IRMS and HPLC-CRIMS have shown promise that they can be combined with stable isotope labeling to produce a simple, rapid, general method for performing MB/MI studies. Neither method has been fully validated, applied to study of a new drug, or received official recognition by a regulatory agency. Perhaps the optimal strategy is to employ both CF-IRMS and HPLC-CRIMS for human SIL tracer MB/ML studies. CF-IRMS would be used to count the total label in a specimen for mass balance and as a check for the completeness of collection and quantitation of the specimen when analyzed by HPLC-CRIMS. This strategy takes advantage of the simplicity and accuracy of CF-IRMS for total label counts and the simplicity and accuracy of HPLC-CRIMS for multiple metabolite detection. Furthermore, the most problematic aspect of HPLCCRIMS, verification of complete detection, is obviated.

ACKNOWLEDGEMENT Supported by the United States Department of Veterans Affairs.

REFERENCES 1. G.E. Von Unruh, D.J. Hauber and D.A. Schoeller et al., Biomed. Mass. Spec., 1 (1974) 345. 2. T.R. Browne, G.K. Szabo and A. Ajami et al., J. Clin. Pharmacol., 33 (1993) 246. 3. T.R. Browne, G.K. Szabo and A. Ajami et al., J. Clin. Pharmacol., 33 (1993) 1003. 4. T.R. Browne, G.K. Szabo and A. Ajami, in J. Allen and R. Voges (eds), 5th International Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds (Wiley, Sussex, 1995). 5. G.K. Szabo, T.R. Browne and A. Ajami in J. Allen and R. Voges (eds), 5th International Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds (Wiley, Sussex, 1995). 6. T.R. Browne, G.K. Szabo and A. Ajami, J. Clin. Pharmacol., 34 (1994) 1029. 7. T.R. Browne, G.K. Szabo and A. Ajami, J. Clin. Pharmacol., 35 (1995) 935. 8. T.R. Browne, G.K. Szabo and A. Ajami, J. Clin. Pharmacol., 35 (1995) 935. 9. Y. Teffera, F.P. Abramson and M. McLean et al., J. Chromatogr., 620 (1993) 89. 10. Y. Teffera and F.P. Abramson, Biol. Mass. Spectrom., 23 (1994) 776. 11. Y. Teffera and F.P. Abramson, Abstracts, 42nd ASMS Conference on Mass Spectrometry 42 (1994) 863. 12. F.P. Abramsom, Mass. Spectrom. Rev., 13 (1994) 341.

233

CHAPTER 12

IDENTIFICATION AND CHARACTERIZATION OF DRUG METABOLITES USING STABLE ISOTOPE TECHNIQUES

SUSAN M. BJORGE Bayer Pharmaceutical Division, 400 Morgan Lane, West Haven, CT 06516

1. INTRODUCTION

The use of stable isotopes to identify and elucidate drug metabolite structures has continued to expand in the last 20 years. Excellent reviews by Baillie (1), Baillie and Rettenmeier (2) and Vandenheuvel (3-4) have been written exploring this area. Recently, however, technologies such as Atmospheric Pressure Ionization-Mass Spectrometry (API/MS) and more powerful NMR capabilities have become available, vastly improving the area of metabolite identification and consequently, the use of stable isotopes in this regard. The purpose of this review is to look at recent examples and methods of drug metabolite recognition using stable isotopes.

2. STABLE ISOTOPE TECHNIQUES

2.1. Isotope Cluster Technique

A major use for stable isotopes in the identification and characterization of metabolites is to allow the analyst to readily identify the compound of interest in a complex biologic sample. By using a 1 : 1 mixture of labeled and unlabeled drugs, a characteristic ion cluster is produced that can readily be identified by mass spectrometry. This procedure, known as the "isotope cluster" technique, is widely used to simplify drug metabolite detection. Optimal results

234 are achieved using isotope clusters that possess m/z values of at least two atomic mass units greater than the unlabeled parent. Computer programs have been developed to identify potential drug metabolites by searching the entire chromatographic run, scan by scan, for the characteristic molecular ion cluster resulting from the isotopically enriched dosing solution (5).

2.2. Isotope Peak Shift Technique The position of the label should be in a metabolically stable part of the molecule, as loss of the isotope cluster through metabolism renders the approach useless. Careful selection of the stable label position in the molecule, however, can assist in metabolite structural elucidation. This technique, known as "isotope peak shift" involves the determination of the metabolic site of the molecule based on loss of the stable label. A recent example of this approach is reported by Poon et al. (6) by using a 1:1 mixture tamoxifendo and its deuterated analog, tamoxifen-ds, with the deuterium in the ethyl side chain. Hydoxylation at the ethyl side chain resulted in metabolites with a four mass unit isotope cluster; metabolism elsewhere in the molecule contained a five mass unit increment. Borel and Abbott (7) identified seven clobazam metabolites in rat bile using tetradeuterated starting material. By labeling the phenyl ring, monohydroxy and catechol metabolites were distinguished by the loss of one, or two, deuteriums from the molecule.

2.3. Stable Isotope Labeled Derivatizing Agent A common application of the isotope peak shift technique is to use a stable isotope labeled derivatizing reagent in the identification and characterization of drug metabolites. The most common reagents used are deuterated (d9 or d18) trimethylsilyl (BSA or BSTFA) and acetic anhydride-d6. With this technique, the sample is divided and derivatized with both unlabeled and labeled reagent. The metabolites derivatized with the labeled reagent produce an identifiable shift in the mass of the molecular ion. Thus, an immediate indication of the number of functional groups that have undergone derivatization is obtained. Analysis of the mass spectral fragmentation patterns greatly simplifies metabolite structural elucidation. Leal et al. (5) used this technique to identify metabolites of CGS 15873 present in human urine using BSTFAd18 and acetic anhydride-d6 derivatizing reagents. Stable isotope derivatization was also used in the identification of over 50 cannabidiol metabolites in dog, rat and man (8). Examination of cannabidiol metabolites by GC/MS comparing unlabeled vs. [2H9]TMS derivatives, Harvey et al. (8) found structur-

235 ally informative fragment ions to determine biotransformation products. Fouda et al. (9) also used deuterated TMS derivatives to deduce the structures of CP-68,722 metabolites using GC/MS analysis. 2.4. "Pseudoracemate" Technique

By placing a unique stable isotope label on one enantiomer of a compound and then mixing equimolar amounts of the labeled enantiomer with the other (unlabelled) enantiomer, a "pseudoracemate" is produced. The metabolic fate of each enantiomer can be determined by selective detection of each enantiomer or its metabolites. In studies conducted by Shinohara et al. (10, 11 ), chiral inversion of suprofen was monitored by GC/MS following a dose of (R)-suprofen-d3 and (S)-suprofen-do in the human and rat. Thus, the individual enantiomers were distinguished and evaluated, including the estimation of chiral inversion, in a racemic mixture. Zhang et al. (12) utilized 2H and 180 to show that metabolic chiral inversion of stiripentol in the rat involved cleavage of the CmO bond at the asymmetric center. Chiral inversion of ibuprofen was also explored using deuterated analogs in isolated rat hepatocytes (13). The pseudoracemate technique has also been used to study drug Pharmacokinetics and drug-drug interactions (see Chapters 16 and 18).

3. ISOTOPES USED IN METABOLITE IDENTIFICATION 3.1. Deuterium

Deuterium continues to be the stable isotope most widely used in metabolic research, due to ease of synthesis and cost effectiveness. It should be kept in mind, though, that deuterium has different physical and chemical properties than hydrogen. Metabolic transformations involving the breaking of a Cm2H bond in the rate-determining step can result in a kinetic isotope effect (see Chapter 2). An isotope effect of significant magnitude can be observed for nonlabeled versus deuterium-labeled compound, with the labeled compound being metabolized to a slower extent. Occasionally, the reduced rate of reaction from the deuterium can result in a change in the metabolism of the drug. Ling and Hanzlik (14) found that stepwise deuteration of toluene in the benzylic position resulted in the expected decreased benzylic hydroxylation. The total oxidation of toluene to other products was increased, however, producing an overall inverse isotope effect. Another physical chemical property that should be noted for deuterium is the effect on chromatographic mobility.

236 Because of the differences in the lipophilicity, deuterium-labeled compounds elute slightly ahead of their unlabeled counterparts. This effect has been observed for both GC and HPLC chromatography (2). Other problems that may occur with deuterium labeling include the possible loss of deuterium through proton exchange, such as keto-enol tautomerism resulting from the oxidation of a carbon adjacent to the site of labeling. Metabolic rearrangement, such as the NIH shift (15), may also occur, complicating the interpretation of results.

3.2. 73Carbon and 75Nitrogen Stable isotopes of carbon (~3C) and nitrogen (~SN) are usually preferred to deuterium for metabolic studies, due to the lack of isotope effects and other consequences observed for deuterium labeling. NMR coupled to ~3C labeling also has advantages in metabolite identification studies, which will be discussed in a later section. 13C labeling was used to identify a ribose metabolite of an antiviral agent (LY 217898) in mouse and rat urine (16), and to examine the biotransformation of an organic nitrate (BM 12.1307)in dogs (17). For nitrogen-containing compounds, it may be feasible to incorporate both ~SN and ~3C to obtain the isotope cluster. Dual nitrogen and carbon labeling was used for identification of trimetrexate metabolites in the dog (18) and for human phenobarbital metabolism studies (19).

3.3. Radioactive Isotopes Radioactive carbon (~4C) tracers are still used to a great extent for identification of drug metabolites (3, 4, 9, 16-18, 20-25). Radioactive flow detectors equipped with solid scintillant cells can be incorporated on-line with LC-MS systems to detect peaks of interest (20). If a compound is labeled with enough specific activity, ~4C can also be used as a stable isotope tracer. Approximately 30 mCi/mmol of ~4C at a single carbon produces an ion cluster of about 1:1 intensity (~2C:~4C), two mass units apart. For a compound with a molecular weight of 400 Daltons, a discernible isotope cluster is observed (1:0.67) when the animal is dosed with 50 ~Ci/mg. Similarly, LC peaks containing drug, or metabolite labeled with stable (~3C,~SN) isotopes, can be identified by combining LC with isotope ratio mass spectrometry (see Chapter 6).

237

3.4. 34Sulfur Although 2H and 13C are commonly utilized stable isotopes for drug metabolite identification, the stable isotope, 34S, has only recently been used in this regard. Weidolf and Covey (27) examined omeprazole metabolism using the stable isotope cluster technique. Following administration of 1:1 omeprazole : [34S]omeprazole, over 40 metabolites were identified from partially purified rat urine, using gradient elution LC combined with ionspray API-MS detection. The authors found that abundant molecular ions were found for sulfate conjugates in the positive ion mode, simplifying the metabolic profiling process. In addition, a "metabolite mass profile" was obtained of the entire TIC trace following background-subtraction of the endogenous matrix. Observed in one chromatographic run were omeprazole cleavage products, reduction and oxidation products, as well as glucuronidation and sulphoxidation conjugates.

3.5. Other Isotopes Other isotopes used in metabolic studies include the naturally labeled elements, such as chlorine and bromine, isotopes of oxygen and other isotopes particular to compounds under study. If the drug contains chlorine or bromine, then the natural isotope cluster arising from 35C1:37CI (1:0.33) or 79Br:81Br (1:1) is already in place for metabolism studies. 180 is used extensively in mechanistic studies involving drug metabolism (see Chapter 15), but it is not common for 180 to be used for metabolite identification purposes. Metabolic or chemical exchange of oxygen in drug metabolism studies is always a concern for the investigator, and the feasibility of 2H or 13C incorporation is preferred to that of 180.

4. INSTRUMENTATION-MASS SPECTROMETRY

4.1. Overview The use of mass spectrometry has been critical to stable isotope research in pharmaceutical science. In the past, mass spectral analysis of drug metabolites has been limited to the extraction and/or purification of the metabolites. Analysis was achieved by direct probe electron impact or chemical ionization mass spectrometry, or derivatization to more lipophilic and volatile components so the metabolites could be analyzed by gas chromatography/mass

238 spectrometry (GC-MS). Within the past decade, first thermospray (20), then atmospheric pressure ionization mass spectrometry (API-MS) (27) has opened the field for on-line liquid chromatography/mass spectrometry (LC-MS) analysis of drug components in various biological matrices with only minimum sample preparation and cleanup (see Chapter 4). In addition, the improvements in chemical reaction interface mass spectrometry (CIR-MS) and Isotope Ratio Mass Spectrometry (IR-MS) has further widened the possibilities for mass spectrometry in stable isotope studies (see Chapter 6). 4.2. GC-MS

Most of the literature examples regarding stable isotopes in drug metabolite elucidation have been conducted by GC-MS, due to the long-standing availability of this technique and the relative low cost of the instrumentation (15, 7-11, 13, 17, 21,28-31). A major disadvantage of GC-MS analysis, however, is the necessity of metabolite extraction from biological matrices and derivatization to more volatile components. Derivatization is usually achieved using trimethylsilyl, trifluoroacetamido or pentylfluroroaryl reagents. Glucuronide and sulfate conjugates can be detected by careful derivatization and analysis, or by extracting the nonconjugated metabolites and treating the aqueous fraction with glucuronidase and/or sulfatase. The hydrolyzed metabolites are then detected by extraction and derivatization. Because of the thermal processes involved with GC-MS analysis, care must be taken to determine if "metabolites" are actually artifacts formed during workup or analysis. For all of its disadvantages, GC-MS analysis still plays an important role in drug metabolism studies. Fragments observed during electron impact ionization often are helpful in metabolite structure elucidation and the use of isotopically labeled derivatizing reagents, as discussed previously, aids in metabolite characterization. Kasuya et al. (28) identified 11 rat urinary metabolites of chlorpheniramine using GC-MS and deuterium ion cluster techniques. The biotransformation of cocaine in rat and humans was also investigated using ion cluster techniques with GC-MS analysis (29). Other examples of this technique include identification of seven rat urinary metabolites of methyapyrilene (30), detection of eight urinary metabolites of Inabenfide in the rat (21) and valproic acid metabolic studies in the rat (31). 4.3. L C-MS with Thermospray Interface

Thermospray was the first commercially available mass spectrometry application that allowed for on-line identification of metabolites using an LC system

239 (see Chapter 4). An example of this technique is the identification of bambuterol metabolites from equimolar mixture of deuterium labeled and unlabeled compound in rat microsomes (22). Stable isotope methods were also used to characterize urinary metabolites of trimetrexate in dogs (18). Thermospray mass spectrometry, however, has a limited mass range; components with molecular weights less than 150 Daltons are difficult to distinguish from solvent ions, and compounds with molecular weights greater than 700 Daltons usually decompose because of thermal processes involved in ionization. Because of the limitations of this technique, it has fallen into disfavor since the introduction and common use of API interfaces in the late 1980s. 4.4. LC-MS with Fast Atom Bombardment Interface

Fast atom bombardment mass spectrometry (FAB-MS) is a useful technique for identification of polar and ionic compounds such as peptides, carbohydrates and nucleosides (see Chapter 4). Because of the low LC flow rate associated with this method, metabolites are usually isolated and purified before analysis. FAB-MS has been used for the identification and structural determination of several glutathione conjugates of deuterated 1,2-dibrome3-chloropropane (DBCP) (25) and N-methylformamide (NMF) (23). In the latter study, detection of glutathoine conjugates by the isotope cluster technique was expanded to evaluate constant neutral loss and daughter ion scanning tandem mass spectrometry (MS-MS). It was concluded that the combined use of stable isotopes with MS/MS techniques represented a powerful approach for the analysis of glutathione adducts. 4.5. L C-MS with Atmospheric Pressure Ionization Interface

Atmospheric pressure ionization (API-MS)is rapidly becoming the mass spectral ionization method of choice for metabolic studies (see Chapter 4). API ionization methods commercially available include electrospray (or ionspray) and heated nebulizer interfaces. With the heated nebulizer interface, the solvent (and compound of interest) is exposed to temperatures as high as 500~ which may cause thermal degradation of the sample. Consequently, electrospray analysis is the preferred ionization mode for metabolism studies. Accommodation of HPLC flow rates up to 1 ml/min is now available for API techniques, allowing for simultaneous chromatography with mass spectral detection. An important advantage of API-MS is the "soft" ionization method involved in analysis, resulting in either the protonated molecular ion, salts of the molecular ion, or multiply-charged molecular ions for compounds with

240 more than one ionization site. Ions of interest can be monitored by MS-MS analysis to assist in structure elucidation. As discussed previously, API-MS was used to identify over 40 [34S]omeprazaole metabolites in one chromatographic run, proving the utility of this technique (27). In a study conducted by Lanting et al. (32), deuterated dextrorphan and levorphanol metabolites from rat liver perfusion experiments were identified and characterized by APIMS-MS. This method was specifically chosen because of its soft ionization, absence of thermal degradation processes and ability to analyze samples without sample cleanup and possible loss of metabolites.

5.

INSTRUMENTATION

-

NMR

NMR analysis is highly complementary to mass spectrometry for metabolite structure elucidation (see Chapter 10). Typically, NMR analysis is required to pinpoint the exact position and stereochemistry of hydroxylation. With the improvement of NMR technology, such as Fourier transform analysis, various pulse techniques and higher magnetic fields, NMR analysis can be performed with less than 50 ~g purified metabolite. When combined with stable isotope labeling studies, 13C-NMR is quite effective in the identification and characterization of purified metabolites following administration of 13C-labelled compounds. In a study by Blanz et al. (33), 13C-labeled mitoxantrone metabolites in minipig urine were characterized by NMR analysis. Structural identity was confirmed in these metabolites by observing the ~3C chemical shift variations at the labeled positions. ~3C-NMR was also used to characterize the covalent binding of Br~3CCI3 to heme during reductive metabolism (34). NMR spectroscopy has been used for determination and characterization of metabolites in biological matrices, such as urine and bile, without the need for prior extraction and chromatographic separations. Although the sensitivity of NMR is a severe limitation (metabolites must be present in millimolar or greater concentrations), compound enrichment with ~3C can enhance metabolite detection sensitivity up to l O0-fold (35). According to Unkefer (35), several important factors should be considered when labeling compounds for in vitro or in vivo NMR experiments. (i) The labeled carbon must be located in a position close enough to the metabolic transformation position so the NMR signals can be resolved. (ii)Ideally, a chemical shift difference should be observed for all metabolites being investigated. (iii) The labeled carbon should be protonated, allowing for a full nuclear Overhauser effect causing an increase in signal sensitivity. (iv) Signals from the labeled metabolites should appear in a region that is free of background resonances. (v) The labeled

241 carbons for all metabolites must have similar spin-lattice relaxation rates. (vi) Finally, the synthetic aspects of the specifically labeled 13C should be considered. Although there are several limitations to using 13C-NMR for metabolite identification in biological matrices, several papers have been published recently using this technique. Akira et al. (36) conducted an NMR study examining the metabolism of benzoic acid to hippuric acid by monitoring nonextracted rat urine. The benzoic acid was selectively labeled to enhance detection based on nuclear Overhauser enhancement and short spin-lattice relaxation time. The lower limit of detection was about 40 nmol in this study. Sumner et al. (37) reported the use of ~3C NMR to detect and quantitate acrylamide metabolites directly in the urine of rats and mice following administration of [1,2,3-~3C]acrylamide. Six metabolites were identified without extraction, chromatography or mass spectrometry. Other examples for direct NMR analysis without sample cleanup include metabolite identification following incubation of N,N-diethyl-~3C-benzamide with rat liver microsomes (38) and identification of rat urinary metabolites of [1,2,3-~3C3]acrylic acid and [1,2,313C3]propionic acid (24).

6. CONCLUSION

Stable isotope labeling of compounds under investigation provides a unique marker for drug metabolite identification studies. Recent advances in mass spectrometry and NMR technology have enhanced the ability to detect and characterize metabolites quickly, with minimal sample preparation before analysis. Combining this new technology with carefully designed stable isotope studies represents a powerful method for metabolite structural elucidation. As more understanding of drug metabolism is expected prior to drug approval, and greater understanding leads to better pharmaceutical compounds, the use of stable isotopes in drug metabolism research should continue to grow.

REFERENCES

1. 2. 3. 4. 5.

T. Baillie, Pharmacol. Rev., 33 (1981) 81. T. Baillie and A. Rettenmeier, J. Clin. Pharmacol., 26 (1986) 481. W. Vandenheuvel, J. Clin. Pharmacol., 26 (1986) 427. W. Vandenheuvel, Xenobiotica, 17 (1987) 397. M. Leal, M. Hayes and M. Powell, Biopharm. Drug Disp., 13 (1992) 617.

242 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

G. Poon, B. Walter and P. Lcnning et al., Drug Met. Disp., 23 (1995) 377. A. Borel and F. Abbott, Drug Met. Disp., 21 (1993) 415. D. Harvey, E. Samara and R. Mechoulam, J. Chromatogr., 562 (1991) 299. H. Fouda, J. Lukaszeqicz and D. Clark et al., Xenobiotica, 21 (1991) 925. Y. Shinohara, H. Magara and S. Baba, J. Pharm. Sci., 80 (1991) 1075. Y. Shinohara, K. Nagao and N. Akutsu et al., J. Pharm. Sci., 83 (1994). K. Zhang, C. Tang and M. Rashed et al., Drug Met. Disp., 22 (1994) 544. S. Sanins, W. Adams and D. Kaiser et al., Drug Met. Disp., 18 (1990) 527. K. Ling and R. Hanzlik, Biochem. Biophys. Res. Comm., 160 (1989) 844. G. Guroff, J. Daly and D. Jerina et al., Science, 157 (1967) 1524. W. Elhardt, W. Wheeler and A. Breau et al., Drug Met. Disp., 21 (1993) 162. C. Zell, R. Neidlein and K. Strein, Arzneim.-Forsch./Drug Res., 44 (1994) 1021. B. Wong, T. Woolf and T. Chang et al., Drug Met. Disp., 18 (1990) 980. T. Browne, G. Szabo and A. Ajami et al., J. Clin. Pharmacol., 33 (1993) 246. S. Bjorge and T. Woolf, LC/GC, 9 (1991) 780. H. Kinoshita, Y. Tohira and H. Sugiyama et al., Xenobiotica, 8 (1987) 925. C. Lindberg, C. Roos and A. Tunek et al., Drug Met. Disp., 17 (1989) 311. T. Baillie, P. Pearson and M. Rashed et al., J. Pharm. Biomed. Anal., 7 (1989) 1351. S. Winter, G. Weber and P. Gooley et al., Drug Met. Disp., 20 (1992) 665. P. Pearson, E. Soderlund and E. Dybing et al., Biochem., 29 (1990) 4971. L. Weiflof and T. Covey, Rapid Comm. Mass Spectrom., 6 (1992) 192. F. Kasuya, K. Igarashi and M. Fukui, Xenobiotica, 21 (1991) 97. S. Jindal and T. Lutz, J. Pharm. Sci., 78 (1989)1009. R. Kammerer, D. Schmitz and M. Lampe et al., Xenobiotica, 18 (1988) 869. A. Rettenmeier, W. Gordon and H. Barnes et al., Xenobiotica, 17 (1987) 1147. A. Lanting, A. Bruins and B. Drenth et al., Biol. Mass Spectrom., 22 (1993) 226. J. Blanz, K. Mewes and G. Ehninger et al., Cancer Research, 51 (1991) 3427. Y. Osawa, R. Highet and A. Bax et al., J. Biol. Chem., 266 (1991) 3208. C. Unkefer, J. Clin. Pharmacol., 26 (1986) 427. K. Akira, N. Takagi and S. Takeo et al., Anal. Biochem., 210 (1993) 86. S. Sumner, J. MacNeela and T. Fennel, Chem. Res. Toxicol., 5 (1992) 81. W. Taylor, T. Hall and D. Vedres, Drug Met. Disp., 21 (1993) 133.

243

CHAPTER 13

ABSORPTION AND BIOEQUIVALENCE

MARK L. POWELL NOVARTUS, 59 Route 10, E. Hanover, New Jersey 07936

1. INTRODUCTION

The earliest use of stable isotopes as tracers in a biological study is generally recognized as 1934 (1). Despite this early use, the lack of sensitive and inexpensive methods of identification and quantification of stable isotopes led to the rapid proliferation of radioisotopes in biomedical research. Liquid scintillation counting, although lacking in specificity since it detects emitted radiation independent of structural characteristics, is both inexpensive and very sensitive. For most radioisotopes, sufficient specific activity can be synthetically incorporated into a drug candidate to allow picogram detection ranges. These positive attributes of radioisotopes helped to shape the early preclinical drug development field, and still remain with us today. Standard preclinical tissue distribution studies are dependent on radioisotopes and the analytical methodology associated with their use. The opposite is increasingly true of clinical studies, however. The potential side effects of radiation, particularly in pregnant women and children, has significantly reduced their use in human drug development studies. The further likelihood is that the future will see a continuing decline in radioisotope exposure to humans. The use of drugs labeled with stable isotopes in clinical biomedical research has rapidly increased over the last 15 years. This growth has directly paralleled the expansion in GC-MS instrumentation and capabilities as well as the growing use of LC-MS and LC-MS-MS. Once the exclusive province of university research centers, they have now found their way into pharmaceutical companies' drug development laboratories. Along with the expanding power of computers, stable isotope applications continue to be elevated to an increasingly practical level. The instrumental requirements still form a significant barrier to routine every day use, but the days of industrial mass spectrometry deficiencies are a thing of the past.

244 This chapter will focus on the use of stable isotopes to investigate drug absorption and elucidate bioavailability and bioequivalence. Throughout the chapter, emphasis will be centered on the perspective of the pharmaceutical industry, which reflects the majority of my experience. Some familiarity with the pharmaceutical drug development process, such as the phases of clinical testing (I to III) leading to a new drug application is also assumed.

2. STABLE ISOTOPE UTILIZATION

The decision to utilize a stable isotope labeled compound in a drug development study is a difficult one to arrive at, and many factors have to be taken into consideration. The synthetic effort required to label a drug with a stable isotope is not a trivial one. Because pharmaceutical companies do not have dedicated stable isotope synthetic groups, the responsibility generally falls on the radiochemistry group who, by the time a drug has moved into Phase I of clinical testing, has usually not been involved with the compound for a year or more. In planning the synthesis, several criteria need to be considered: (1) the site of incorporation needs to be metabolically stable so that the stable isotope label will be retained; (2) more than one isotopically labeled atom should be incorporated in the drug, in order to minimize the deconvolutions required by the significant signal at M + 1 due to the naturally occurring 13C abundance; and (3) the synthesis needs to be as simple as possible and produce a reasonable quantity of pure drug. With this in mind, deuterium incorporation has been utilized more than that of lSN, 13C or 180, the other leading candidates for organic molecules. Deuterium labeling is done frequently by simple exchange reactions or catalytic hydrogenation under a deuterium atmosphere. Incorporation of 180 is also achievable via exchange, but suffers from the problem of losing the label in an aqueous environment. Incorporation of ~SN and ~3C requires specific structural characteristics and the availability of labeled intermediate chemicals for synthesis. Nevertheless, all three of these stable isotope labels are used, although much less than deuterium. See Chapter 4 for further discussion of synthetic considerations.

3. PHARMACOKINETIC APPLICATIONS

The many and varied uses of stable isotopes in clinical pharmacokinetic research have been addressed in several review articles (2-7). Although the

245 pharmacokinetic applications of stable isotopes fall into many different categories, this section will focus only on bioavailability/bioequivalency studies and stereoselective pharmacokinetic studies.

3.1. Bioavailability Studies Bioavailability investigations are generally concerned with defining either absolute or relative bioavailability. Absolute bioavailability studies use an intravenous dose as the reference against which the test dosage form (e.g. oral, transdermal, rectal, buccal, etc.) is compared. Assuming that solubility characteristics permit the development of an intravenous dosage form, an absolute bioavailability study is normally done sometime during Phase I1-111 of clinical development. In relative bioavailability studies, the test formulation is compared with a nonintravenous dosage form (e.g. commercial tablet, oral solution, etc.). Relative bioavailability studies are performed in support of formulation development efforts throughout clinical development, and beyond. During early clinical development, these studies often take the form of pilot investigations to confirm acceptable performance of a developmental dosage form for further extensive clinical use. During Phase III of development, definitive bioavailability studies are performed with the final formulation(s) intended for marketing. Definitive bioavailability/bioequivalency studies require a fairly large number of subjects to achieve the necessary statistical power to confirm equivalence. Although the actual number of subjects required for any given study is a function of the intrinsic properties of the test drug and formulation, experience suggests that 18-24 subjects are minimally required. The greater the inter- and intra-subject variability, however, the larger the number of subjects that will be required to demonstrate bioequivalency between two dosage forms, for example. Because bioavailability/bioequivalency studies are normally conducted using a standard "cross-over" design with two or more drug treatment phases, in addition to the large number of subjects involved, the clinical durations of the studies can be long. The validity of cross-over study results is based on the assumption that a drug's clearance remains constant as a function of time. For most drugs this is a valid assumption. For drugs that undergo extensive first pass metabolism, however, this assumption may not be valid. An excellent article has been published detailing the relationships between intra-individual kinetic parameters and various metabolic influences for many drugs (8). For drugs that are extensively metabolized, systemic clearance can be strongly influenced by factors such as posture, food ingestion, blood flow changes to eliminating organs, etc. Where these factors cannot be controlled, or exactly replicated, the assumption of

246 constant clearance may no longer be valid and a simple cross-over design is neither appropriate nor practical. In these cases, the use of stable isotopes provides a convenient means for conducting bioavailability or bioequivalency studies.

3.1.1. Absolute bioavailability The absolute bioavailability of a given drug formulation can be determined in a study by the simultaneous administration of an intravenous stable isotope labeled drug solution and the unlabeled comparative formulation. One of the earliest reported studies of this nature utilized N-acetylprocainamide (NAPA) (9). This drug exhibits considerable variability in pharmacokinetic parameters between and within individuals, which prompted the use of a stable isotope approach. In this study, 13C-NAPA was given intravenously at the same time as an equivalent dose of unlabeled NAPA, in capsule form. The simultaneous GC-MS determination of labeled and unlabeled NAPA plasma levels resulted in a calculated absolute bioavailability of 87.2 percent with a range of 79.2 to 93.0 percent for the NAPA capsules, results that were not otherwise achievable using conventional techniques. A similar study was utilized to determine the absolute bioavailability of methadone (10). Eight patients received 20 mg of D3-methadone, intravenously, at the same time as an equivalent dose of unlabeled methadone tablets (2 x 10 mg). Simultaneous determination of methadone concentrations from both formulations resulted in a calculated mean absolute bioavailability of 79 percent (range 41 to 99 percent). This calculated bioavailability was significantly higher than the previously reported 45 percent which was based on a conventional cross-over study design (11). The differences in these estimates are of considerable significance when switching from oral to parenteral methadone therapy.

3.1.2. Relative bioavailability The relative bioavailability of the widely prescribed antiarrhythmic drug, verapamil, has been examined using stable isotope techniques (12, 13). Although verapamil is nearly completely absorbed, its bioavailability is very low due to extensive first pass metabolism. In addition, large differences in the extent of first pass metabolism have been observed between different individuals. When the relative bioavailability of the tablet formulation was investigated in a pilot study using a conventional cross-over design (solution versus a commercial tablet), different results were obtained for the same subjects

247 during different study periods, leading to the conclusion that a statistically significant cross-over study for this drug could not be undertaken without using a prohibitively large number of subjects. The use of stable isotope methodology, however, provided a practical means of definitively determining relative bioavailability. Six subjects received commercial 80-mg verapamil tablets (Isoptin | 80) along with an equal dose of D3-verapamil solution. Serially collected blood samples were analyzed for both labeled and unlabeled verapamil. The relative bioavailability of the tablet averaged 108 percent with a 95 percent confidence interval of 89.1 to 127.1 percent. The six subjects used in the stable isotope study were the same as those used in the pilot conventional cross-over study, emphasizing the significant advantages of this approach. The area under the curve (AUC) data from the stable isotope study for the solution was highly variable and deviated by 67 to 268 percent from the AUC in the conventional study. With each subject serving as their own control, however, acceptable relative bioavailability data was generated from only 6 subjects in a single treatment phase. The same technique has been applied, successfully, to a relative bioavailability study with a sustained release verapamil tablet (14). The relative bioavailability of maprotiline tablets has been determined using a stable isotope labeled maprotiline solution with a limited number of subjects (15). Unlike verapamil, maprotiline averages approximately 15 percent first pass metabolism. In this study, the relative bioavailability of the tablet ranged from 94.5 to 108.3 percent. Although six subjects were used in the study, statistical calculations suggested that only three were actually required to demonstrate with at least 95 percent confidence that the AUC for the tablet formulation did not differ by more than 10 percent from that for the solution. The relative bioavailability of two different imipramine tablet formulations has also been investigated using stable isotope methodology, placing particular emphasis on the statistical power of the bioavailability test (16). The two tablet formulations were given in different treatment phases, but each subject simultaneously received an equivalent dose of D2-imipramine solution in each treatment phase. This study design allowed for a comparison of the relative bioavailability parameters calculated with the conventional design to be compared with the same parameters calculated from the stable isotope technique. The two formulations were shown to be bioequivalent by both methods. However, the conventional cross-over study design would ultimately have required 36 subjects to demonstrate, with at least 95 percent confidence, that the AUCs of the two tablet formulations did not differ by more than 15 percent. The stable isotope approach could meet this same criteria with only 4 subjects. Numerous other examples of bioavailability studies using stable

248 isotopes have been reported for many drugs including trazodone (17), phenytoin (18, 19), metaproterenol (20), 17e-methyltestosterone (21), mefloquine (22), terodiline (23), methoxsalen (24), benoxaprofen (25) and theophylline (26). Stable isotopes have proven to be of significant importance at Ciba-Geigy in investigating the absorption and pharmacokinetics of two drugs which are typically delivered transdermally. The absorption and bioavailability of both nicotine and nitroglycerin have been studied using this methodological approach. Representative studies with each drug will be described in sufficient detail so the complexities of study design and implementation can be appreciated. For both drugs, the results using stable isotope methodologies could not have been easily obtained with conventional cross-over study designs.

4. NICOTINE

Transdermal delivery systems containing nicotine are readily available as an adjunct to smoking cessation therapy. The relationship between drug release from the delivery system and blood levels is well characterized and the need for maintaining continual blood levels of nicotine is considered necessary for the desired therapeutic effects (27-30). The determination of the absolute bioavailability of nicotine delivered transdermally, however, has been a significant challenge. The amount of drug delivered across skin can be estimated using different approaches. One simple approach is to calculate the difference between the amount of drug remaining in a used transdermal system and the amount originally in the system, and to assume that this amount represents the dose of drug which passed through the skin. These types of calculations, based on residual systems however, don't take into consideration the possibility of drug left on the skin surface, skin metabolism, problems with poor system adhesion, or other general types of system losses. The more conventional approach of comparing blood levels of nicotine delivered transdermally versus intravenously, in a cross-over study suffers from the subject treatment periods being separated by time, as previously described. An alternative approach to determining absolute bioavailability is to co-administer stable isotope labeled nicotine intravenously, along with unlabeled nicotine administered transdermally. Benowitz et al. (31) administered nicotine to 14 healthy adult male smokers by the intravenous and transdermal route, simultaneously, to determine the absolute bioavailability and absorption kinetics of a new nicotine patch. The

249 nicotine used for intravenous administration had been labeled with two deuterium atoms (D2-nicotine). The transdermal nicotine systems (experimental systems under clinical development at Ciba-Geigy) were applied on the lower abdomen and kept in place for 24 h, while an intravenous infusion of D2-nicotine was initiated. The transdermal patches were 30cm 2 and contained 52.5mg of nicotine. The intravenous dose was infused into the nondominant arm for 24 h (total infused dose approximately 21 mg). The intravenous dosing regimen was designed to approximate that of the transdermal delivery system. Therefore, any changes in systemic clearance with time on the days of the study, would identically effect the kinetics of the drug after both transdermal and intravenous delivery. Blood samples were taken at frequent intervals for 24 h post transdermal system application and for an additional 8 h after the patch was removed. Plasma samples were analyzed for both labeled and unlabeled nicotine and

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25O the metabolite cotinine using GC/MS. By measuring the amount of nicotine remaining in the unused transdermal systems, the absolute bioavailability of the transdermal system could be calculated assuming that the amount of nicotine released from the system is the actual dose available for absorption into the systemic circulation. Figure 1 shows the mean (SD) plasma nicotine concentrations after simultaneous administration of both intravenous and transdermal nicotine. The results of this study showed that an average of approximately 44 percent of the nicotine originally in the transdermal patches was released during 24 h, and that the absolute bioavailability, based on the amount of nicotine released from the patch, averaged 82 percent. The study provided the first determinations of the absolute bioavailability of transdermal nicotine and demonstrated that good data could be obtained from a small amount of subjects due to the stable isotope approach.

5. NITROGLYCERIN

Another drug whose intrinsic properties make it particularly appropriate for a stable isotope study is nitroglycerin. Ciba-Geigy is evaluating new transdermal dosage forms of the widely used anti-anginal drug, nitroglycerin. It has been our experience that the intrinsic variability in historically generated nitroglycerin data has been so high that we have never been able to generate acceptable statistical power in any of our bioavailability/bioequivalency studies with nitroglycerin patches. The extreme nature of nitroglycerin's kinetic behavior results in major fluctuations in plasma levels associated with changes in posture, physical activity, ingestion of food, etc. which cannot be controlled in conventional cross-over study designs. Additionally, the designs of these studies have been such that the variability in plasma levels could not be attributed specifically to changes in systemic clearance as opposed to changes in drug delivery, with time. The use of a stable isotope approach had the potential to permit the demonstration of bioequivalence between test and commercial formulations with sufficient statistical power in a small number of subjects.

5.1. Pilot Bioavailability/Bioequivalency Study A pilot study was designed to compare the in vivo performance of two new developmental transdermal nitroglycerin systems with a commercially available system (Transderm-Nitro 10| as a reference. Stable isotope labeled

251 nitroglycerin was synthesized with an 15N label. The study design allowed for lSN-nitroglycerin to be infused at a constant and known rate to provide a point-by-point estimate of systemic clearance. The developmental or reference transdermal systems containing unlabeled nitroglycerin were simultaneously applied to a selected site on the body and the delivery rate was calculated from the plasma level data for unlabeled drug together with the instantaneous clearance obtained from the stable isotope infusion data. The study illustrates the significant advantages offered by the use of stable isotopes in the biopharmaceutic evaluation of transdermal formulations of nitroglycerin. This pilot study utilized six healthy male volunteers and followed a threeway randomized cross-over design. Each phase consisted of the application of one of two new test transdermal systems or the commercial system, and a simultaneous intravenous infusion of lSN-nitroglycerin. The stable isotope solution was infused for a duration of 12 h, while the transdermal systems were applied for a period of 24 h. Blood samples were drawn at intervals over 26 h to characterize the respective plasma profiles. A representative plasma level versus time curve for a typical subject is shown graphically in Figure 2. The bottom curve represents the lSN-nitroglycerin infusion, while the top curve is from one of the transdermal nitroglycerin test systems. The transdermal plasma profile exhibited significant fluctuations in levels which are typical of those observed in previous studies. However, similar fluctuations can be seen for the intravenous infusion profile despite the high level of control on the delivery rate. The strikingly close agreement in the two profiles (fluctuations) provides a very clear indication that the fluctuations observed for the transdermal system reflect variations in systemic clearance with time, rather than changes in the delivery profile. The benefits of the stable isotope approach are most clearly illustrated by the data presented in Figure 2. During the 12 h period of simultaneous intravenous infusion and transdermal application, the fluctuations observed in the transdermal profile can be accounted for. In the period after the infusion was stopped (e.g. after 12 h) we can only surmise that the fluctuations were related to clearance changes, but have no direct proof for this observation. By considering the ratio of the intravenous and transdermal plasma levels, it is possible to obtain a point-by-point estimate of the input rate from the transdermal system, allowing for a detailed analysis of its performance as a function of time. In addition, the absolute bioavailability of nitroglycerin delivered by the transdermal route can be determined from the ratio of the AUC values and the relative amounts delivered by the two routes. The cross-over design of the study with stable isotope intravenous infusion,

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allowed for a comparison of the data from the three transdermal systems to be made with and without correcting for the random fluctuations due to clearance changes. The variability in overall mean transdermal delivery rates was reduced by 5-fold when the plasma levels were "corrected" using the associated infusion data. For a drug like nitroglycerin, with which it is nearly impossible to achieve acceptable statistical power in a bioavailability/bioequivalency study due to the number of subjects which would be required, this technique is particularly attractive. Based on the results of this pilot study, a definitive study of similar design was employed to compare the bioavailability/bioequivalency of three commercially available transdermal systems: Transderm-Nitro | Nitrodisc | and Nitro-Dur II| The overall objective of this study was to achieve statistically significant results in a definitive bioavailability/bioequivalency study using a practical number of subjects.

5.2. Definitive Bioavailability/Bioequivalency Study Eighteen healthy adult male subjects were each given a single 14-h application of one of the three comparative nitroglycerin formulations in a three-way

253 cross-over study design. A 14-h intravenous infusion of ~SN-nitroglycerin was administered simultaneously to correct for changes in systemic clearance. An initial 2-day "wash-in" tolerance phase was used for each subject to decrease the dropout rate of subjects due to the severity of the common nitrate headaches which were seen in the previously described pilot study. Two of the three commercially available systems were 20 cm 2 in size with a delivery rate of 0.4 mg/hr (Transderm-Nitro | and Nitro-Dur I1| The third system was 16cm 2 in size with a delivery rate of 0.4mg/hr (Nitrodisc| ~5N-labeled nitroglycerin (98 percent isotope purity) solution in ethanol at a concentration of 13 mg/ml was diluted to 7.1 mg/ml and was infused at a rate of 35 ml/hr (0.25 mg/hr) over 14 h. Nitroglycerin is rapidly metabolized to it's pharmacologically active metabolites, 1,2-glyceryl dinitrate and 1,3-glyceryl dinitrate (32), which are known to achieve higher concentrations in plasma and are cleared slower than nitroglycerin (33). Therefore, we had considerable interest in also quantitating metabolite levels as well as parent drug for bioavailability/bioequivalency determinations and comparisons. Blood samples were collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 14 (before system removal and infusion termination), 14.08, 14.17, 14.5, 15 and 16 h after transdermal system application. Plasma samples were analyzed for nitroglycerin, 1,2-glyceryl dinitrate, 1,3-glyceryl dinitrate and their stable isotope analogs using GC/MS. As seen in the pilot study, plasma level time curves for nitroglycerin, although showing considerable fluctuations, were directly paralleled by those of the intravenously administered stable isotope labeled drug. This was true for both nitroglycerin and its two metabolites, as shown in a representative subject (Figure 3). The calculated AUC values for nitroglycerin for each system exhibited a typically large range (1621 21,500 pg. hr/ml). Without any stable isotope corrections, a statistically significant demonstration of bioequivalence was estimated from this data to have required between 80 to 115 total subjects. After the stable isotope corrections for clearance changes, however, the 90 percent confidence intervals for AUC for Nitro-Dur II| and Nitrodisc | compared to Transderm-Nitro | were between 85 and 116 percent for nitroglycerin. The corresponding data for the 1,2-glyceryl dinitrate metabolite was 86 to 103 percent and for the 1,3-glyceryl dinitrate metabolite was 88 to 110 percent. The data in this study demonstrated a unique application of stable isotopes to demonstrate bioequivalency with nitroglycerin transdermal systems using only 18 subjects. Additionally, nitroglycerin metabolite levels were also used successfully to demonstrate bioequivalency. To our knowledge, this is the first statistically significant demonstration of bioequivalency for commercially available transdermal nitroglycerin systems, and it has the potential to influence future regulatory requirements.

254

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255 6. STEREOSELECTIVE PHARMACOKINETIC STUDIES

In addition to bioavailability/bioequivalency applications, stable isotopes have also been used to investigate stereospecific absorption and pharmacokinetics. More than 50 percent of all therapeutic agents available by prescription are marketed as racemic mixtures. Economic and synthetic considerations have historically limited the development of a single enantiomer in preference to a racemic mixture, despite the fact that the desired pharmacological activity may predominantly reside in one enantiomeric form. Additionally, the stereospecific nature of active binding site interactions can result in stereoselective metabolism/elimination such that the pharmacokinetic parameters which are routinely derived from a racemic mixture may not reflect those of the pharmacologically active component. If the racemate is already on the market, however, individual enantiomeric information, while of scientific interest, has limited clinical significance since the safety and tolerability profile has been established for the racemic mixture rather than for one of the enantiomers. However, in the current international regulatory environment where progressively greater emphasis is being placed on the development of the enantiomer with the desired pharmacological activity, knowledge of the absorption and disposition of individual enantiomers following dosing with racemic mixtures is becoming increasingly important. It appears that a valid therapeutic rationale supported by detailed enantiomeric information (pharmacological, toxicological, metabolism, pharmacokinetic, etc.) will become a prerequisite for obtaining approval from any regulatory agency to market a racemic mixture in the future. There is, therefore, a rapidly expanding interest in stereospecific analytical techniques as well as the alternative use of "pseudo-racemic" mixtures in pharmacokinetic studies. The use of these mixtures, in which one enantiomer has been labeled with a stable isotope, allows the absorption and disposition of both enantiomers to be determined when both are simultaneously administered. They also encompass the effects of one enantiomer on the disposition of the other or on its own kinetic parameters, as well as the potential for enantiomeric interconversion or racemization. Their current applications have been primarily confined to research studies on marketed drugs. Pseudo-racemic mixtures have been used in the investigation of a number of drugs, including propranolol. A pseudo-racemic mixture of propranolol was administered to dogs in a study designed to look at stereoselective changes in bioavailability during chronic dosing (34). The R-(+)-propranolol enantiomer was labeled with two deuterium atoms, while the S-(-)-enantiomer was labeled with six deuterium atoms. Data following single dose

256 administration of this pseudo-racemic mixture demonstrated a significant difference in the enantiomeric disposition of propranolol in the dog, with a 2.8-fold higher oral bioavailability for R-(+)- compared with S-(-)-propranolol. On chronic dosing, however, the oral bioavailability for S-(-)-propranolol increased by 167 percent, while that for R-(+)- was essentially unchanged. Stereoselective clearance and distribution of propranolol has also been demonstrated in man following intravenous administration of a pseudo-racemic mixture consisting of R-(+)-D6-propranolol and unlabeled S-(-)-propranolol (35). Plasma levels of the S-(-)-enantiomer were shown to be higher than those for its R-(+)- counterpart. Similar techniques have been utilized to investigate several other drugs including verapamil (36) and methadone (37). The importance of evaluating the kinetics of one enantiomer in the presence of the other is clearly illustrated by the data obtained for propoxyphene (38), where significantly higher plasma levels of the d-form were obtained following administration of a pseudo-racemic mixture as compared to a dose of the pure enantiomer. The implication of this result is that the pharmacokinetic behavior of the d-form is influenced or modified by the presence of the/form. Although pseudo-racemic mixtures may continue to be used in the future as enantiomeric studies become a standard part of racemic mixture drug development, general stereospecific analytical techniques will eventually replace them.

7. DRUGS WITH NONLINEAR PHARMACOKINETICS

For drugs with nonlinear pharmacokinetic properties, the AUC produced by a given test dose of drug will vary directly with the background plasma drug concentration (39). In cross-over studies performed at steady-state plasma concentrations, differences in background plasma drug concentrations can result in differences in measured AUC values for products with identical bioavailability. This problem can be eliminated by the simultaneous administration of two stable isotope labeled forms of the drug (19).

8. PRO-DRUGS

Pro-drug formulations typically consist of a parent drug which is relatively insoluble in water (e.g. phenytoin) with a moiety attached which renders it more soluble (e.g. phosphate) (19). After gaining access to the circulation, the attached moiety is cleft off (e.g. phosphate by phosphatase), liberating the

257 parent drug. Because of differences in water and lipid solubility, direct renal excretion of pro-drug while present in the plasma may be different from (usually greater than) direct renal excretion of parent drug. Thus, if equimolar amounts of pro-drug and parent are simultaneously infused intravenously, the AUC values for parent drug derived from the pro-drug may be lower than those obtained from administration of the parent drug alone (19). The "missing" portion of AUC for parent drug derived from pro-drug can be accounted for by placing unique stable isotope labels on parent drug derived from pro-drug and unchanged parent drug and measurement of urinary excretion of the pro-drug, the parent drug derived from pro-drug and the unchanged parent drug derived from parent drug.

9. CONCLUSIONS The use of stable isotopes in the drug development process has expanded steadily in recent years, but the full potential of this approach has yet to be realized. The applications which have been described in absorption and bioavailability/bioequivalency studies have been exciting and innovative. However, the most significant barrier to broader applications of stable isotopes in drug development continues to be the timely availability of appropriately labeled compounds. Most drug companies have not made stable isotope synthesis a regular part of their synthetic effort. More extensive applications of the techniques described in this chapter, however, are likely to change this situation. Pharmacokinetic applications, although more specialized, are also beginning to be more widely used since they allow simultaneous administration of a drug by different routes, thus avoiding traditional cross-over study designs. In addition to savings of both time and money, the advantages for drugs such as nitroglycerin, whose clearance is not constant with time, are enormous. Pseudo-racemic mixture studies have also found their way into drug development programs for some drugs. The historical interest in enantiomeric pharmacokinetics will probably diminish, however, with the continuing trend towards developing active enantiomers in preference to racemic mixtures. Pseudo-racemic mixture studies may still play a small role in the development of enantiomers, but will probably be replaced by general stereospecific analytical techniques. The use of radioisotopes in humans, although declining, will continue to be a useful tool, and along with the use of stable isotopes will provide

258 considerable flexibility in the design of developmental studies. Given the increasing a m o u n t of resources being devoted to stable isotope applications, the future will undoubtedly see additional innovative uses in the pharmaceutical industry.

REFERENCES 1. G. Heresy and E. Hofer, Klin. Wochenschr., 13 (1934) 1524. Cited in H. Craig, S.L. Miller and G.J. Wasserburg (eds), Isotopic and Cosmic Chemistry, (North-Holland Publishing Company, Amsterdam, 1964). 2. T.A. Baillie, Pharmacol. Rev., 33 (1981) 81. 3. D.R. Knapp and T.E. Gaffney, Clin. Pharmacol. Ther., 13 (1972) 307. 4. P.J. Murphy and H.R. Sullivan, Ann. Rev. Pharmacol. Toxicol., 20 (1980) 609. 5. M. Eichelbaum, G.E. von Unruh and A. Somogyi, Clin. Pharmacokinet., 7 (1982) 490. 6. T.R. Browne, J. Clin. Pharmacol., 26 (1986) 485. 7. T.R. Browne, Clin. Pharmacokinet., 18 (1990) 423. 8. A.P. Alvares, A. Kappas, J.L. Eiseman, K.E. Anderson, C.B. Pantuck, E.J. Pantuck, K.-C. Hsiao, W.A. Garland and A.H. Conney, Clin. Pharmacol. Ther., 26 (1979) 407. 9. J.M. Strong, J.S. Dutcher, W.-K. Lee and A.J. Atkinson Jr., Clin. Pharmacol. Ther., 18 (1975)613. 10. U. Meresaar, M.-I. Nilsson, J. Holmstrand and E. Anggard, Eur. J. Clin. Pharmacol., 20 (1981) 473. 11. W.T. Beaver, S.L. Wallenstein, R.W. Houde and A. Rogers, Clin. Pharmacol. Ther., 8 (1967)415. 12. M. Eichelbaum, H.J. Dengler, A. Somogyi and G.E. von Unruh, Eur. J. Clin. Pharmacol., 19 (1981) 127. 13. M. Eichelbaum, A. Somogyi, G.E. von Unruh and H.J. Dengler, Eur. J. Clin. Pharmacol., 19 (1981) 133. 14. M. Marvola, A. Kannikoski, J. Taskinen and P. Ottoila, J. Pharm. Pharmacol., 37 (1985) 766. 15. D. Alkalay, W.E. Wagner, S. Carlsen, L. Khemani, J. Volk, M.F. Bartlett and A. LeSher, Clin. Pharmacol. Ther., 27 (1980) 697. 16. H. d'A. Heck, S.E. Buttrill Jr., N.W. Flynn, R.L. Dyer, M. Anbar, T. Cairns, S. Dighe and B.E. Cabana, J. Pharmacokinet. Biopharm., 7 (1979) 233. 17. R.E. Gammans, A.V. Mackenthun and J.W. Russell, Br. J. Clin. Pharmacol., 18 (1984) 431. 18. Y. Kasuya, K. Mamada, S. Baba and M. Matsukura, J. Pharm. Sci., 74 (1985) 503. 19. T.R. Browne, G.K. Szabo, C. McEntegart, J.E. Evans, B.A. Evans, J.J. Miceli, C. Quon, C.L. Dougherty, J. Kres and H. Davoudi, J. Clin. Pharmacol., 33 (1993) 89. 20. F. Hatch, K. McKellop, G. Hansen and T. MacGregor, J. Pharm. Sci., 75 (1986) 886. 21. Y. Shinohara, S. Baba, Y. Kasuya, G. Knapp, F.R. Pelsor, V.P. Shah and I.L. Honigberg, J. Pharm. Sci., 75 (1986) 161. 22. S. Looareesuwan, N.J. White, D.A. Warrell, I. Forgo, U.G. Dubach, U.B. Ranalder and D.E. Schwartz, Br. J. Clin. Pharmacol., 24 (1987) 37. 23. B. Hallen, O. Guilbaud, S. Stromberg and B. Lindeke, Biopharm. Drug Dispos., 9 (1988) 229. 24. J. Schmid, A. Prox, H. Zipp and F.W. Koss, Biomed. Mass Spectrom., 7 (1980) 560.

259 25. R.L. Wolen, R H. Carmichael, A.S. Ridolfo, L. Thompkins and E.A. Ziege, Biomed. Mass Spectrom., 6 (1979) 173. 26. J.R. Koup, S.B. Walker, G.G. Shapiro, W.N. Howald, W.E. Pierson, C.W. Bierman and C.T. Furukawa, J. Allergy Clin. Immunol., 78 (1986) 752. 27. J.P. Dubois, A. Sioufi, P. Muller, D. Mauli and P.R. Imhof, Meth. and Find. Exp. Clin. Pharmacol., 11 (1989) 187. 28. S.C. Mulligan, J.G. Masterson, J.G. Devane and J.G. Kelly, Clin. Pharmacol. Ther., 47 (1990) 331. 29. T. Abelin, P. Muller, A. Buehler, K. Vesanen and P.R. Imhof, Lancet, 1 (1989) 7. 30. J.E. Rose, E.D. Levin, F.M. Behm, C. Adivi and C. Schur, Clin. Pharmacol. Ther., 47 (1990) 323. 31. N.L. Benowitz, K. Chan, C.P. Denaro and P. Jacob III, Clin. Pharmacol. Ther., 50 (1991) 286. 32. M. Gumbleton and L.Z. Benet, Br. J. Clin. Pharmacol., 31 (1991) 211. 33. D.K. Yu., R.L. Williams, L.Z. Benet, E.T. Lin and D.H. Giesing, Biopharm. Drug Dispos., 9 (1988) 557. 34. S.A. Bai, M.J. Wilson, U.K. Walle and T. Walle, J. Pharmacol. Exp. Ther., 227 (1983) 360. 35. L.S. Olanoff, T. Walle, U.K. Walle, T.D. Cowart and T.E. Gaffney, Clin. Pharmacol. Ther., 35 (1984) 755. 36. B. Vogelgesang, H. Echizen, E. Schmidt and M. Eichelbaum, Br. J. Clin. Pharmacol., 18 (1984) 733. 37. K. Nakamura, D.L. Hachey, M.J. Kreek, C.S. Irving and P.D. Klein, J. Pharm. Sci., 71 (1982)40. 38. R.L. Wolen, B.D. Obermeyer, E.A. Ziege, H.R. Black and C.M. Gruber, Jr., Stable Isotopes (Macmillan Press, London, 1978), p. 113. 39. T.R. Browne, G.K. Szabo, G.E. Schumacher, D.J. Greenblatt, J.E. Evans and B.A. Evans, J. Clin. Pharmacol., 32 (1992) 1141.

261

CHAPTER 14

DRUG DISTRIBUTION AND "DEEP POOL EFFECT"

THOMAS R. BROWNE Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center

1. MEASUREMENT OF DISTRIBUTION HALF-LIFE AND VOLUME OF DISTRIBUTION

Distribution half-life and volume of distribution can be determined for a given drug using conventional or stable isotope labeled (SIL) tracer techniques (16). However, it is sometimes desirable to perform serial studies of these parameters during uninterrupted chronic administration to determine presence or absence of changes in these parameters (especially volume of distribution) associated with chronic administration, differences in plasma concentration, or drug interactions. Stable isotope tracer studies are ideally suited to such serial studies because it is not necessary to discontinue drug or to expose the patient to radioactivity during serial studies (see Chapter 16 for details). Using serial stable isotope tracer methods, Browne et al. (3, 4) demonstrated absence of change in volume of distribution for phenytoin or phenobarbital associated with chronic administration or changes in plasma concentration (Table 1). Similarly, Browne et al. (5, 6) demonstrated absence of change in volume distribution for phenytoin after addition of phenobarbital or carbamazepine (Table 1).

2. MEASUREMENT OF RATE OF ENTRY OF DRUG INTO TISSUES OTHER THAN BLOOD ("STAGGERED STABLE ISOTOPE ADMINISTRATION TECHNIQUE")

Traditionally, drug distribution has been studied by administration of a single dose of drug followed by serial collections of tissue. Using a series of uniquely

262 TABLE 1. Serial Measurements of Drug Volume of Distribution During Monotherapy or Combined Therapy Determined with Stable Isotope Tracer

Drug

Week 01

Week 42

Week 122

1.2 _+0.5 0.63 _+0.06

5.5 -+ 2.5 0.68 _ 0.09

10.3 _+6.1 0.73 _+0.09

1.2 _+0.1 0.62 _+0.05

12.5 _+3.0 0.61 _+0.04

13.1 +_4.5 0.61 _+0.04

Phenytoin (after adding phenobarbital) Cs, (~g/ml) 13.2 _ 5.4 VD (L/kg) 0.69 _+0.06

13.2 _+5.7 0.64 -+ 0.09

15.0 _+5.6 0.60 _+0.05

Phenytoin (after adding carbamazepine) Css (l~g/ml) 13.2 _+6.6 VD (L/kg) 0.65 _+0.08

13.4 _+5.6 0.69 -+ 0.09

17.8 _+7.1 0.63 _+0.08

Phenytoin (monotherapy) Cs, (l~g/ml) 3 VD (L/kg)" Phenobarbital (monotherapy) Cs, (kg/ml) VD (L/kg)

1Single dose study prior to beginning chronic therapy (monotherapy); tracer dose study of drug given alone prior to adding second drug (combined therapy). 2Tracer dose study performed 4 or 12 weeks after beginning chronic therapy or after adding second drug. 3Mean steady state plasma concentration. 4Volume of distribution. Based on data from Refs 3-6 with permission.

labeled drug analogues it is possible to obtain the same information by administering different labeled drug analogues at different times prior to obtaining a single tissue specimen ("staggered stable isotope administration technique") (7).

2.1. Procedures Serial injections of SIL drug with different labels are administered at predetermined times prior to collection of a single tissue specimen such as cerebrospinal fluid, liver or kidney (Figure 1) (7). The tissue concentration of each SIL form of the drug is determined. Drug-tissue entry rate constant and entry half-life are determined from tissue concentration versus time relationships (7).

263

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Phenobarbital (PB) CSF-free plasma concentration ratio versus time data points. (O) from four sets of specimens collected 5, 15, 30 and 60 min after infusion of +0 PB. (r-I) from single set of plasma and CSF specimens collected 5 min after infusion of +0 PB, 15 min after infusion of +3 PB, and 30 min after infusion of +5 PB. (x) from single set of specimens collected 15 min after infusion of +0 PB, 25 min after infusion of +3 PB, and 40 min after infusion of +5 PB. (A) from single set of specimens collected 30 min after infusion of +0 PB, 40 min after infusion of +3 PB, and 55 min after infusion of +5 PB, CSF = Cerebrospinal fluid. From Evans et al. (7) with permission.

2.2. Assumptions

This method assumes that (1) the tissue collection methods employed (e.g. lumbar puncture, liver biopsy) do not alter drug distribution, and (2) the staggered stable isotope technique yields results similar to the conventional multiple-specimen collection technique (see below). The second assumption can be verified (Figure 1) (7). 2.3. Alternative Methods

Serial samples of tissue are obtained after the test drug is administered. Tissue concentration of drug is determined by conventional techniques (e.g.

264

gas or liquid chromatography). Drug-tissue entry rate constant and entry halflife are determined from tissue concentration versus time relationships.

2.4. Advantages The requirement for only a single tissue specimen greatly reduces subject morbidity in comparison with the conventional multiple-specimen technique. Furthermore, it is often possible to justify collection of a single sample of tissue as part of a routine diagnostic procedure (e.g. lumbar puncture, renal biopsy), while multiple collections of tissue may raise questions regarding the comfort and safety of the patient. The staggered stable isotope technique also requires analysis of only a single tissue specimen per patient.

2.5. Disadvantages The use of multiple stable isotopes results in higher than average synthetic costs for an SlL study. Furthermore, the presence of multiple ions of the same drug in a tissue creates the potential for ion overlap errors in mass spectrometric analysis.

3. "DEEP POOL EFFECT"

If a portion of administered drug is distributed into a "deep" peripheral compartment, the drug's actual elimination half-life during the terminal exponential phase of elimination may be longer than determined by a single dose study, or a tracer dose study if the study is not carried out for a sufficient length of time (usually due to insufficient assay sensitivity) to detect low plasma concentrations of drug at later times after administration (Figure 2). This has been called "deep pool effect" and can lead to an overestimation of actual clearance in single dose studies and in tracer dose studies because of factitiously small values for apparent elimination half-life and apparent area under the plasma concentration versus time curve (Figure 2). Biphasic plasma concentration versus time relationships similar to those shown in Figure 2 have been reported after single and multiple doses of unlabeled gentamicin and tobramycin, and the predicted (by a two-compartment model) tissue concentration of these two drugs has been found in postmortem tissue (8-12). A biphasic plasma concentration versus time relationship similar to that shown in Figure 2 also has been reported after single doses of procainamide, presumably due to a deep tissue compartment (11).

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Figure 2. Tracer dose plasma concentration versus time relationships in presence and absence of deep peripheral compartment ("deep pool effect"). From Browne et al. (17) with permission.

All of these studies used analytical methods with a lower limit of sensitivity less than 0.05 i~g/mL. Although the theoretical possibility of deep pool effect is one of the most frequently raised criticisms of stable isotope tracer methods, the presence or absence of deep pool effect upon stable isotope tracer studies of drug

266 clearance has received little empirical study. A deep pool for calcium has been shown to exist in bone with stable isotope studies of calcium metabolism using multiple compartment analysis (12). The technique of coadministration of single doses of labeled and unlabeled drug used to detect "metabolic isotope effect" (1, 2) also can be used to detect deep pool effect, since drugs with known deep pools exhibit biphasic plasma concentration versus time curves in single dose studies (8-11). However, such coadministration studies typically are performed early in the development of a stable isotope-labeled drug for tracer studies and typically use low doses of labeled and unlabeled drug because the studies are performed on volunteers. Often coadministration studies utilize simple chemical extraction/gas chromatographic-mass spectrometric (GC-MS) analytic methods whose lower limit of sensitivity for plasma drug concentration is no better than 0.1 i~g/mL because of interference from endogenous compounds (e.g. Refs 13, 14). In the reported single dose studies of gentamicin, tobramycin, and procainamide showing biphasic plasma concentration versus time curves, the break points in the curves all occurred at plasma concentrations less than 0.1 i~g/mL and at times greater than 12 hr after drug administration. The deep pool effect for these drugs would have been missed using an analytical method with a lower limit of sensitivity of 0.1 i~g/mL. Following the washout of drug after chronic administration can also demonstrate the presence or absence of deep tissue compartments and has the advantage that plasma concentration versus time relationships can be followed for a longer period of time, in comparison with single dose or tracer studies, before plasma concentration values fall below the lower limit of assay sensitivity (9, 10, 15, 16). This method has three drawbacks: (1) discontinuation of chronically administered drug is not always safe in patients (e.g. antiepileptic drugs); (2) chronic administration of a drug to volunteers may be unsafe and/or impractical; and (3) a special study must be performed to determine the presence or absence of deep pool effect. Our group (17) described two simple methods of testing for "deep pool effect." These methods were applied to phenytoin, a "worst case" drug with nonlinear pharmacokinetic properties. 3.1. Mathematical Background of Two Methods to Test for Deep Pool Effect 3.1. I. First method

As a first method to test for deep pool effect, single dose or tracer dose plasma concentration versus time relationships during the elimination phase

267 can be inspected visually for linearity on semilog plots and evaluated statistically for semilog linearity. Deviation from linearity would be suggestive of multiple compartments. Semilog (exponential) regression analysis can be performed using the following equation" CoB = ae -bt

(1)

where COB =observed plasma concentration; a = initial COB va~ue (time=O intercept); e = constant 2.718282; b = elimination rate constant; and t = time. Linearity with this semilog function can be evaluated by squared correlation coefficient (r 2) calculated by a procedure described elsewhere (18, 19). This procedure is the same as that used for linear regression analysis, except the natural log of the dependent variable is substituted for the dependent variable. Note that we have demonstrated that the expected semilog regression line by this analysis will be linear for drugs with linear pharmacokinetics regardless of actual total (labeled and unlabeled) plasma concentration during the period of study and also will be linear for drugs with nonlinear pharmacokinetics if total drug plasma concentration is held constant during the period of study (18). Demonstration of semilog linearity of elimination of a single dose or a tracer dose of drug cannot exclude the possibility of a deep peripheral compartment of a drug which cannot be detected in a short-term study (Figure 2). Definitive demonstration of absence of deep pool effect upon single dose clearance or tracer dose clearance requires demonstration that apparent single dose or tracer dose clearance is equal to clearance of drug at steady state plasma concentration ("steady state clearance"). 3.1.2. S e c o n d m e t h o d

The second method of testing for deep pool effect meets the requirement of direct comparison of single dose or tracer dose drug clearance with steady state clearance. Steady state clearance can be calculated utilizing drug dosing rate (D), fraction of drug absorbed (F), observed mean steady state plasma concentration (C~), and the following equation" steady state clearance = (D x F)/C~

(2)

For drugs with linear pharmacokinetic properties, single dose clearance, tracer dose clearance at steady plasma concentration, and steady state clearance (calculated with Eq. (2)) will be the same if no deep pool effect is present. For

268

drugs with nonlinear pharmacokinetic properties, single dose clearance and clearance at steady state plasma concentration will be different (Eq. (1)). However, tracer dose clearance at steady state plasma concentration should be the same as simultaneously determined steady state clearance if Css remains relatively constant during the test period (Eq. (1)). Again, if tracer dose clearance is greater than steady state clearance, deep pool effect is suggested. 3.2. Example of Application of Methods to Test for Deep Pool Effect 3.2.1. General

Detailed descriptions of patients, drug administration, specimen collection, analytical methods and pharmacokinetic methods have been published elsewhere (3, 5, 6, 17, 20). Briefly, 15 patients on stable (>45 days) regimens of phenytoin monotherapy were studied with intravenous tracer doses of 150 mg of 13ClSN2-1abeled sodium phenytoin. Labeled and unlabeled phenytoin plasma concentrations were followed for 48 hr after each infusion. 3.2.2. First method

Elimination phase plasma concentration versus time relationships of labeled phenytoin were inspected visually and tested statistically for semilog linearity using procedures described previously. 3.2.3. Second method

Tracer dose clearance and steady state clearance computed with Eq. (2) were determined for each patient as described previously. Differences between tracer dose clearance and steady state phenytoin clearance values were tested by the Student's paired t-test (i.e. probability of a type I or alpha error was computed). The probability of failing to detect a truly significant difference of 20 percent or greater in clearance values was determined by the method of Glenberg (i.e. the probability of a type II or beta error was computed) (21). 3.2.4. Results of first method

The elimination phase plasma concentration versus time relationships for ~3C~SN2-phenytoin after each infusion appeared linear on semilog plots. The

269 TABLE 2. First Method: Correlation Coefficients tration Versus Time Relationships

Patient number

r2

(p)

1 2 3 4 5 6 7 8

0.994 0.999 0.998 0.977 0.994 0.998 0.990 0.981

(< 0.001 ) (<0.001) (<0.001) (<0.001) (<0.001) (<0.001) (<0.001) (<0.001)

(r 2) for Phenytoin Tracer Dose Plasma ConcenPatient number

r2

(p)

9 10 11 12 13 14 15

0.985 0.998 0.984 0.908 0.993 0.933 0.952

(< 0.001 ) (<0.001) (<0.001) (<0.001) (<0.001) (<0.001) (<0.001)

From Browne et al. (17) with permission.

lower limit of sensitivity of the GC-MS analytical method employed to measure plasma concentration of phenytoin and 13C15N2-phenytoin was 0.1 i~g/mL (3). The plasma concentration versus time plots of three patients have been published elsewhere (3, 5, 6). All 15 of the correlation coefficients (r 2) for tracer dose plasma concentration versus time relationships were greater than 0.9 and showed significant (p < 0.001) agreement with the semilog linear model described previously (Table 2). These results suggest elimination of tracer doses of phenytoin was semilog linear over the 48-hr study interval and provide no evidence for additional compartments (deep pools). However, this analysis cannot definitively exclude the possibility of a deep pool (discussed previously). 3.2.5. Results of second m e t h o d

Individual patient phenytoin tracer dose clearance and phenytoin steady state clearance were published elsewhere (20). Statistical analysis of these values for determination of the presence or absence of deep pool effect is shown in Table 3. No significant difference between tracer dose clearance and steady state clearance was found (P - probability of type I error = 0.67). The probability of missing a truly significant difference of 20 percent or greater in clearance values (probability of type II error) was less than 0.01. These results indicate equivalence of clearance of tracer doses of ~5N~3C2-phenytoin with clearance of chronically administered unlabeled drug. Moreover, these results indicate

270 TABLE 3. Second Method: Statistical Analysis of Phenytoin Clearance Values (mL/min/kg) 1 ,,,

Probability of error Type 13

Tracer dose (N= 15) Steady state (N= 15)

Mean

SD2

0.313

0.183

0.307

(P)

Type II4

0.67

0.01

0.185

1Individual patient values published in Browne et al. (20). 2Standard deviation. 3By paired Student's t-test. 4probability of missing a truly significant difference of 20 percent or greater by method of Glenberg (21). Browne et al. (17) with permission.

deep pool effect is either absent or pharmacokinetically insignificant for 15N13C=-phenytoin tracer doses.

3.2.6. Discussion Assumptions of First Method. Use of semilog plots of tracer dose venous

plasma concentration versus time relationships depends upon the following assumptions: (1) total (labeled and unlabeled) drug plasma concentration remains relatively constant during the time period studied (for drugs with nonlinear pharmacokinetic properties, verified for phenytoin in Ref. 20); (2) absence of "isotope effect" on tracer drug pharmacokinetics (verified for labeled phenytoin used in this study in Ref. 1); and (3) absence of significant arteriovenous difference in drug plasma concentration (see Chiou (22) for discussion, verified by Boiholm et al. (23, 24) for phenytoin).

Assumptions of Second Method. Use of tracer dose venous plasma concentra-

tion versus time relationships to determine clearance depends upon the three assumptions discussed for the First Method. Use of Eq. (2) to compute steady state clearance relies upon the following

271 assumptions: (1) patients have been completely compliant in taking medications; (2) assumed value for fraction of drug absorbed is correct for all patients; and (3) mean plasma concentration has arrived at steady state value at the time it is measured. These three assumptions have been discussed for the patients in this study in Ref. 20.

"Last-in, First-out Phenomenon". Colburn and Matthews (25) reported that some drugs with multiple compartment pharmacokinetics exhibit a phenomenon whereby semilog tracer dose plasma concentration versus time plots show less extensive distribution and more rapid elimination after chronic administration of a drug than similar plots following a single dose of drug to naive patients ("last-in, first-out phenomenon"). For drugs cleared by a single enzyme, clearance is determined by Vmax, Kr,,, and C. If these remain constant over time, clearance will remain constant. Clearance (CL) can also be defined as:

CL = SLOPETx Vd

(3)

where SLOPET is the slope of the terminal portion of a semilog plot of plasma concentration versus time and Vd is volume of distribution. If CL remains constant (linear pharmacokinetics or nonlinear pharmacokinetics with constant plasma concentration, Eq. (1), or constant dosing rate, Eq. (2)), a decrease in Vd will result in an increase in SLOPET. However, the product of SLOPET x Vd will remain constant and will measure accurately CL at the time of the test. The considerations of the previous paragraph demonstrate that the presence or absence of "last-in, first-out phenomenon" does not invalidate the basic premises of the two reported methods: the terminal, post distribution phase(s) slope of a semilog plot of tracer dose plasma concentration versus time will be linear (First Method) and will predict accurately simultaneous steady state clearance (Second Method). On the other hand, differences in SLOPET and volume of distribution between single dose studies and tracer studies performed during chronic administration can be used to infer the possible presence of "last-in, first-out phenomenon". The "last-in, first-out" phenomenon does not appear to be significant for phenytoin for two reasons. First, the original study of Colburn and Matthews (25) tested for "last-in, first-out phenomenon" for phenytoin in rats and found it did not occur. Second, in humans, semilog plots of phenytoin plasma concentration versus time do not show a decrease in volume of distribution

272 or an increase in slope before and after chronic administration of phenytoin (21). 3.3. Conclusions Regarding Methods to Test for Deep Pool Effect 3.3. I. First method This method can establish the presence, but not the absence, of a deep pool effect. 3.3.2. Second method The second method represents the only reported method to test for equivalence of clearance of stable isotope labeled tracer doses of drug with clearance of chronically administered drug while maintaining a steady state drug plasma concentration. 3.3.3. Pharmacokinetic interference If the single dose clearance value and the clearance value computed at steady state plasma concentration using Eq. (2) agree, one can infer that the drug has linear pharmacokinetic properties (18). If the single dose clearance value is greater than the value computed with Eq. (2), one must consider the possibility of deep pool effect upon the single dose clearance value and/or nonlinear pharmacokinetics. If the single dose clearance value is less than the value computed with Eq. (2), one must consider the possibility of enzyme induction. If single dose clearance and SLOPET values are the same as tracer dose clearance and SLOPETvalues, one can infer that the drug has linear pharmacokinetic properties (18) and "last-in, first-out phenomenon" is not present (described previously). If single dose clearance and tracer dose clearance are the same, but tracer dose SLOPET is greater than single dose SLOPET and tracer dose volume of distribution is less than single dose volume of distribution, one must consider the possibility of linear pharmacokinetics with "lastin, first out phenomenon" present (25). If the single dose clearance value is greater than the tracer dose clearance value, one must consider deep pool effect upon the single dose value and/or nonlinear pharmacokinetics. If the tracer dose clearance value is greater than the single dose clearance value, one must consider deep pool effect upon the tracer dose value and/or enzyme induction.

273 If tracer dose clearance values and clearance values computed at steady state plasma concentration using Eq. (2) agree, one can infer that the tracer dose clearance values were computed using the actual value of SLOPET and all distribution effects have been accounted for. If the tracer dose clearance value is greater than the value computed with Eq. (2), one must consider the possibility of a deep pool effect upon the tracer dose value. 3.3.4. S i m p l i c i t y

of methods

The data necessary for the First and Second Methods are collected routinely in the performance of tracer studies and require no additional patient inconvenience or analytical work. Thus, these tests can be included as part of the routine performance of such studies.

ACKNOWLEDGMENT Supported by the United States Department of Veterans Affairs.

REFERENCES 1. T.R. Browne, A. Van Langenhove and C. Costello et al., Clin. Pharmacol. Ther., 29 (1981) 511. 2. T.R. Browne, A. VanLangenhove and C. Costello et al., J. Clin. Pharmacol., 22 (1982) 309. 3. T.R. Browne, J.E. Evans and G.K. Szabo et al., J. Clin. Pharmacol., 25 (1985) 43. 4. T.R. Browne, J.E. Evans and G.K. Szabo et al., J. Clin. Pharmacol., 25 (1985) 51. 5. T.R. Browne, G.K. Szabo and J.E. Evans et al., Neurology, 38 (1988) 639. 6. T.R. Browne, G.K. Szabo and J.E. Evans et al., Neurology, 38 (1988) 1146. 7. J.E. Evans, T.R. Browne and P.L. Kasdon et al., J. Clin. Pharmacol., 25 (1985) 309. 8. M. Andelman, E. Evans and J.J. Schentag, Antimicrob. Agents Chemother., 22 (1982) 800. 9. J.J. Schentag, W.K. Jusko and M.E. Plant et al., J.A.M.A., 238 (1977) 327. 10. J.J. Schentag, G. Lasezkay and J. Cumbo et al., Antimicrob. Agents Chemother., 13 (1978)649. 11. F. Jamali, R.S. Alballa and R. Mehuear et al., Ther. Drug Monit., 10 (1988) 91. 12. A.L. Yengey, N.E. Vieira and D. Covell, et al., in Synthesis and Applications of Isotopically Labeled Compounds 1985, R.R. Muccino (ed) (Elsevier, Amsterdam, 1986), p. 343. 13. A. Van Vangenhove, C. Costello and J.E. Biller et al., Biomed. Mass Spectrom., 9 (1982) 202. 14. A. Van Vangenhove, C. Costello and J.E. Biller et al., Biomed. Mass Spectrom., 7 (1980) 576. 15. Y. Kasuya, K. Mamadu and S. Baba et al., J. Pharm. Sci., 74 (1985) 503.

274 16. H.R. Sullivan, P.G. Wood and R.E. McMahon, Biomed. Mass Spectrom., 3 (1976) 212. 17. T.R. Browne, A.J. Greenblatt and G.E. Schumacher, J. Clin. Pharmacol., 30 (1990) 680. 18. T.R. Browne, A.J. Greenblatt and J.S. Harmatz et al. J. Clin. Pharmocol., 25 (1985) 59. 19. T. Colton, Statistics in Medicine (Little Brown, Boston, 1974). 20. T.R. Browne, D.J. Greenblatt and G.K. Szabo et al., J. Clin. Pharmacol., 30 (1990) 488. 21. A.M. Glenberg, Learning from Data, An Introduction to Statistical Reasoning (Harcourt Brace Jovanovich, New York, 1989). 22. W.L. Chiou, Clin. Pharmacokinet., 17 (1989) 275. 23. S. Bojholm, O.B. Paulson and H. Flachs, Clin. Pharmacokinet., 17 (1989) 5. 24. S. Bojholm and O.B. Paulson, Clin. Pharmocol. Ther., 22 (1982) 475. 25. W.A. Colburn and H.B. Matthews, Toxic Appl. Pharmacol., 44 (1979) 387.

275

CHAPTER 15

BIOTRANSFORMATION AND EXCRETION: MECHANISTIC STUDIES: DEUTERIUM ISOTOPE EFFECTS AS A PROBE OF THE CATALYTIC PROPERTIES OF THE CYTOCHROMES P450

WILLIAM F. TRAGER

Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195

1. INTRODUCTION

A particularly powerful methodology for elucidating the mechanistic details by which organic chemical reactions occur has been through the use of isotope effects. Since so many reactions involve either the loss or rearrangement of carbon-hydrogen bonds the insight provided by deuterium isotope effects has been particularly powerful. While isotope effect methodology has been very successful in its application to problems of traditional physical organic chemistry, historically, it has been far less successful in the realm of biochemistry in revealing the mechanistic details of enzymatically mediated reactions. The reason for the difference in success rates resides in the inherent differences that prevail between the two systems during the course of a reaction. For example, enzymatically mediated reactions in general, unlike most simple chemical reactions, involve a number of binding and de-binding steps that can contribute to the overall rate of the reaction. The overall effect of these additional partial rate limiting steps is to lower, or mask, the experimentally observable deuterium isotope effect, (kH/ko)obs, relative to the deuterium isotope effect associated specifically with the bond breaking step, i.e. the intrinsic isotope effect, (kH/ko). Thus, an experimentally observed deuterium isotope effect, (kH/kD)ob~, for a carbon-hydrogen bond cleavage reaction

276 can only be equal to, or less than, the magnitude of the intrinsic deuterium isotope effect, kH/kD, for the reaction. It can never be greater. The chemical events leading to attainment of the transition state and the subsequent steps that result in formation of products are the descriptors of a chemical reaction mechanism. An intrinsic deuterium isotope effect reports directly on the nature and symmetry of the transition state, the key element of the reaction mechanism, and it is for this reason that it is potentially so powerful in revealing the chemical nature of the reaction. If a deuterium isotope effect is to be meaningfully used in establishing a reaction mechanism the relationship between (kH/kD)obs and kH/ko must be understood, since by definition experiment can only yield (kH/ko)obs. AS indicated above, this is particularly relevant for biochemically mediated reactions because, in general, they are complex multi-step processes. Fortunately, work by Northrop (1-3) and Cleland (4) has done much to clarify the relationship between (kH/ko)obs and kH/ko and open the way to understanding how partially rate limiting steps, other than the bond breaking step, lead to the masking of the intrinsic isotope effect by lowering the magnitude of the observable isotope effect. The interested reader is referred to their excellent book on the subject (2). While the new understanding of isotope effects, provided by the work of Northrop and Cleland, is applicable to enzymatically mediated reactions in general, the focus of this chapter will be on their application to studies in drug metabolism, particularly as probes to gain insight into the fundamental catalytic properties of the cytochromes P450. The cytochromes P450 are a superfamily of heine containing enzymesthat are found essentially in every living organism from bacteria to man. Together with cytochrome P450 reductase, they have the capacity to activate molecular oxygen to a heme-iron bound perferryl oxo species that can catalyze the oxidation of virtually any lipophilic organic compound irrespective of whether it is of exogenous or endogenous origin (Figure 1). The primary determinant in this regard appears to be whether or not the compound in question reaches the active site of the enzyme. The use of oxygen to convert foreign and potentially toxic compounds to more water

O

r~---pS ]l~,Fe+x~ + 02 + H+ +

NADPH

P450 reductase , ~

/ N\-~/14 ] Fe+~ NI x N

+

H20

Figure 1. Formation of the active perferryloxy heme oxidizing species of cytochrome P450.

277 soluble species, thereby promoting their elimination from the organism, suggests that host defense is one of the primary functions of this enzyme system. In general, enzymes achieve their catalytic power by binding substrate in a geometry that specifically promotes attainment of the transition state of the reaction to be achieved. The cytochromes P450 are unusual in that most of the energy required for activation of the system does not directly involve substrate but rather involves the generation of the highly reactive perferryloxy heme (oxene) species. Because of the inherent reactivity of the oxene species, specific substrate binding is of relatively minor importance in achieving catalysis. However, substrate binding can play a major role as the regioselective determinant of oxidative attack on the substrate. That is, the reactive oxygen atom once generated is transferred to the substrate at the molecular site(s) dictated by the steric interactions between the substrate and the active site architecture of that specific cytochrome P450. Because the energy required for catalysis is embodied within the oxene species the individual isoforms of cytochrome P450, unlike most enzymes, possess broad and overlapping substrate specificity and generally have the capacity to catalyze the formation of multiple products from a single substrate. This class characteristic has the effect of broadening the impact of any new discovery. Thus, when some fundamental property is revealed about a specific isoform, perhaps through the use of isotope effects, it often will have meaning for the P450s as a class of enzymes.

2. INTRAMOLECULAR ISOTOPE EFFECTS

As mentioned above, a major complication in using deuterium isotope effects to study enzymatically mediated reactions is the fact that under most circumstances the observed isotope effect for the reaction is rarely equal to the intrinsic isotope effect associated with the bond breaking step. The general problem of inequality between the magnitudes of (kH/ko)obs and kH/ko can often be circumvented by conducting isotope effect studies in cytochrome P450 catalyzed reactions using experiments of intramolecular competitive design (5-8). In such an experiment, a substrate is chosen that is susceptible to enzymatic attack at two symmetrically equivalent sites. One site is deuterated while the second retains its normal complement of hydrogen. Thus, the enzyme has the choice of attacking either a deuterated or an equivalent protio site within the same molecule. The observed isotope effect reflects the intramolecular competition between these two sites. Under the conditions of

278

CH30~OCH3

kH r

CH30@OH kH/kD = 2

CD30~OCD3

KD

CD30@OH

Figure 2, The deuterium intermolecular noncompetitive isotope effect experiment used to determine (kH/kD)obsfor O-demethylation when para-methoxyanisole and paraditrideuteromethoxybenzene were used as the substrates.

CD30~OCH3

kH ;~,

CD30~OH

/

kH/kD =

11

CH30~OH Figure 3. The deuterium intramolecular competitive isotope effect experiment used to determine (kH/kD)obsfor O-demethylation when para-trideuteromethoxyanisole was used as the substrate.

this kind of experiment essentially all of the masking factors fall out of the rate equations and (kH/ko)obs approaches kH/ko (5-8). A classic example of the effect can be seen with the deuterium isotope effects reported for the Odemethylation of para-methoxyanisole (9). When para-methoxyanisole and para-ditrideuteromethoxybenzene were used as the substrates, in a typical intermolecular noncompetitive experiment, (kH/ko)obs was 2 (Figure 2). However, when the isotope effect for the same reaction was determined using para-trideuteromethoxyanisole as the substrate, in an experiment of intramolecular competitive design, (kH/ko)obs was 11 (Figure 3).

279

2. I. Factors Affecting the Magnitude of an Intramolecular Isotope Effect and tthe Relationship Between (kH/kD)obsand kH/kD-- Rapid Equilibration (Rotation) Kinetically, the relationship between the observed isotope effect and the intrinsic isotope effect is given by Eq. (1) (10). In this equation ESH and ESD denote the

(kH/kD)obs =

kH" [ESH] kD" [ESD]

(1)

enzyme-substrate complexes that yield products resulting from cleavage of a C~H and C~D bond, respectively. As indicated above, the quantity (kH/kO)obs is the term defining the observed isotope effect, while kH/ko is the term defining the intrinsic isotope effect associated exclusively with the bond breaking event. It is the unique design of the experiment that allows determination of the approximate intrinsic isotope effect for the reaction. According to the equation, one feature that may significantly affect the magnitude of the observed isotope effect is the rate at which the two symmetrical oxidation sites (protio versus deutero), equilibrate within the active site of an enzyme, relative to bond breaking (10). If the rate of equilibration is not sufficiently rapid to maintain the ratio of the enzyme-substrate complexes (ESH and ESD) at unity, the magnitude of (kH/kD)obs will decrease relative to the magnitude of kH/kD in direct relation to the degree of departure of ESH/ESD from unity. That is, the intrinsic isotope effect will be masked in an intramolecular isotope effect experiment whenever the concentration of ESD exceeds the concentration of ESH. Consistent with this expectation, Jones et al. (10) found that (kH/kD)obs for hydroxylation of the methyl group ((ohydroxylation) of the linear hydrocarbon, octane-l,2,3-2H7, was only 4 (see Figures 10 and 11), even though kH/ko for the reaction was found to be close to 1 1.8 (10). The results suggest that equilibration between the terminal methyl and trideuteromethyl groups of octane-l,2,3-2H7 within the active site of the enzyme is slow enough to lead to significant masking of kH/kD. Similarly, the intramolecular deuterium isotope effects associated with a series para substituted (H, CI, CN and NO2) N-methyI-N-(trideuteromethyl) aniline analogs were found to be either identical to, or significantly lower than, the intramolecular isotope effects determined for the respective N,Nbis(dideuteriomethyl) anilines (11). According to Eq. (1), the observation of an intrinsic deuterium isotope effect for the N-demethylation of the N-methylN-(trideuteromethyl) aniline analogs requires that rotational equilibration of

280

_

X

-% //-- N

kH,,,~' J

S H3

@

/H

X

NNcD3

X

/ CH3 N\

kH/k D = 1.8, X = H; 2.0, X = CI; 3.1, X = CN; 3.5, X = NO2

Figure 4. The intramolecular deuterium isotope ettects associated with a series para substituted (H, CI, CN and NO2) N-methyI-N-(trideuteromethyl) aniline analogs.

N-methyl and N-trideuteromethyl groups be fast relative to bond breaking (Figure 4). A similar observation for the N,N-bis(dideuteriomethyl) aniline analogs also requires rapid equilibration between catalytically susceptible and symmetrical carbon-hydrogen and carbon-deuterium bonds. But in this case it is more easily attained since deuterium and hydrogen equilibration is achieved by the independent rapid rotation of the individual methyl groups (Figure 5). The smaller isotope effects actually observed for the trideuteromethyl compounds were attributed to masking as a consequence of a slow rate of methyl group equilibration arising possibly from hydrogen bond formation between the amine nitrogen and a hydrogen bond donor in the active site of cytochrome P450. A final example of the effect of an active site concentration imbalance

xC

/H N\ z/

~ CHD2

X

'/~NNcHD2

kH/k D = 2.5, X = H; 2.7, X = CI; 3.2, X = CN; 3.6, X = NO2

Figure 5. The intramolecular deuterium isotope effects associated with a series para substituted (H, CI, CN and NO2) N,N-bis(dideuteriomethyl) aniline analogs.

281 CH 3

I

CH 3

~

I

2.2

J

(R)

13CH3

(R)

CHzOH 1.0

I

(S)

Figure 6. Stereoselective hydroxylation of the pro (R)-methyl group of cumene catalyzed by rat liver microsomes.

between ESH and ESD on the magnitude of (kH/kD)obs can be found in the whydroxylation of the prochiral substrate cumene (12). Specifically labeling one of the methyl groups with ~ZC so that the two methyl groups can be distinguished from each other, reveals that rat liver microsomes selectively hydroxylate the pro (R)-methyl over the pro (S)-methyl by 2.2-fold (Figure 6). The observed stereoselectivity can be attributed directly to the difference in concentration between the two conformations of cumene that orient either the pro (R)-methyl orthe pro (S)-methyl group for catalysis (12). The difference in concentration between these two conformations arises, presumably, from a difference in the degree of steric hindrance encountered by each conformation in its interaction with the amino acid residues defining the active site of the enzyme. If one of the methyl groups is specifically trideuterated then the magnitude of the observed isotope effect for the hydroxylation reaction will not only reflect the isotope effect associated with carbon (deuterium) bond cleavage but also the active site concentration imbalance between trideutero and normal methyl groups that is driven by stereoselectivity. Thus, the observed isotope effect for hydroxylation of (S)-l,l,l-trideutero-2-phenylpropane would be expected to be less than the isotope effect associated exclusively with the bond breaking event because hydroxylation of this speci-

282

~

H2OH

CD3

(F

H3

CD3

kH ~ ,

J

(s)

k H / k D = 4.2

~

H3

(s) CD2OH

(R)

Figure 7. The intramolecular deuterium isotope effect observed for hydroxylation of (S)-l,l,l-trideutero-2-phenylpropane. The stereochemistry assigned to the hydroxylated product is opposite that assigned to the substrate. In this regard it is important to note that the reversed stereochemical assignments are a result of the absolute configurational nomenclature rules and not because of a reversal of absolute stereochemistry. This is also why the 1,1,1-trideuteromethyl group of (S)-l,l,l-trideutero-2phenylpropane is designated as the pro (R)-methyl group.

fic methyl group (pro (R)-methyl) is stereoselectively favored (higher active site concentration) over hydroxylation of the alternate or pro (S)-methyl group (Figure 7). Conversely, the observed isotope effect for hydroxylation of (R)1,1,1-trideutero-2-phenylpropane would be expected to be greater than the isotope effect associated exclusively with the bond breaking event because hydroxylation of this specific methyl group (pro (S)-methyl) is not stereoselectively favored (lower active site concentration) over hydroxylation of the alternate or pro (R)-methyl group (Figure 8). Consistent with these expectations, (kH/kD)obs for hydroxylation of (S)-l,l,l-trideutero-2-phenylpropane was found to be 4.2, while (kH/ko)obs for (R)-l,l,l-trideutero-2-phenylpropane was found to be 15.6 (12).

2.2. Factors Effecting the Magnitude of an Intramolecular Isotope Effect and tthe Relationship Between (kH/kO)obs-and kH/ko-Branched Pathways While the ratio of the enzyme-substrate complexes (ESH and ESD) must be maintained at unity in order for (kH/ko)obs to be equal to kH/ko, rapid

283

~D3

C~'"H

[ ~

~D3

CH2OH (R) kH/kD = 15.6

~D2OH (R)

(S) Figure 8. The intramolecular deuterium isotope effect observed for hydroxylation of (R)-l,l,l-trideutero-2-phenylpropane. The stereochemistry assigned to the hydroxylated product is opposite that assigned to the substrate. In this regard it is important to note that the reversed stereochemical assignments are a result of the absolute configurational nomenclature rules and not because of a reversal of absolute stereochemistry. This is also why the 1,1,1-trideuteromethyl group of (R)-l,l,l-trideutero-2phenylpropane is designated as the pro (S)-methyl group.

equilibration of equivalent, but isotopically distinct, intramolecular oxidation sites is not the only means by which this condition can be met. If a reaction path is branched, i.e. multiple products arise from an enzyme-substrate complex, formation of an alternate product when a site of oxidation is suppressed by being deuterated can serve to unmask the isotope effect associated with hydroxylation at the deuterated site. Inequality between ESH and ESD as a consequence of build-up of the enzyme-substrate in which the deuterated site is poised for catalysis can be relieved by branching of the reaction to oxidize a nondeuterated site to generate an alternate product. A kinetic model that expresses the effect of an isotopically sensitive branched pathway is presented in Figure 9. In this model the products P1 and P2 are assumed to be formed irreversibly and arise from kinetically indistinguishable enzymesubstrate complexes, the combination of which can be expressed by ESH or ESD. It is further assumed that a single rate constant, k2, describes the fractionation of ES to ESN and ESD and a single rate constant k-2 describes the formation of ES from ESH and ESD. Thus, increased formation of P2 from ESD under conditions where the overall rate of conversion of ESD to ESH is

284

[E]

+

kH

[ESH]

[E] + [S]

~

kl~

[P2]

[E]

+

[P1]

kD ~--~ I-E]

+

[P1]

X.

~

[ES] ..k2

k_l -~k~.2 [ES D]

[E]

+

[P2]

Figure 9. A kinetic model for an enzymatically catalyzed reaction that includes the possibility isotopically sensitive branched pathway,

slow relative to P1 product formation, prevents the expected concentration build-up of ESD and the observation of a masked kH/kD. A general equation, Eq. (2), that quantitatively describes the effect of branching on the isotope effect was derived assuming steady state kinetics (10). (kH/kD)ob s -

kH/kD + kH/(k-2 + k3,) (k-2 + k3)/(k-2 + k 3 , ) + kH/(k2 + k3,)

(2)

If kz = k3, the equation simplifies to Eq. (3)"

(kH/kD)obs =

kH/kD + kH/(k-2 + k3,) 1 + kill(k-2 + k3,)

(3)

Unmasking of the magnitude of an intrinsic isotope effect by a branched reaction pathway was a major result to emerge from a study of the o~-hydroxylation of a series of selectively deuterated n-octanes (10). As indicated above, the (kH/kO)obs for the ~hydroxylation octane-l,2,3-2H7, was only 4 because of the relatively slow rate of equilibration between the two terminal

285

~

D-CD3

kH , ~ HOCH2.,- v A ~ CH3." v v

v

Eo

~,,,,D -CD3 UD

kH/kD = 4.0

~ ~ ~"D~,cD2OH CH3~ v v

D'"D Figure 10. The intramolecular deuterium isotope effect observed for the ~-hydroxylation of octane-l,2,3-2H7.

kH ,,~ HOCH2~

C

D

3 kH/kD = 11.8

CH3~

C

D

3 ~kD

CH3~

C

D

z

O

H

Figure 11. The intramolecular deuterium isotope effect observed for the w-hydroxylation of octane-l-2H3.

methyl groups and the catalytic site of the enzyme (Figure 10). By contrast, the (kH/kD)obs for the w-hydroxylation of octane-l-2H3 was 11.8 (Figure 11). Since both deuterated substrates would have identical rates of equilibration between the terminal methyl groups, the large isotope effect for the w-hydroxylation of octane-l-2H3 must be the consequence of some other factor. The other factor is branching of the hydroxylation reaction from the w to the w - 1 position. The difference in magnitude of (kH/kO)obs between the two deuterated substrates is because the hydroxylation of octane-l,2,3-2H7 is suppressed at both the w and ( o - 1 sites by the presence of deuterium, which leads to an imbalance between ESH and ESo and results in a masked kH/kD. In contrast, w - 1 hydroxylation of octane-l-2H3 is promoted by the presence of deuterium at the ~, site. Thus, a buildup of an imbalance between ESH and ESD is prevented and the result is that (kH/kD)ob~ approaches kH/ko.

2.3. Determination of kH/ko and the Establishment of a Maximum kH/ko for Carbon-Hydrogen Bond Oxidation An additional unexpected, but significant, finding to emerge from the octane study was that it provided a new method for determining intrinsic isotope

286 effects. If Eq. (3) is rearranged to yield Eq. (4) some interesting properties emerge when limits are considered. As k-2 approaches infinity, (kH/kD)obs approaches kH/ko.

k H / k D : (kH/kD)ob s Jr- [(kH/(kD)obskH/(k-2 4- k3')] - k H / ( k - 2 + k3,)

(4)

Thus, at very large values of k-2, (kH/ko)obs should provide a very good estimate of kH/kD. However, the problem is that there is no easy way to evaluate k-2. Therefore, there is no easy way to determine when this condition (large k-2) is being met, or to establish what the value of kH/kD might be, for the specific reaction being studied. Fortunately, the existence of a branched reaction pathway makes an independent estimate of kH/kD possible when k_2 approaches 0. If k3, is assumed to be approximately equal to k3,, substitution of k_2 into Eq. (4) reveals that kH/kD can be estimated from (kH/kD)obs and the ratio of products obtained from the nondeuterated substrate (10). The measured ratio of 2-0ctanol to 1-0ctanol obtained from the P4502B1 catalyzed hydroxylation of octane was 23:1, while (kH/kD)obs, as indicated above, was 11.8. Substitution of these values into Eq. (4) indicates that kH/kD for the reaction is 12.2. The observed isotope effect of 11.8 for octane-l-2H3 is a combination of a primary and two secondary isotope effects (Figure 11). Based on the fact that isotope effects are multiplicative, Hanzlik et al. (13) proposed a method for separating the composite effects of both primary and e-secondary deuterium isotope effects by analyzing the isotope effects obtained from a series of appropriately deuterated substrates. Using their method, the primary isotope effect for the o~-hydroxylation of n-octane was found to range between 7.6 (microsomes) and 9.2 (P450b or CYP2B1 in the new nomenclature) while that for the secondary isotope effect was found to range between 1.09 (microsomes) and 1.14 (P450b/CYP2B1) (14). A primary isotope effect falling within this range is consistent with the maximum value of 8.58 (15) to 9 (16) theoretically predicted for the kH/kD associated with the oxidation of a carbon-hydrogen bond. The correspondence between the values of the experimentally determined isotope effects and the theoretical maximum implies that the reaction has a highly symmetrical transition state. Perhaps even more importantly, the determination of a maximal kH/kD for the ~o-hydroxylation of noctane sets a limit, excluding tunneling effects, for the maximum primary isotope effect that can be observed for the oxidative cleavage of any carbonhydrogen bond catalyzed by cytochrome P450.

287

2.4. Factors Affecting the Magnitude of kH/kD Having established that the maximum kH/kD value for a cytochrome P450 catalyzed hydroxylation of a carbon-hydrogen bond is 9 and is attained in the hydroxylation of the primary carbon of a normal alkane sets an important standard for aiding interpretation. Knowing that a symmetrical transition state characterizes the carbon-hydrogen bond cleavage of an unsubstituted primary carbon atom enables the investigation of factors which lead to less symmetrical transition states and lower values of kH/kD. The variability in magnitude of kH/ko defines a bell-shaped curve that correlates with the symmetry of the transition state in reactions involving carbon-hydrogen bond cleavage (17-20) (Figure 12). The maximum primary kH/kD is achieved when the transition state for the reaction is symmetrical, and minimum values are approached when the transition state approaches the limits of being either more reactant-like or more product-like. Since cytochrome P4502B1 catalyzed hydrogen atom abstraction from a terminal methyl group of n-octane to form a primary carbon radical was found to be symmetrical (10), replacing a hydrogen on the primary carbon of a substrate with any substituent, if it has any effect at all, can only lead to a less symmetrical transition state by either stabilizing or destabilizing the carbon radical that is formed as an intermediate relative to the carbon radical that would be formed in the absence of the substituent. This conclusion suggests that the magnitudes of the primary kH/kD associated with carbon hydroxylation of secondary and tertiary alkyl carbons and e-carbon substituted alkanes should all be less than the theoreti-

0 ,---,-

/ F+30__.H___C ......

\

.._.,

o

F+30-H Reaction Coordinate

/ + .C---

\

r

Figure 12. The magnitude of kH/ko for hydrogen atom abstraction from carbon as a function of reaction coordinate symmetry.

288 cal maximum value of 9. The actual value should reflect the degree to which the substituent on the carbon being oxidized causes the transition state to become either more reactant-like or more product-like. While systematic studies with cytochrome P450 and various substituted alkanes specifically exploring this effect have not appeared there are a number of studies that while focused on other questions provide data that strongly support this hypothesis and several examples will be considered shortly. A second factor, besides substrate structure, that can at least potentially effect the transition state symmetry of hydrogen atom abstraction from a primary carbon is the enzyme itself. This leads to the fundamental question of whether different P450s can catalyze the same oxidative reaction with different kH/kos. If different P450s can catalyze the same reaction with different kH/kos, two different explanations could account for the observation. The first would simply be that the various P450s catalyze the same reaction by different mechanisms. The second would be that the symmetry of the transition state and, therefore, kH/kD is altered by differences in the stability of the [FeO] 3+substrate complex of the various P450s as a consequence of differences in active site architecture. Since substantial evidence exists that supports the hypothesis that cytochrome P450 catalyzed carbon hydroxylation reactions occur by a hydrogen atom radical recombination mechanism (21), the first explanation that different P450s catalyze carbon hydroxylation by different mechanisms is unlikely. The second explanation that the symmetry of the transition state might be highly sensitive to active site architecture is a seemingly strong possibility. However, it is less compelling when considered in the context of the chemical nature of the cytochrome P450s. As indicated earlier the cytochromes P450 are unlike most enzymes in that catalysis is primarily dependent on the inherent reactivity of the oxene-heme complex rather than substrate binding. Thus, the symmetry of a transition state for an oxidation reaction catalyzed by the P450s might not be as sensitive to active site architecture as might be expected, based on the behavior of other enzymes, and kH/ko might be a reasonably constant value for specific reaction types, particularly those that occur with either highly symmetrical or highly unsymmetrical transition states.

2.5. Sensitivity of kH/ko to Enzyme Structure To explore the question of whether, or not, different P450s can catalyze the same oxidative reaction with different kH/kos, the isotope effects for the (ohydroxylation of octane with purified P451A1, P4502B1 and P4502B4 were determined (22). Since the three enzymes chosen represent members from

289 different species (rat and rabbit), and different P450 genetic families (1A and 2B), they presumably have very different active site amino acid composition and architecture. However, while the three enzymes catalyzed the (o and o)-1 hydroxylation of octane in very different ratios and with very different efficiencies, all three gave a virtually identical primary isotope effect of 9.0 to 9.1 for ~o-hydroxylation. Similar findings were reported for N-demethylation in a series of para substituted (H, CI, CN, NO2) dimethylanilines catalyzed by CYP1A2, CYP2B1, CYP4B1, or the bacterial isoform CYP101 (23). While the intrinsic isotope effect was found to vary with substituent in this study, the magnitude of the isotope effect for any given substrate was found to be essentially invariant across the four different P450 isoforms. The observation that active site architecture has no discernible effect in two very different P450 catalyzed oxidative reactions (aliphatic hydroxylation and N-dealkylation) implies that kH/kD for a cytochrome P450 catalyzed oxidative carbonhydrogen bond cleavage reaction is a reasonably rugged value.

2.6. Sensitivity of kH/ko to Electronic Effects in Substrate While kH/kD appears to be insensitive to active site architecture, and presumably steric effects in general, there is evidence that strongly indicates that kH/kD is more sensitive to electronic effects within the substrate which impinge directly on the energetics of the bond breaking event. This was alluded to above where it was stated that several examples would be presented in support of the hypothesis that substitution of a primary carbon would lead to a lower kH/ko for its hydroxylation proportional to the reactions departure from a symmetrical reaction coordinate. For example, because of the possibility of resonance stabilization, cytochrome P450 catalyzed formation of a benzylic radical should be exothermic relative to the ~o-hydroxylation of n-octane, its transition state more reactant-like and the reaction should occur with a kH/ko less than 9. In accord with these expectations, Hanzlik and Ling (24) report that the primary deuterium isotope effect for the benzylic hydroxylation of ortho and para xylene derived from a series of selectively deuterated analogs of each substrate is 5.25 _+ 0.16 and 5.58-+ 0.49, respectively. The values of 5.25 and 5.58 for ortho and para xylene indicate that the isotope effect for benzylic radical formation lies on the ascending portion of the bell-shaped curve illustrated in Figure 12 and, therefore, should be reasonably sensitive to further structural alteration, e.g. aromatic substitution. Electron withdrawing substituents would make the transition state less reactant-like and, therefore, expected to move the substrate up the curve (more symmetrical, a larger kH/kD), while electron donating groups would be ex-

290 pected to make the transition state even more reactant-like and, therefore, expected to move the substrate down the curve (less symmetrical, a smaller kH/ko). Whether, or not, these expectations are met remains to be tested. A second example can be found with cytochrome P450 catalyzed N-dealkylation reactions. A large number of studies have measured the deuterium isotope effects associated with these reactions as a means of exploring the reaction mechanism. With few exceptions, the magnitude of the isotope effects found are relatively low and fall within the range, 1.5-3.0. Because cytochrome P450 carbon atom hydroxylation and O-dealkylation reactions are generally associated with large deuterium isotope effects which are believed to reflect the initial removal of a hydrogen atom, the relatively small isotope effects associated with N-dealkylation reactions were thought to be inconsistent with a similar mechanism, i.e. initial hydrogen atom abstraction from the carbon atom a to nitrogen. The mechanism believed to be the most consistent with the magnitude of the isotope effect was thought to involve the initial transfer of a nonbonded electron from nitrogen to the enzyme to form an enzyme stabilized hydroxy radical (25-29) (Figure 13). After electron transfer, a proton would be lost from the adjacent carbon, then a carbonbased radical would form upon electron reorganization. Recapture of the initially formed hydroxy radical by the carbon-based radical to generate an unstable carbinolamine intermediate would be the penultimate step to final product formation. As indicated above, the belief that initial electron transfer is the general mechanism for cytochrome P450 catalyzed N-dealkylation reactions rests on two critical assumptions concerning the magnitude of deuterium isotope effects. The first is that deuterium isotope effects of low magnitude are consistent with, and expected of, the initial electron transfer mechanism. The second is that an initial hydrogen atom abstraction mechanism should proceed with large isotope effects based on those found for O-dealkylation and carbon hydroxylation. However, both assumptions appear to be flawed. Dinnocenzo and Banach (30) report that the deprotonation of the known amine radical cation, N,N-(di-para-anisyl)-N-methylaminium ion proceeds with deuterium isotope effects ranging in value from 6 to 9 depending on the strength of the base used to catalyze the deprotonation. Their results unambiguously establish that the magnitude of a deuterium isotope effect associated with N-dealkylation via an electron transfer mechanism is not inherently small but depends on reaction conditions. As to the second assumption, dealkylation reactions like benzylic hydroxylation would be, in general, exothermic relative to the (o-hydroxylation of n-octane, since the presence of an a nitrogen atom would tend to stabilize radical formation. The

291

R2.N.--CH3

)

+

H

R2N--

[ R2N--CH 2 ]

R2N--CH 2

N"., ,.XN

:N H+

J

/Fe+3/ Figure 13. N-dealkylation via the electron transfer mechanism.

transition state for such a reaction, therefore, would be expected to be more reactant-like and, as a consequence, associated with an inherently smaller intrinsic primary isotope effect. Evidence that this is indeed the case was recently provided by Dinnocenzo and coworkers (11, 23) who found that the demethylation of a series of substituted dimethylanilines catalyzed by the hydrogen atom abstracting species, tert-butoxy radical, was characterized by kH/kD values ranging from 2.5 to 3.9. Since it now appears that both electron transfer and hydrogen atom abstraction mechanisms can be associated with kH/kos, having either low or high values, it is reasonable to conclude that the magnitude of a kH/ko for any given P450 catalyzed N-dealkylation reaction by itself cannot distinguish between mechanisms. As alluded to earlier, the intramolecular isotope effects associated with the cytochrome P450 catalyzed N-demethylation of several para substituted N,Ndimethylaniline analogs varied with the electronegativity of the substituent (23). In these substrates, each of the N-methyl groups was substituted with two deuterium atoms to minimize masking effects and insure that (kH/kD)obs

292 approached kH/kD. The values of (kH/kD)obs actually found for deuterated N,Ndimethylaniline and three deuterated analogs with expressed CYP2B1 were: N,N-dimethylaniline, 2.5; p-chloro, 2.7; p-cyano, 3.7; and p-nitro 3.9. While (kH/kD)obs varied with electronegativity (kH/kD)obs for any individual substrate was virtually invariant (maximum variation of 0.4 isotope effect units) across four different P450s (CYP2B1, CYP4B1, CYP1A2 and CYP102).

2.7. Distance as a Factor That Masks kH/kD The apparent lack of sensitivity of the intrinsic primary isotope effect to active site architecture has the potential of opening up a new methodological approach to studying P450 active site structure. It suggests that the masking effect might be exploited to provide a unique approach to obtaining information on the relative three-dimensional spatial capacities of the active sites of various P450 isoforms. The more open (greater free space) an active site, the less substrate motion would be hindered, and the more likely the equivalent protio and deuterio sites in a symmetrical substrate would be to equilibrate with the active oxygen prior to reaction. This in turn would lead to a fuller expression of the intrinsic isotope effect. Thus, a set of substrates in which the distance between equivalent protio and deuterio sites differed between members of the set, might be expected to have a range of (kH/ko)obs values whose magnitude varied as a function of the differences in distance. Similarly, a correlation between the dimensions of free space within an active site, and changes in the magnitudes of (kH/kO)obs, would also be expected. Preliminary studies in our laboratory with a series of progressively longer n-alkanes and isomeric xylenes suggests that this is indeed the case.

3. INTERMOLECULAR ISOTOPE EFFECTS

The discussion thus far has stressed the notion that utilization of isotope effect experiments of intramolecular design can be a powerful methodology for simplifying and clarifying the meaning of the magnitude of an observed isotope effect. Unfortunately, the structural symmetry requirement (two identical intramolecular catalytic sites) limits the kinds of compounds that can be studied using this approach. For example, because of the lack of appropriate substrate symmetry, ring hydroxylation of a steroid would fall into this category. Thus, for some problems, experiments of intermolecular isotope effect design are the only approach possible, despite the limitations that can result from masking. Even with the inherent problems, cytochrome P450

293 experiments of intermolecular isotope effect design can still be rich with information because of the possibility of forming multiple products via shunting to branched reaction pathways. The theoretical basis for the interpretation of the observed isotope effects for enzymatically catalyzed processes which can generate more than one product from a single substrate has been established (10, 31, 32). Knowledge of how branched reactions can effect the magnitude of an observed isotope effect has provided new insights. Indeed it clarifies why an isotope effect is ever observed with a P450 catalyzed reaction. If formation of the heme-oxene-substrate complex (EOS) is irreversible, as it is believed to be, then an isotope effect should never be observed since the isotopically sensitive reaction occurs after irreversible formation of EOS. However, the possibility of shunting EOS back to an enzyme substrate complex (ES), by reduction of the oxene to water, is the phenomenon that allows isotopic discrimination and the observation of isotope effects in the absence of the formation of more than a single product. This increased understanding has led to the development of new approaches that allow information to be gained from intermolecular deuterium isotope effect experiments that could not be gained without the new insight. Recently, Gillette et al. (33) published a theory that describes the isotope effects to be expected from three different kinetic mechanisms by which a single substrate, single enzyme, multiple product system can operate. The three mechanisms are" (1) the parallel pathway mechanism; (2) a nondissociative mechanism; and (3) a dissociative mechanism which define three different ways in which the substrate can interact with the active site of the enzyme. In the parallel pathway mechanism, a substrate that is converted to more than a single product binds to the enzyme in distinct ways that leads to the formation of a different EOS complex for each product that is ultimately formed. The complexes are rigid and stable and not free to convert to EOS complexes with any orientation other than the one with which they were initially formed. Once formed, the EOS complexes can only go on to generate the products dictated by their respective orientations. In the nondissociative mechanism, the EOS complexes are not rigid and have sufficient lifetime and freedom to interconvert while the substrate is in the active site of the enzyme. Finally, in the dissociative mechanism the EOS complexes have sufficient lifetime to dissociate to enzyme.oxene (EO) and substrate (S) and then recombine to form a new EOS complex with either the same or different orientation than the original. The theory states that the three kinetic mechanisms can be distinguished by measuring the magnitude of the isotope effect associated with product formation from a nondeuterated site both competitively and noncompetitively. Inthe competitive experiment, the ratio of products formed

294

TABLE 1. The Deuterium Isotope Effects Expected via Competitive and Noncompetitive Experiments for Cytochrome P450 Catalyzed Hydroxylation of a Substrate at a Nondeuterated Site by Three Possible Kinetic Mechanisms: Parallel Pathway, Nondissociative or Dissociative ,,

Mechanism

Parallel pathway Nond issociative Dissociative

Isotope effect Competitive VH/Vo

Noncompetitive ( V/KH)/(V/KH)D

1.0 < 1.0 1.0

1.0 < 1.0 < 1.0 to > 1.0

at the nondeuterated site from a 50:50 mixture of normal and deuterated substrate is measured to determine the isotope effect. In the noncompetitive experiment, the isotope effect is determined from the ratio of products formed at the nondeuterated site when normal and deuterated substrates are independently incubated with enzyme. The value of the isotope effects to be expected for each combination of experimental design and mechanism are given in Table 1. A value of 1.0 for the product formation rate ratio between normal and deuterated substrates, VH/VO, in the competitive experiment, but <1.0 to > 1.0 for the ratio of V/Ks between normal and deuterated substrates, (Vrnax/Krn)H/(Vmax/Krn)D in the noncompetitive experiment is diagnostic for a dissociative mechanism. Values of 1.0 in both kinds of experiment are diagnostic for a parallel path mechanism while values of <1.0, again in both kinds of experiments, are diagnostic for a nondissociative mechanism. Gillette's theory has been applied to the CYP2D6 catalyzed oxidation of sparteine by Ebner et al. (34). These workers report that the formation of 5,6-didehydrosparteine from either 2S-[2H]-sparteine (Figure 14), or 2,2-[2H2] sparteine, proceeds with isotope effects of <1.0 in experiments run under both competitive and noncompetitive conditions. When oxidation at the 2 position is retarded by the presence of deuterium, formation of the 5,6didehydrosparteine metabolite is enhanced resulting in the observation of an inverse isotope effect for the formation of this metabolite. The isotope effect results are consistent with a nondissociative kinetic mechanism, Table 1, indicating that sparteine has sufficient freedom of motion within the active site of CYP2D6 to allow formation of either the 2,3-didehydrosparteine, or the 5,6-didehydrosparteine, but not sufficient freedom to allow its complete

295 }-]

Figure 14. The P4502D6 catalyzed formation of 2S-[2H]-5,6-didehydrosparteine and 2,3-didehydrosparteine from 2S-[2H]-sparteine.

dissociation from the active site followed by reassociation prior to the first irreversible step leading to product formation. In summary, it appears that the unique catalytic characteristics, that result from the inherent reactivity of the P450s, makes the application of deuterium isotope effects to their study particularly powerful both in terms of delineating the mechanisms of the oxidative reactions they catalyze and in revealing subtle properties of their active site structure. The potential of using deuterium isotope effects to gain a new understanding of the factors that dictate the catalytic behavior of the cytochromes P450 would seem to be limited only by our imaginations.

ACKNOWLEDGMENTS The preparation of and part of the research described in this chapter were supported by Grant GM-36922 from the National Institutes of Health. I also wish to acknowledge the major contributions of my research associates and former students, particularly Drs. Jeff Jones and Ken Korzekwa, whose insights, imagination and hard work formed the foundation of the work to come from my laboratory.

296

REFERENCES 1. D.B. Northrop, Biochemistry, 14 (1975) 2644. 2. D.B. Northrop, Isotope Effects on Enzyme Catalyzed Reactions, W.W. Cleland, M.H. O'Leary and D.B. Northrop (eds) (University Park Press, Baltimore, 1978), p. 122. 3. D.B. Northrop, Biochemistry, 20 (1981) 4056. 4. W.W. Cleland, CRC Crit. Rev. Biochem., 13 (1982) 385. 5. L.M. Hjelmeland, L. Aronow and J. R. Trudell, Biochem. Biophys. Res. Commun., 76 (1977)541. 6. G.T. Miwa, W.A. Garland, B.J. Hodshon, A.Y.H. Lu and D.B. Northrop, J. Biol. Chem., 255 (1980) 6049. 7. M.H. Gelb, D.C. Heimbrook, P. Malkonen and S.G. Sligar, Biochemistry, 21 (1982) 370. 8. J.R. Lindsay Smith, N.E. Nee, J.B. Noar and T.C. Bruice, J. Chem. Soc., Perkin. Trans., 2 (1984) 255. 9. A.B. Foster, M. Jarman, J.D. Stevens, P. Thomas and J.H. Westwood, ChemicoBiol. Interact., 9 (1974) 327. 10. J.P. Jones, K.R. Korzekwa, A.E. Rettie and W.F. Trager, J. Am. Chem. Soc., 108 (1986) 7074; and correction, J. Am. Chem Soc., 110 (1988) 2018. 11. J.P. Dinnocenzo, S.B. Karki and J.P. Jones, J. Am. Chem. Soc., 115 (1993) 7111. 12. K. Sugiyama and W.F. Trager, Biochemistry, 25 (1986) 7336. 13. R.P. Hanzlik, K. Hogberg, J.B. Moon and C.M. Judson, J. Am. Chem. Soc., 107 (1985) 7164. 14. J.P. Jones and W.F. Trager, J. Am. Chem. Soc., 109 (1987) 2171; and correction, J. Am. Chem Soc., 110 (1988) 2018. 15. P. Shea, S.D. Nelson and G.P. Ford, J. Am. Chem. Soc., 105 (1983) 5451 16. R.P. Bell, Chem. Soc. Rev., 3 (1974) 513. 17. L. Melander, Isotope Effects on Reaction Rates (Ronald Press, New York, 1960). 18. F.H. Westheimer, Chem. Rev., 61 (1961) 265. 19. G.S. Hammond, J. Am. Chem. Soc., 77 (1965) 334. 20. W.A. Pryor and K.G. Kneipp, J. Am. Chem. Soc., 93 (1971) 5584. 21. P. Ortiz de Montellano, Cytochrome P-450, Structure, Mechanism, and Biochemistry, P. Ortiz de Montellano (ed.) (Plenum Press, New York, 1986) p. 235. 22. J.P. Jones, A.E. Rettie and W.F. Trager, J. Med. Chem., 33 (1990) 1242. 23. S.B. Karki, J.P. Dinnocenzo, J.P. Jones and K.R. Korzekwa, J. Am. Chem. Soc., 117 (1995) 3657. 24. R.P. Hanzlik and K-H.J. Ling, J. Am. Chem. Soc., 115 (1993) 9363. 25. R.P. Hanzlik and R.H. Tullman, J. Am. Chem. Soc., 104 (1982) 2048. 26. G.T. Miwa, J.S. Walsh, G.L. Kedderis and P. F. Hollenberg, J. Biol. Chem., 258 (1983) 14445. 27. F.P. Guengerich and T.L. Macdonald, Acc. Chem. Res., 17 (1984) 9. 28. L.T. Burka, F.P. Guengerich, R.J. Willard and T.L. Macdonald, J. Am. Chem. Soc., 107 (1985) 2549. 29. F.P. Guengerich, Chem. Res. Toxicol., 3 (1990) 21. 30. J.P. Dinnocenzo and T.E. Banach, J. Am. Chem. Soc., 111 (1989) 8646. 31. K.R. Korzekwa, W.F. Trager and J.R. Gillette, Biochemistry, 28 (1989) 9012. 32. J.R. Gillette and K. Korzekwa, Adv. Exp. Med. Biol., 283 (1991) 87. 33. J.R. Gillette, J.F. Darbyshire and K. Sugiyama, Biochemistry, 33 (1994) 2927. 34. T. Ebner, C.O. Meese and M. Eichelbaum, Mol. Pharmacol., 48, (1995) 1078.

297

CHAPTER 16

BIOTRANSFORMATION AND EXCRETION: PHARMACOKINETIC STUDIES

THOMAS R. BROWNE Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center

1. INTRODUCTION

Clearance (CL) and elimination half-life (tl/2) are the parameters typically measured in clinical pharmacokinetic studies. Single dose CL and tl/2 can be determined readily without using stable isotope labeled (SIL) tracer techniques. SIL tracer techniques are useful for determining pharmacokinetic values at steady state plasma concentration (single tracer dose studies) and the presence or absence of concentration-dependent (nonlinear) or timedependent pharmacokinetic properties or drug interactions (multiple tracer dose studies).

2. METHODS

An SIL tracer dose of drug is administered by the intravenous (i.v.) route. A number of plasma samples are taken, and the concentration of SIL and unlabeled drug is determined for each sample by gas chromatography-mass spectrometry (GC-MS). Volume of distribution (Vd), CL and tl/2 are calculated from plasma concentration versus time relationships. A single SIL study can be used to determine pharmacokinetic values at typical steady state plasma concentrations; such a study performed before, and one or more tracer studies performed during, long-term therapy can be combined to determine the presence of concentration-dependent or time-dependent pharmacokinetics. Alternatively, SIL studies performed at two different steady state plasma concentration values can be used to determine the presence or absence

298 of concentration-dependent pharmacokinetics. Finally, performing SIL tracer studies of drug 1 before and after adding drug 2 can determine the presence or absence of pharmacokinetic drug-drug interactions (see Chapter 18). If test doses are administered orally, it is possible to measure directly only the elimination half-life, tl/2 and area under the plasma concentration-time curve (AUC); CL and Vd cannot be measured, although CL can be estimated using an assumed value for Vd. In new drug development the usual questions are whether a drug has linear, concentration-dependent, or time-dependent pharmacokinetic properties and how these properties effect therapeutic usage. The findings of SIL tracer doses in each of the three types of pharmacokinetics will be discussed in detail below.

3. TYPES OF PHARMACOKINETICS

The following discussions assume a drug is metabolized by a single enzyme with a maximum velocity of Vrnax and a Michaelis constant of Km. Drug clearance (CL) can be defined as: CL = Vrnax/(Km + C)

(1)

where C is drug plasma concentration. Drugs whose value for C is small in relation to Km and whose values for Vmax and Kr, do not change over time will exhibit linear pharmacokinetics (i.e. clearance does not vary with plasma concentration or time). Drugs whose value for C is similar to, or greater than, Kr, will exhibit concentration-dependent pharmacokinetics (i.e. clearance will vary inversely with plasma concentration). Drugs whose values for Vrnax and/or Km vary with time will exhibit time-dependent pharmacokinetics (e.g. enzyme induction may result in increased values for Vrnax and CL). Tracer dose elimination rate constant (k) is the actual quantity measured in SIL tracer dose studies. Elimination rate constant, tl/2, and CL are related as follows: tl/2 = 0.693/k

(2)

CL = k x Vd

(3)

We have shown (1) that the elimination rate constant of labeled tracer drug can be expressed by the equation,

299 k = V=,x/(Km + Cu = CL)

(4)

where CL iS the plasma concentration of labeled drug, and Cu is the plasma concentration of unlabeled drug. Plasma concentration of labeled drug versus time (t) relationships can be expressed by the equation, CL = Co e -kt

(5)

where Co is the initial plasma concentration of labeled drug immediately after a rapid bolus infusion (the time = 0 intercept) (1). Equations (4) and (5) assume the total (labeled and unlabeled) plasma concentration of drug is relatively constant during the test period and that C, is small in comparison with Kr,,. Equations 4 and 5 predict: (1) tracer-dose plasma concentration versus time plots will appear linear on semilog plots whether the plasma concentration is in the linear or the concentration-dependent (nonlinear) portion of a drug's dose versus steady state plasma concentration relationship; (2) the elimination rate constant and elimination half-life of tracer doses of drug will not vary with plasma concentration in drugs with linear pharmacokinetics; (3) the elimination rate constant of tracer doses of drug will vary inversely, and the elimination half-life directly, with plasma concentration in drugs with concentration-dependent pharmacokinetics; and (4) the elimination rate constant of tracer doses of drug will vary directly with Vmax and inversely with Km as Vmax and Kr,, change over time in drugs with time-dependent pharmacokinetics. Thus, the presence or absence of change in tracer dose elimination rate constant (or half-life) with changing total drug plasma concentration and time can be used to determine the presence or absence of concentration-dependent or time-dependent pharmacokinetics (1).

4. LINEAR PHARMACOKINETICS

As an example of a drug with linear pharmacokinetics, we determined the k values of phenobarbital using SIL tracer dose methods before (Week 0) and 4 and 12 weeks after beginning monotherapy in six subjects (Table 1, Figure 1) (1, 2). Phenobarbital tracer dose plasma concentration versus time plots were tested for semilog linearly using mathematical procedures described in detail elsewhere (1). As predicted by the above considerations, the plots showed a high degree of semilog linearly (r 2 = 0.791 - 0.996, p < 0.005). Phenobarbital elimination rate constant did not correlate significantly with phenobarbital plasma concentration (r 2 = 0.19, N.S.) and did not change significantly over 12 weeks. Thus, phenobarbital failed to show concentration-

300 T A B L E 1. Pharmacokinetic Monotherapy

Data f r o m SlL T r a c e r Doses of P h e n o b a r b i t a l in Six Patients D u r i n g

Week 0 Mean total plasma concentration (l~g/ml) 1

Week 4

Week 12

1.2 _+ 0.1

12.5 _ 3.0

Volume of distribution (L/kg)

0.624 +_ 0.045

0.610 _+ 0.044

0.605 _+ 0.0402

Elimination rate constant (1/hr)

0.007 +_ 0.002

0.008 -+ 0.003

0.007 +_ 0.0022

Elimination half-life (hr)

111.5 +_ 35.8

99.9 - 35.8

109.0 _+ 42.02

0.0709 _+ 0.0268

0.0767 _+ 0.0226

0.0703 _+ 0.02112

Total clearance (ml/min/kg)

13.1 _+ 4.5

1Labled and unlabeled plasma concentration. 2Difference among values for weeks 0, 4, 12 not significant by analysis of variance. Tracer dose study performed 4 or 12 weeks after beginning chronic therapy. From Browne e t al. (2) with permission. z 0

5 4

-/

i o-----o.,

/

I

3 _

x-.-------.K W E E K I 2

0

0-0"

.8 IE 2~ w

.6 _ l 0

, 24

. HOURS

, ............. 48

AFTER

i ............ 72

I 96

DOSE

Figure 1. Tracer dose serum concentration versus time relationships for SIL tracer

dose molecules of phenobarbital after 90 mg tracer infusions in a patient performed before (Week O) and 4 and 12 weeks after beginning chronic administration. From Browne e t al. (2) with permission.

301 dependent or time-dependent changes in k and meets the criteria for linear pharmacokinetics.

5. CONCENTRATION-DEPENDENT (NONLINEAR) PHARMACOKINETICS

As an example of drug with concentration-dependent pharmacokinetics, we determined the k values of phenytoin using SIL tracer methods before (Week 0) and 4 and 12 weeks after beginning monotherapy in six subjects (Table 2, Figure 2) (1, 3). Phenytoin tracer dose plasma concentration versus time plots were tested for semiiog linearly using mathematical procedures described in detail elsewhere (1). The plots showed a high degree of semilog linearly (r 2 = 0.988-0.999, p < 0.01). This illustrates the prediction of Eqs. (4) and (5) that tracer dose plasma concentration versus time relationships will be linear for drugs with concentration-dependent pharmacokinetics if total drug concentration is held constant during the test interval. The elimination rate constant decreased significantly (p < 0.01) as mean plasma concentration rose over 12 weeks and showed significant inverse correlation (r 2 = 0.76, p < 0.001) with total phenytoin plasma concentration. Thus, phenytoin tracer dose plasma

TABLE 2. Pharmacokinetic Data from SIL Tracer Doses of Phenytoin in Six Patients During Monotherapy

Week 0 Mean total plasma concentration (l~g/ml) 1 Volume of distribution (L/kg) Elimination rate constant (1/hr) Elimination half-life (hr) Total clearance (ml/min/kg)

Week 4

Week 12

1.2 _+0.5

5.5 +_0.5

10.0 _+6.12

0.63 _+0.06

0.68 _+0.09

0.73 +_0.09

0.056 _+0.015

0.040 _+0.009

0.031 _+0.0132

13.2 _+3.6

18.4 +_5.0

25.9 _+9.72

0.587 +_0.149

0.456 _+0.147

0.387 _+0.1872

1Labled and unlabeled plasma concentration. 2Difference among values for weeks 0, 4, 12 significant (p < 0.05) by analysis of variance. Differences between weeks 0 and 12 significant (p < 0.05) by Bonferroni ttest. From Browne et al. (3)with permission.

302

I0-

5

WEEK

0

X. . . .

X

WEEK

4

o--..

o

WEEK

12

2-

::t.

I

p.

Z O [.., <

[., Z r.~ c,) Z 0 c.)

PHENYTOIN

.7

-

4

- t

0

1

1

i

i

I0

20

30

40

J

50

2F

= I

O3 (

5

J

''-K--_X___X. ""

,-

I

X

p.-.

-0-----

• -

~ • O

~

. 0----

- O ~ .

"

p-HPPH l

-(~ -t

,

0

TIME AFTER

l

,

IO

,

l

,

20

PHENYTOIN

L

,.

30

t

,,

40

INFUSION

•

50

(hours)

Figure 2. Tracer dose serum concentration versus time relationships for SIL tracer dose molecules of phenytoin and p-HPPH (prinicpal metabolite of phenytoin) tracer infusions in a patient performed before (Week O) and 4 and 12 weeks after beginning chronic administration. From Browne et al. (3)with permission.

303 concentration versus time plots show the findings typical of concentrationdependent pharmacokinetics. Once it is determined that a drug has concentration-dependent pharmacokinetics, there are five additional questions to answer in order to have a complete picture of how the concentration-dependent pharmacokinetics will effect clinical usage: (1) what are the drug's Km and Vm,x values; (2) how does concentration-dependent pharmacokinetics effect the drug's dosing rate, dosing interval, and time to reach steady state plasma concentration; (3) which metabolic pathway is responsible for the observed concentration-dependent pharmacokinetics; (4) does product inhibition contribute to the drug's concentration-dependent pharmacokinetics; (5) are there additional metabolic pathways for the drug which do not exhibit concentration-dependent pharmacokinetics. Using only data from the six-patient study described above, we have shown (4) how each of these questions can be answered (see below). This demonstrates the large volume of useful data which can be obtained from a SIL tracer study of only a modest number of subjects.

5.1. What are the Drug's Km and V~x Values? It is possible to estimate a drug's Km and Vm,x by utilizing the elimination rate constants (k~ and/<2) of tracer doses of drug at two different known total plasma concentrations of drug (Cu~ + CL~) and (Cu2 + CL2) and the following equations (5), Km = [k2 x (Cu2 + CL2)- kl × (Cul + CL1)]/(kl -/<2)

(6)

Vmax = kl × (Kin + Cu1 + CL~)= k2 × (Kin + Cu2 + CL2)

(7)

Data from a single-dose study and a tracer study at one plasma concentration also may be utilized in Eqs. (6) and (7). Values of Km and Vrnax for the six study subjects are shown in Table 3.

5.2. How does Concentration-Dependent Pharmacokinetics Affect the Drugs Dosing Rate, Dosing Interval, and Time to Reach Steady State Concentration Drug mean steady state plasma concentration Css is related to dosing rate (R) as follows: (~ss = (R x Km)/(Vmax- R)

(8)

304 TABLE 3. Phenytoin K,. and

Vma x

Values

Patient number

1

2

3

4

5

6

Mean

Range

Kr, (~g/ml)

5.4

6.1

4.9

8.1

8.2

30.7

10.6

4.9-30.7

0.417

0.311

0.452 0.575 0.438

Vrnax

(#g/ml/hr)

1 . 3 6 1 0.592 0.311-1.361

From Browne et al. (5) with permission.

Thus, once a patient's values for Krn and Vmax are determined, it is possible to predict Css at any given dosing rate (4). This was done for our six subjects in Table 4. Note the nonlinear relationship of R and (~s~ within patients and the wide range of possible values for Cs~ at any given R. Drug tl/2 is the key determinant of dosing interval. Drug tl/2 can be expressed as (6),

tl/2 = 0.693 X (Kr,, + C)Nmax,

(9)

Note that tl/2 will vary directly with C. One may construct a table predicting tl/2 at given plasma concentrations using Eq. (9), Kn,, and Vma× (Table 4). The time (t) to reach a given serum concentration (Ct) after initiating chronic drug administration can be expressed as (7), t=

VdCt

VdKmVmax (R

(R - Vmax) (R

In

-

Vmax)

2

V m a x ) C t -{- RKm

RKm

(10)

Time to reach 95 percent of (~ss at three dosing rates was calculated for our 6 patients using Eq. (10) (Table 4). Note time to reach Css varies linearly with C~s and nonlinearly with R.

5.3. Which Metabolic Pathway is Responsible for the Observed Concentration-Dependent Pharrnacokinetics Drug clearance via production of a given metabolite (CVP) can be expressed as,

CVP = Vmax × percent administered drug excreted as metabolite/(Kr,, + C) (11)

305 TABLE 4. Range of Expected Values for Phenytoin Steady State Plasma Concentration, Elimination Half-Life, and Time to Reach Steady State Plasma Concentration Patient number 1

2

3

4

5

6

Mean

Range

Steady state plasma concentration (l~g/ml) at selected dosing rates of sodium phenytoin 1 2.5 mg/kg/d 5.0 mg/kg/d 7.5 mg/kg/d

1.9 6.2 20.4

4.5 34.2 0o2

2.1 7.1 38.2

2.3 6.3 15.4

3.1 11.8 43.3

2.8 6.1 10.2

2.8 12.0 -

1.4-4.5 6.2-34.1 10.2-0o2

12.8 25.8 40.2 69.1

9.3-16.6 21.4-33.8 26.6-54.8 37.1-96.8

Elimination half-life (hr) at selected plasma concentration values 3 1 i~g/ml 10 #g/ml 20 p,g/ml 40 #g/ml

10.3 24.8 40.9 73.2

14.9 33.8 54.8 96.8

9.3 23.5 39.2 70.7

11.5 22.8 35.4 60.6

14.5 28.7 44.4 75.9

16.6 21.4 26.6 37.1

Time (days) to reach 95 percent of steady plasma concentration at selected dosing rates of sodium phenytoin 1 2.5 mg/kg/d 5.0 mg/kg/d 7.5 mg/kg/d

2.1 5.2 16.1

9.0 91.8 0o2

3.8 7.2 11.9

3.3 5.1 8.2

3.6 6.9 0o2

6.2 6.8 10.0

4.67 20.5 -

2.1-9.0 5.1-91.8 8.2 -0o2

1Assuming fraction absorbed = 0.86. 2Dosing rate similar to Vmax. From Browne et al. (4)with permission.

Thus, CVP (and rate of metabolite formation) for the metabolic pathway responsible for a drug's concentration-dependent pharmacokinetics will vary inversely with C. in our patients, rate of urinary excretion of labeled p-HPPH metabolite varied inversely with C (r = - 0 . 6 4 0 , p < 0.005) (3). This implies that production of p-HPPH occurs via the enzyme system responsible for phenytoins concentration-dependent pharmacokinetics (presumably cytochrome P450 isoform 2C9) see Refs (3) and (8) for more details.

5.4. Effects of Substrate Saturation and Additional Pathways with Linear Pharmacokinetics We have shown these questions can be answered by an analysis of plots of Css versus 1/drug clearance (Figure 3) (9). If only one enzyme is operative, the y intercept of the plot will always be KmNmax. The slope of the plot will

306

14 13-

/

J

/ s

J

12-

t

j J f

11

-

A

~m

z

::::) >l:z: < n,,, l-m nr, <:

.,,,~, ,

10-

:4"

9-

"°"g5 ,

8-

~ /

oO /

7-

UJ

0 z < (3:: <

6-

IJJ

,

.,,J

£,0 ::3 n,O

..~~oO~C'

,..,./ ,~7

4-

~o

~ K m

3-"--

v msx

. . . . . . ". . . . . . . .

.............. g.ttCEh~

2-

• ...... $EGOt4D I-

0

I

I

I

1

2

3

I

4

I

i

I

I

5

6

7

8

-

I

9

10

DRUG S T E A D Y S T A T E SERUM C O N C E N T R A T I O N ( A R B I T R A R Y UNITS) Figure 3. Expected y intercepts, slopes and shapes for plots of drug plasma concentration versus 1/clearance in the absence of product inhibition and in the presence of competitive, noncompetitive, and uncompetitive inhibition by a drug metabolic product whose plasma concentration varies with drug plasma concentration. The bottom curve shows the expected plot in the absence of product inhibition and the presence of two independent enzymes, one with linear and one with concentration-dependent pharmacokinetic properties. From Browne et ai. (9)with permission.

307 be 1Nmax and the shape of the plot will be linear in the absence of product inhibition or a second linear pathway. The slope will always be greater than 1Nmax in the presence of product inhibition. The y intercept will be less than Kn~Nm~x and the slope will be less than 1Nmax if a second linear pathway is present. We applied this method to our six phenytoin patients and found no evidence of product inhibition or a second pathway with linear pharmacokinetics (9). 5.5. Drugs Other than Phenytoin The above discussion utilized phenytoin as an example of a drug with concentration-dependent kinetics. Denaro et al. (10) determined the presence and mechanism of concentration-dependent kinetics for caffine using nine subjects.

6. TIME-DEPENDENT PHARMACOKINETICS

Bertilsson et al. (11) measured carbamazepine clearance with SIL tracers before and during chronic therapy and demonstrated a time-dependent increase (Figure 4). This is presumed to be due to due to enzyme induction with a resulting increase in Vmax (12).

7. SPECIAL PHARMACOKINETIC SITUATIONS

7.1. Pediatrics SIL methods offer several advantages for pediatric studies. First, the safety of stable isotope labeling is unquestionable, while administration of radioactive tracers to children is forbidden by many institutions because of safety concerns. Second, the great sensitivity of GC-MS and HPLC-MS methods allows quantitation of drug in small specimens of plasma or urine. Third, breath analysis allows noninvasive quantitation of metabolism of some drugs. Fourth, the ease and safety of SIL methods facilitates repeated studies to determine changes in drug pharmacokinetics with growth and development. Pediatric applications of SIL methods are reviewed in detail in Chapter 19.

308 Clearance of C B Z - D 4

007-1

Lx kg-lx hr"

Patient C

0o6 Patient A Oos Pat ient B 0.04

0.03

0.02 -

0.01 -

O i

0

,

,

,

,

10

20

30

40

//

,

140

,

,

I

160

Days

Figure 4. Plasma clearance of carbamazine (CBZ)-D4 when given as a single oral dose at different times before and during maintenance CBZ treatment in three patients with recently discovered epilepsy. Multiple dosing was started on day 5. From Bertilsson et al. (11 ) with permission.

7.2. Racernates

By selectively labeling each of the enantiomers of a racemic drug with a unique stable isotope label, it is possible to determine simultaneously the pharmacokinetics of each enantiomer within an individual in the presence of the other enantiomer. Such data are very valuable and very difficult to obtain by techniques other than SIL. The "pseudoracemate" technique has been applied successfully to the study of the pharmacokinetics of several drug

309 TABLE 5. Mean (SEM) Pharmacokinetic Parameters after Intravenous (IV) and Oral (PO) 80-mg Doses of Cibenzoline to Healthy Subjects and to Patients with Renal Failure Parameter

Route

Normal

Renal failure

t-test

trn~x (hr)

PO

1.75 (0.05)

1.18 (0.08)

p < .01

Cm~x (ng/mL)

i.v. PO i.v. PO i.v. PO i.v.

573 (48) 250 (19) p < 0.01 7.3 7.4 NS 1905 (72) 1578 (91) p < .01 707 (27)

673 (52) 365 (26) p < 0.01 22.4 23.9 NS 6646 (752) 5961 (737) p < .01 224 (31)

NS p < p < p < p < p < p <

i.v. i.v. i.v. i.v. PO -

344 (24) 363 (29) 4.05 (0.49) 39 (2.8) 34 (2.8) p < .01 83

224 (31) 5.33 (0.39) 90

p < .01 p < .01 NS NS

t-test tl/2 (hr)* t-test AUC (ng-h/mL) t-test CLp (mL/min) CLr (mL/min) CLnr (mL/min) Vdss (L/kg) Amount in urine (mg)

t-test Bioavailability (%)

0.05 .01 .01 .01 .01 .01

*Harmonic mean. A b r e v i a t i o n s : trnax = time to maximum plasma concentration, Cmax maximum plasma concentration, tl/2 - elimination half-life, CLp = plasma clearnace, CLr = renal clearance, CLnr = nonrenal clearance, Vdss = volume of distribution at steady state. From Arnoff et al. (17) with permission.

i n c l u d i n g : i b u p r o p h e n (13), n i m o d i p i n e (14), s u p r o f e n (15) and w a r f a r i n (16). The p s e u d o r a c e m a t e t e c h n i q u e has been e m p l o y e d also to s t u d y the drug i n t e r a c t i o n s of r a c e m i c d r u g s (see Chapter 18).

7.3. R e n a l I m p a i r m e n t

The effects of renal i m p a i r m e n t on d r u g a b s o r p t i o n , renal clearance and n o n r e n a l clearance can be studied by s i m u l t a n e o u s a d m i n i s t r a t i o n of d i f f e r e n t stable isotopes of d r u g via the i n t r a v e n o u s and oral routes to subjects w i t h n o r m a l and i m p a i r e d renal f u n c t i o n (Table 5) (17).

310 8. ASSUMPTIONS OF SlL TRACER METHODS

Use of SIL tracer methods is based upon three assumptions: (1) absence of isotope effect; (2) absence of "deep pool effect"; and (3) safety of SIL labels.

8.1. Isotope Effects A bond involving a heavy isotope and another atom will be stronger than the same bond between the corresponding light isotope and that atom, because of the mass difference between the heavy and light isotopes. In a chemical reaction in which breaking this bond is the rate-limiting step, the reaction will proceed more slowly for the molecule with the heavy isotope ("metabolic isotope effect"). The presence or absence of isotope effects on the pharmacokinetic parameters of a labeled drug can be determined by simultaneous administration of the labeled and the unlabeled forms of the drug and simultaneous measurement of the pharmacokinetic parameters to be studied (e.g. 18, 19). See Chapter 2 for a complete discussion of isotope effect.

8.2. Deep Pool Effect If a portion of a drug in long-term administration is distributed to a deep peripheral compartment, the actual elimination half-life (tl~=) of the drug during the terminal exponential phase of elimination may be longer than the value determined by a single dose or tracer study, if the study is not carried out for a sufficient length of time to detect the deep compartment (usually due to insufficient assay sensitivity to detect low plasma concentrations of tracer drug at later times after administration) (20). This has been called "deep pool effect" and could lead to an overestimatation of actual clearance in single-dose tracer studies because of factitiously small values for apparent t1~2and area under the plasma concentration curve. Two methods of checking for deep pool effect using only data routinely obtained in the course of performing clinical tracer studies are available (20). See Chapter 14 for a complete discussion of deep pool effect.

8.3. Safety of Stable Isotope Labeling The safety of stable isotope labeling is above question. See Chapter 1 for a complete discussion.

311 9. ALTERNATIVE M E T H O D S

9.1. Measurement of Plasma Drug Concentration Steady state CL and t~/2 can be estimated using measured mean steady state plasma drug concentration, standard equations, dosing rate and assumed values for compliance, fraction absorbed and Vd (21). Unfortunately, this method is dependent upon no less than seven assumptions, six of which are often untrue, are seldom verified, and have the potential to introduce significant errors in estimated values of CL and tl/2 (Table 6) (21).

9.2. Stopping Drug Administration Long-term administration of a drug can be stopped temporarily and serial plasma specimens obtained for conventional analysis of plasma concentration. The tl/2 during long-term administration can be determined from plasma concentration versus time relationships; CL can be estimated from tl/2 and an assumed value for Vd (21). However, this method creates several problems. First, patients may be exposed to risk because of being deprived of necessary medication (e.g. antiepileptic drugs). Second, tl/2 data without Vd may yield misleading information when utilized to make inference regarding CL (21). Third, use of this method for drugs with concentration-dependent pharmaco-

TABLE 6. Assumptions of Measurement of Drug Plasma Concentration Method

All subjects have been completely compliant in taking their medications, or degree of noncompliance is known. Plasma concentration value measured is mean value (not trough, maximum or random). 3.

Plasmaconcentration value is measured at steady state.

4, 5. Fraction of drug absorbed and drug volume of distribution are known exactly for each subject. 6, 7. Fraction of drug absorbed and drug volume of distribution do not change in the presence of other drugs. See Browne et al. (21) for detailed discussion.

312 kinetics is problematic since the t1~2 at low plasma concentrations is lower than at therapeutic plasma concentrations (3).

10. ADVANTAGES OF SlL TRACER METHODS

10.1. Safety Traditional pharmacokinetic methods of measuring biotransformation and excretion have involved temporarily stopping maintenance administration of a drug to measure tl~= or the administration of radio-labeled drug. The former technique is problematical (see above). Although the amount of radiation exposure risk to patients in studies using radiolabeled drugs can often be demonstrated to be trivial, such studies are difficult to perform in many countries because of the concerns of institutional review boards and difficulties with the disposal of radioactive waste. SIL tracer methods have none of these problems.

10.2. Assumptions Verifiable SIL tracer studies are based on assumptions that are generally true and can be verified (see above). Alternative methods are based upon assumptions which often are not true, and seldom are verified (21).

10.3. High Statistical Power Random "noise" is minimized in SIL tracer studies by: (1) the high precision and accuracy of GC-MS analytical methodology; (2) the ability to coadminister drugs and avoid crossover errors; and (3) the absence of a need to use assumed values for compliance, fraction absorbed, Vd and mean steady state plasma concentration (21). This results in high statistical power and the ability to produce statistically significant results with a small number of subjects. For example, using only six subjects per drug, the author was able to show with a high degree of statistical probability that in humans phenytoin possesses concentration-dependent pharmacokinetic properties and phenobarbital does not possess either concentration-dependent or time-dependent pharmacokinetic properties (1-3).

313 10.4. Economic Savings The high statistical power of SIL studies reduces the number of subjects and specimens for analysis necessary to obtain results with adequate statistical power for acceptance by regulatory agencies. The economic savings of performing pharmacokinetic studies with SIL methods are discussed in detail in Chapter 24. 10.5. Urinary Data can be Collected for Additional Analysis Serial urine specimens can be obtained and analyzed with GC-MS for concentrations of SIL drug and its metabolites. These data can be used to determine rates of urinary excretion and time-dependent changes in urinary metabolite profile (3, 5). Analysis of total urinary excretion per unit time of drug and metabolites using non-GC-MS methods can also provide useful data. Clearance via production can be computed using SIL tracer plasma clearance and measured urinary metabolite profiles (22). Serial measurements of total urinary excretion of drug per unit time can be used to determine changes in compliance or fraction absorbed. 10.6. Comprehensive and Efficient Evaluation of Nonlinear and Time-Dependent Pharmacokinetics As demonstrated above, it is possible to obtain a comprehensive picture of how concentration-dependent or time-dependent pharmacokinetics will effect a drugs dosage and administration using SIL tracer studies and a small number of subjects.

11. DISADVANTAGES OF SIL TRACER METHODS

The two disadvantages of SIL tracer methods are: (1) the need to verify assumptions; and (2) the high cost of GC-MS analytical determinations. Assumptions can be verified with a modest amount of work (see above). The high cost of GC-MS analytical determinations may be reduced in the future using newly-developed table-top mass spectrometers or isotope ratio mass spectrometry (Chapter 6). There is growing evidence that deuterium labeled analogues of many drugs can be separated from nonlabeled analogues and quantitated with inexpensive high-performance liquid chromatography/ultraviolet detection instrumentation (Chapter 7).

314

ACKNOWLEDGMENT Supported by the United States Department of Veterans Affairs.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

T.R. Browne, G.K. Szabo and J.E. Evans et al., J. Clin. Pharmacol., 25 (1985) 59. T.R. Browne, J.E. Evans and G.K. Szabo et al., J. Clin. Pharmacol., 25 (1985) 51. T.R. Browne, J.E. Evans and G.K. Szabo et al., J. Clin. Pharmacol., 25 (1985) 43. T.R. Browne, G.K. Szabo and G.E. Schumacher et ai., in Synthesis and Applications of Isotopically Labeled Compounds, T.A. Baillie and J.R. Jones (eds) (Elsevier, Amsterdam, 1989), p. 157. T.R. Browne, D.J. Greenblatt and J.E. Evans et al., J. Clin. Pharmacol., 27 (1987) 321. T.R. Browne, D.J. Greenblatt and J.E. Evans et al., J. Clin. Pharmacol., 28 (1988) 318. G. Lam and W.L. Chiou, J. Pharmacokinet. Biopharm., 7 (1979) 227. T.R. Browne and B. LeDuc, in Antiepileptic Drugs, R.H. Levy, R.H. Mattson, B.S. Meldrum (eds) (Raven, New York, 1995), p. 283. T.R. Browne, G.K. Szabo and C.T. Walsh et al., J. Clin. Pharmacol., 30 (1990) 578. C.P. Denaro, C.R. Brown and M. Wilson et al., Clin. Pharmacol. Ther., 48 (1990) 277. L. Bertilsson, G. Tybring and T. Tomson, J. Clin. Pharmacol., 26 (1986) 459. J.W. Faigle and K.F. Feldman, in Antiepileptic Drugs, R.H. Levy, R.H. Mattson, B.S. Meldrum (eds) (Raven, New York, 1995), p. 499. A.C. Rudy, P.M. Knight and D.C. Brater et al., J. Pharmacol. Exp. Ther., 259 (1991) 1133. C. Fischer, F. Schonberger and W. Muck et al., J. Pharm. Sci., 82 (1993) 244. Y. Shinohara, H. Magra and S. Baba, J. Pharm. Sci., 80 (1991) 1075. R.A. O'Reilly, W.F. Trager and C.H. Motley et al., J. Clin. Invest., 65 (1980) 746. G. Aronoff, M. Brier and M.L. Mayer et al., J. Clin. Pharmacol., 31 (1991) 38. T.R. Browne, A. Van Langenhove and C. Costello et al., Clin. Pharmacol. Ther., 29 (1981) 511. T.R. Browne, A. Van Langenhove and C. Costello et al., J. Clin. Pharmacol., 22 (1982) 309. T.R. Browne, D.J. Greenblatt and G.E. Schumacher et al., J. Clin. Pharmacol., 30 (1990) 680. T.R. Browne, D.J. Greenblatt and G.E. Schumacher et al., in Antiepileptic Drug Interactions, W. Pitlick (ed.) (Demos, New York, 1989), p. 1. T.R. Browne, G.K. Szabo and J.E. Evans et al., Neurology, 38 (1988) 1146.

315

CHAPTER 17

BIOTRANSFORMATION AND EXCRETION: QUANTITATIVE STUDIES OF TISSUE METABOLISM

RAYMON DURSO Department of Neurology, Boston University School of Medicine/Boston Veterans Administration Medical Center

1. INTRODUCTION

With many drugs metabolism occurs exclusively or chiefly in the liver and the concentration of active drug and metabolites in plasma and at sites of action is determined by hepatic metabolism. However, for some drugs metabolism in nonhepatic tissues is the critical factor in determining the concentration at sites of action. Deficient production of dopamine in the central nervous system ("central" production) is a major feature of Parkinson disease. Because dopamine does not cross the blood-brain barrier, Parkinson disease often is treated with the dopamine precursor levodopa. Levodopa crosses the blood-brain barrier and is converted to dopamine by the enzyme dopadecarboxylase. Dopamine produced by this enzyme in brain improves the symptoms and signs of Parkinson disease. Dopamine produced by dopadecarboxylase in peripheral tissues causes a variety of autonomic side effects. Peripheral dopa-decarboxylase (and side effects) can be reduced by administering carbidopa, a dopa-decarboxylase inhibitor which does not cross the blood-brain barrier. Dopamine produced in the brain or peripheral tissues is further metabolized to homovanillic acid. A number of very important pharmacologic issues have been inadequately studied in Parkinson disease because of a previous inability to determine reliably in vivo central and peripheral levodopa metabolism. For example, the capacity of individual Parkinsonian patients to convert levodopa to dopamine centrally has rarely been investigated despite its possible crucial role in determining clinical benefit derived from the medication. Second, the dose of

316 carbidopa required to maximize inhibition of peripheral ievodopa metabolism is essentially unknown. Such information would be of vital importance to patients who suffer potentially lethal peripheral side effects (e.g. cardiac arrhythmias) related to peripheral dopamine production. We have recently been granted an FDA IND for a new stable isotope labeled form of levodopa and have begun experiments investigating such clinical problems in Parkinson disease. We have had access to this labeled drug for only a short time; nevertheless, it is clear that this technology will open many new areas of pharmacologic research in Parkinson disease and other diseases where tissue metabolism of drug is important. Based on preliminary data, it would also appear that some of our existing assumptions regarding levodopa and dopamine metabolism/distribution will need to be significantly altered. Our studies give an example of how stable isotope techniques can be utilized to study human in vivo tissue metabolism of drug. Stable isotope techniques have not been used extensively for this purpose in the past. In this chapter, I will briefly discuss two particular pharmacologic problems in Parkinson disease which we are currently investigating with stable isotope labeled levodopa. I will present the rationale for applying stable isotope technology over other existing pharmacologic methods. I will next relate our preliminary experience and discuss the implications of our work. The data discussed in this chapter has been presented elsewhere (see Refs. 39-41).

2. APPLICATION OF STABLE ISOTOPE TECHNOLOGY TO PHARMACOLOGIC RESEARCH IN PARKINSON DISEASE

2.1. Statement of Clinical Problems 2.1.1. The importance of central levodopa metabolism in determining levodopa efficacy in Parkinson disease A number of reports examining populations of levodopa treated Parkinson disease patients are unable to find a correlation between levels of levodopa attained in blood and degree of global improvement obtained from the drug (1-5). In addition, levodopa infusion studies examining response to increasing infusion rates indicate that once clinical improvement is noted in advanced Parkinson disease, the magnitude of that response cannot be improved by simply increasing the levodopa infusion rate. Nutt (6) has commented on this observation stating that the "magnitude of response appears to be largely all

317 or nothing". Nutt and Woodward reported (7) that doubling a clinically effective rate of levodopa infusion led to little further improvement. Such an observation is consistent with the central nervous system as a limiting factor in determining magnitude of response to levodopa. One possible explanation could be that levodopa, once inside striatum (the portion of brain comprising the caudate and putamen where dopamine is produced in axon terminals), is not adequately converted to dopamine. There is good reason to believe that the central pharmacokinetics of levodopa metabolism are altered in Parkinson disease. Autopsy studies of Parkinson disease patients demonstrate that dopa-decarboxylase activity in putamen and caudate of affected individuals are about 7 percent and 15 percent, respectively, of controls (8). A study by Spenser and Wooten (9) demonstrating altered pharmacokinetics of central levodopa metabolism in rat striata ipsilateral to 6-hydroxydopamine substantia nigra (the brainstem area which contains the cell bodies of origin for striatal axon terminals) lesions, further supports the hypothesis that central levodopa metabolism is altered in Parkinson disease. They found that peak striatal levels of dopamine and homovanillic acid after systemic carbidopa/levodopa were markedly reduced in striatum deprived of dopaminergic innervation. More recent studies in mice (10) and primates (11) exposed to MPTP demonstrate that in severe substantia nigra lesions there is a significant reduction in the ability to convert levodopa to dopamine in striatum. Thus, it may well be that in late-stage disease the conversion of levodopa to dopamine by dopa-decarboxylase becomes the kinetically rate limiting step influencing clinical response.

2.1.2. What dose of carbidopa is needed to maximally inhibit peripheral levodopa metabolism? The peripheral decarboxylation of levodopa to dopamine after sole levodopa administration is responsible for significant side effects which include nausea, vomiting and potentially fatal cardiac arrhythmias. Carbidopa blocks this peripheral conversion by acting as a competitive inhibitor of dopa decarboxylase. Since it does not cross the blood-brain barrier, there is no interference with central decarboxylation of levodopa to dopamine (a step required for therapeutic action). Thus, the effect of carbidopa when coadministered with levodopa is to substantially reduce systemic side effects of levodopa as well as make more systemic levodopa available for central conversion to dopamine (12). How this is accomplished is uncertain but the best studies to date employing unlabeled levodopa suggest that both inhibition of systemic dopa-

318 decarboxylase with a resultant decrease in plasma levodopa clearance and inhibition of gastrointestinal decarboxylase activity increasing bioavailability of levodopa may be most important (13, 14). A review of the literature reveals little information on how increasing doses of carbidopa directly affect peripheral levodopa pharmacokinetics. Existing studies employing unlabeled levodopa have been most successful in demonstrating effects of carbidopa on levodopa bioavailability (14, 15). A major gap exists, however, in understanding how carbidopa alters plasma levodopa clearance (i.e. inhibition of peripheral levodopa catabolism). Even more specifically, the dose of carbidopa needed to maximize inhibition of peripheral levodopa metabolism is uncertain. The primary problem in attempting such studies with unlabeled levodopa has been an inability to separate newly produced homovanillic acid (representing metabolized levodopa) from existing or baseline homovanillic acid blood concentrations. To date, the best relevant unlabeled levodopa study suggests that peripheral levodopa metabolism may be maximally inhibited at 100mg/dy since greater carbidopa doses do not lead to higher levodopa levels in cerebrospinal fluid (15). More sophisticated studies employing radioactive levodopa tracer doses and measuring exhaled labeled CO2 indicate that increasing carbidopa doses do indeed have progressively greater effects on peripheral levodopa LD decarboxylation (16). However, the dose of carbidopa that has maximal effects on peripheral levodopa metabolism cannot be ascertained from this study because of its indirect methodology. In addition, the use of tracer amounts of labeled levodopa make interpretation difficult (i.e. tracer levodopa doses likely result in an underestimation of peripheral metabolism). A better understanding of the effectiveness of carbidopa in inhibiting peripheral levodopa decarboxylation is a critical step in addressing peripheral levodopa toxicity.

2.2. Limitations of Existing Techniques to Examine Levodopa Central and Peripheral Metabolism In vivo techniques have not existed previously which could adequately address the forementioned pharmacologic problems in Parkinson disease. Previous methodologies tracking peripheral and central levodopa catabolism have involved the administration of radioactive labeled levodopa with analysis of blood and cerebrospinal fluid samples for labeled metabolites at multiple times following labeled drug administration (17, 18). However, such techniques are no longer employed due to the health hazards of radioactive substances. In addition, these studies were always limited by the necessity

319 of giving only tracer amounts of labeled drug to humans. PET scanning, a more recent technology, has also demonstrated limitations with regard to analyzing central levodopa metabolism. Specifically, when increased activity on PET scan is noted in striatum after [18F] fluorodopa, the exact source of that activity cannot be ascertained. The likely possibilities are [18F] fluorodopa itself or one of its metabolites, notably fluorinated dopamine, homovanillic acid, dihydroxyphenylacetic acid or 3-O-methyldopa. Studies in primates would favor most activity being derived from fluorinated dopamine (19). In any individual study, however, one cannot be certain what percentages of activity represent unmetabolized flourodopa or one of its metabolites- an issue critical to analyzing central levodopa metabolism as a function of levodopa-induced clinical response. What is needed is a methodology by which fully labeled pharmacologic doses of levodopa can be traced through various metabolic pathways in a manner that is safe and without the previously discussed limitations of other methods. It would appear that stable isotope technology now provides that methodology.

2.3. Use of Stable Isotopes to Examine Central Levodopa/Dopamine Metabolism in Parkinson Disease 2.3.1. Use of stable isotopes in pharmacology Stable isotopes are naturally occurring isotopes of elements that usually differ from their parent atom by the addition of one neutron. These substances are not radioactive. They are usually one atomic mass unit greater than the parent atom (the most abundant isotope form of the element). Examples of such stable isotopes are 13C and 15N (as compared to usually occurring 12C and 14N). 180 represents a stable isotope 2 mass units greater than the more abundant 160 isotope. The use of stable isotopes as a pharmacologic tool has been expandedconsiderably in recent years (20). The primary rationale is that a drug labeled with such isotopes can be quantitated along with its metabolites using gas chromatography/mass spectrometry (GC-MS). The absence of radioactivity from stable isotope labeled compounds make them a preferable choice over radionucleotides with regard to safety for the investigation of drug bioavailability and metabolism in humans. The stable isotope labeling of any drug must take into consideration that the isotopically labeled substance will have sufficiently higher mass than unlabeled drug in order for the two substances to be accurately measured by

mass spectrometry (21). In general, an increase of 2-3 mass units over the unlabeled drug, or a difference of 2-3 mass units between different isotopes (if multiple isotopes are to be measured), is most desirable although differences of 1 mass unit can also be detected. The second requirement is that an “isotope effect” be absent. The latter refers to the possibility that stable isotope labeling of a substance may alter it pharmacologically. The absence of an “isotope effect” is assured by labeling at nonmetabolized areas of the compound and by using 13C, I80 or 15N and avoiding 2H (deuterium) labeling (21).

2.3.2. Previous use of stable isotopes metabolism in Parkinson disease

to examine

central

dopamine

The author is aware of one study examining central dopamine metabolism in Parkinson disease using stable isotopes. The details of this research are published in part (22) as well as known to the author through personal communication (23). Six Parkinsonian patients and two controls were given I80 via inhalation. Lumbar punctures were performed in each subject 3, 6, 9, 12 and 15 hr after inhalation (total of 5 lumbar punctures). 180 became incorporated into dopamine metabolism during inhalation resulting in a percentage of the cerebrospinal fluid homovanillic acid concentration in each tap being homovanillic acid present in labeled with 180. The amount of 180-labeled each cerebrospinal fluid sample was used as a marker for central dopamine metabolism. In Parkinson disease patients the labeled I80 form of homovanillit acid expressed as a percentage of the total homovanillic acid concentration was 3, 5.5, 5.3,4, and 3 percent for times 3, 6, 9, 12 and 15 hours, respectively. In controls the percentages were 1, 2.5, 2.7, 2.5 and 2 percent. Thus, central dopamine metabolism was reported as significantly increased in Parkinson disease patients as compared to controls. Increased central dopamine metabolism in surviving dopamine neurons of Parkinson disease patients (24) and hyperactivity of remaining dopaminergic neurons in animal Parkinsonian models (25) are well described. Thus, this I80 data would appear to support the measurement of stable isotope labeled compounds in lumbar cerebrospinal fluid as a reliable method for determining rate of central levodopa and dopamine metabolism. It is now apparent, however, that the labeling of homovaniilic acid in lumbar cerebrospinal fluid is much higher than reported in these 180 studies when our more direct methodology is used (administration of stable isotope labeled levodopa).

321

2.3.3. Stable isotope labeled levodopa In collaboration with Cambridge Isotope Laboratories (Andover, MA), we have synthesized a stable isotope labeled levodopa form where all carbons within the ring a r e labeled with 13C. The latter results in a c o m p o u n d which is 6 mass units greater than unlabeled levodopa. Its metabolites are easily distinguished from background levodopa/dopamine metabolites via gas GCMS (Figure 1). This form (L-DOPA ring 1', 2', 3', 4', 5', 6' 13C) along with the critical metabolism which must be considered when investigating central and peripheral levodopa metabolism in Parkinson disease is shown in Figure 2.

2 5 0 0 0 0

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Figure 1. The displayed GCMS printout is derived from a single GCMS run. It represents the analysis of a lumbar cerebrospinal fluid sample from a subject who received an intravenous infusion of 150 mg of L-DOPA (ring 1', 2', 3', 4', 5', 6' 13C) 6 hr prior to a lumbar puncture. Within this sample two distinct homovanillic acid (HVA) peaks can be seen. The dotted line represents homovanillic acid which is 6 mass units greater than unlabeled metabolite. It was derived solely from the stable isotope labeled levodopa infusion. The solid line represents background homovanillic acid synthesized from other nonlabeled central dopamine sources. These two peaks were manually superimposed on each other using GCMS analytical software in order to better visualize the comparison.

322 levodopa (ring 1',2',3'.4',5',6'-13C)

dopamine (ring 1',2',3',4',5',6-13C) H2

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(ring 1',2',3',4',5',6"J3C) H2 CH30 C~I 3OOH

C~iH 2

HO

HO Monoamine oxidase CatechoI-O-methyltransferase

Figure 2. The represented stable isotope labeled levodopa form has all carbons within the ring replaced with 13C making it 6 mass units greater than unlabeled levodopa. These "heavy" carbons are marked by an asterick. The major catabolic route of levodopa pictured involves metabolism occuring completely outside the ring structure. Thus, all rnetabolites derived from the labeled form will also be 6 mass units greater and detectable from background metabolite concentrations via GCMS.

3. CURRENT RESEARCH WITH LEVODOPA (RING 1', 2', 3', 4', 5', 6' 13C) IN PARKINSON DISEASE 3.1. Clinical Methodology 3.1.1. Patient characteristics

A total of nine patients participated in our infusion studies. Subjects were all diagnosed with idiopathic Parkinson disease by the author and at least one other neurologist. All patients had experienced previous benefit to levodopa except for one. This latter patient had been newly diagnosed and his first levodopa exposure was the stable isotope labeled levodopa given in our

323 study. The mean (SD, range) age of our patient group was 61.4 years (12.3, 44-75) and they averaged 8.1 years duration of disease (4.9, 0.5-15). They were taking a mean of 475 mg (307, 0-1000) of levodopa per day in the form of Sinemet.

3.1.2. Drug preparation for clinical studies On the morning of each infusion study (approximately 6:30 am) under a laminar flow hood 165 mls of 5 percent Dextrose Injection U.S.P. (pH = 4.5) was drawn from a 500-ml sterile pouch and placed in a previously autoclaved beaker with stirring bar. To the beaker was added 165 mg of L-DOPA (ring 1', 2', 3', 4', 5', 6' ~3C) giving a concentration of 1 mg/ml. This labeled levodopa was dissolved using gentle 5-min stirring (on a magnetic stirrer) at room temperature. The resulting solution was then drawn up into three 60-ml sterile, single use, amber Luer-Lok syringes (55 ml/syringe) equipped with sterile 18-gauge needles. Using sterile procedure, the needles were removed and nonpyrogenic, single-use 0.22 micron filter units (Millex-GS/Millipore) were connected to the syringes via the Luer-Lox connections. To the other end of the filter unit of each syringe was attached another 60-ml sterile, single-use amber Luer-Lok syringe via a sterile, nonpyrogenic, single-use fluid dispensing connector (Burron Medical Inc.). Thus, when the levodopa solution was pushed through the 0.22 micron filters, it directly passed into new sterile 60-ml syringes. Any air in the newly filled syringes was flushed out and the remaining volume in each syringe was 50m1. These syringes were then capped with sterile Leur tip caps (Becton, Dickinson and Company) and were ready for injection into patients.

3.1.3. Methodology for clinical infusion studies After signing informed consent in clinic, the patient was admitted to the hospital on the afternoon prior to the day of study. Food and all medications were held from 12 midnight on the day of study. At 7 a.m. the subject was connected to a cardiac monitor and two heparin locks (one for blood drawing and one for levodopa infusion) were placed in arm veins (one lock in each arm). To one lock a 500-ml bag of 5 percent Dextrose Injection U.S.P. solution (pH = 4.5) was connected at a "keep open" rate (20 drops/min). The patient received 50 mg of carbidopa orally at 7 a.m. At 8 a.m. a serum sample was drawn and the subject was examined using a modified Unified Parkinson

324 Rating Scale. The subject was then given an infusion of 150 mg (1 mg/ml) over 12 min of stable isotope labeled levodopa (L-DOPA (ring 1', 2', 3', 4', 5', 6' 13C6)) via the sequential intravenous injection of three 50-ml boluses (from the three previously prepared syringes) using Harvard Infusion pumps (Harvard Apparatus Syringe Infusion Pump 22). Two infusion pumps were used. Repeat examinations (using the modified Unified Parkinson Rating Scale) and blood drawings were done every 30 or 60 min for a duration of 360 min. Finally, at time 360 min (6 hr after the stable isotope labeled levodopa infusion), a lumbar puncture was performed using standard lumbar puncture technique. Twenty-five cc's of spinal fluid were withdrawn sequentially into five numbered polypropylene tubes (5 cc's/tube) containing 0.1 percent cysteine, immediately placed on dry ice and then transferred to a freezer (-70°C) on completion of the lumbar puncture. The drawing of cerebrospinal fluid into sequential tubes with subsequent laboratory analysis of similarly numbered tubes was employed to minimize patient variability based on the cerebrospinal fluid gradient for homovanillic acid which exists along the brain-spinal cord axis (26).

3.2. Animal Methodology One large (10 kg) male rhesus monkey underwent a 3-hr intravenous infusion of stable isotope labeled homovanillic acid (ring 1', 2', 3', 4', 5', 6' 13C) with concurrent sampling of cerebrospinal fluid and blood for 3 hr to determine the degree to which homovanillic acid crosses the blood-brain or bloodcerebrospinal fluid barrier. The animal was anesthetized with telazol (6 mg/kg) and a percutaneous cannula was inserted into the lumbar space for cerebrospinal fluid sampling. In addition, a right saphenous vein catheter was placed for the infusion and a left saphenous vein catheter for venous sampling. Afterward, the animal was placed in a primate restraining chair and allowed to regain consciousness. Labeled homovanillic acid was infused at a rate of 250 i~g/kg/hr. The labeled homovanillic acid solution had been prepared in 10 ml 5 percent dextrose in water using sterile technique. The solution was then filtered through a 0.22 i~m filter and, in sterile fashion, injected into a 500-ml bag of 5 percent dextrose in water. This was infused into the animal at a rate 125 ml/hr for 3 hr. Blood samples (1 ml) were drawn at time 0 and every 30 rain for 3 hr. Samples were immediately spun and resultant serum pipetted into polypropylene tubes and then frozen (-70°C). Lumbar cerebrospinal fluid (0'3 ml)was taken at the same time blood samples were drawn,

325 placed into polypropylene tubes and then similarly frozen. After participation, the animal was returned to his previous habitat.

3.3. Laboratory Methodology The laboratory methodologies involved the use of high-performance liquid chromatography for quantitation of total homovanillic acid concentrations (labeled and unlabeled) in blood and cerebrospinal fluid. The GCMS methodology was used primarily for determining the ratios of labeled to unlabeled homovanillic acid. High-performance liquid chromatography was used to assay serum levels of levodopa.

3.3.1. High-performance liquid chromatography methodology The assay for homovanillic acid employed a model 6000A solvent pump and 712 automatic injector, both from Waters Associates (Milford, MA). An LC-4C amperometric detector with a glassy carbon working electrode and AG/AgCL reference electrode from Bioanalytical Systems Inc. (W. Lafayette, IN) served as the detector system. The chromatographic column consists of a partisil-5 ODS-3 (5.0 i~m particle size, 4.6 mm ID x 25 cm) from Whatman Inc. (Clifton, NJ). For HVA quantitation, a Canon AS-100 computer using software designed by Binary Systems (Newton, MA) and an analog digital computer interface from Quasitronic Inc. (Houston, PA) was used. The mobile phase was a mixture of 20 percent methanol in 0.75 M KH2PO4 adjusted to a pH of 2.5. It was prepared by filtration through a millipore membrane filter (Millipore, Bedford, MA) type HA, pore size 0.45 ~m. The column temperature was kept at ambient room temperature. The flow-rate was 1.8 ml/min with the working electrode operated at 1.0 V. The detector was set at 1 nA sensitivity. On the day of assay, patient cerebrospinal or serum samples were thawed. To a 16 x 150 mm disposable glass test tube capped with inert Tainertops (Fisher Scientific), 500 ~1 of internal standard solution (HVACN 100 ng/ml) was added to 5001~1 of cerebrospinal fluid or serum. The samples were then acidified with approximately 100 i~1 of 10 N hydrochloric acid to a pH less than 1.0. Homovanillic acid and internal standard were extracted by adding 5 ml of methylene chloride to samples, shaking for 10 min on a horizontal shaker and then centrifuging for 2 min to separate the liquid phases. The upper aqueous and interphase layers were then aspirated and discarded. The lower organic layer was evaporated to dryness under a gentle stream of dry nitrogen in the absence of heat. Subsequently, samples were reconstituted with 200 ~1 of mobile phase and 25 i~1 were used to inject onto the column. In the analysis

326 of serum, the reconstituted samples were centrifuged prior to injection in order to remove any particulate matter.

3.3.2. Gas chromatography~mass spectrometry methodology The ratios between labeled and unlabeled forms of homovanillic acid were measured by the method of DeJong et al. (27). This procedure utilizes fusedsilica capillary GC with electron capture negative ion MS (EC-NIMS) to obtain the high sensitivity necessary for measurement of the low levels of labeled homovanillic acid encountered in cerebrospinal fluid. Using this procedure an O-acetyl carboxypentafluorobenzyl ester derivative of homovanillic acid is formed which yields an intense [M-PFB]- that retains all the oxygen and carbon atoms of homovanillic acid. This procedure provides a limit of detection lower than 0.2 pg injected when the [M-PFB]- ion is selectively monitored. The isolation and derivatization of homovanillic acid from cerebrospinal fluid and serum is accomplished by the following steps. The cerebrospinal fluid or serum sample (500 ~1) is added to 1.5 ml of redistilled/deionized water, mixed vigorously for 1 min with 2 ml of 1 M phosphate buffer (pH 7.7) and 60 ~1 of acetic anhydride. Then 0.5 ml of 1 M K2HPO4-saturated K2CO3 (1:1) pH 10.5 is added and the mixture allowed to react for 10 rain at room temperature. The resulting phenolic O-acetyl derivative of homovanillic acid is extracted twice with 4 ml of ethyl acetate after being acidified to pH 3 with 0.8 ml of 0.6 M HCL. The combined extracts are evaporated with a stream of nitrogen. Finally, the pentafluorobenzyl ester is formed (the anhydrous acetylation step is not required for HVA) by treating the residue with 5 i~1 of pentafluorobenzyl bromide and 10 ILl of triethylamine in 50 i~1 of acetonitrile at room temperature for 5 min. Then 3 ml of ethyl acetate is added and the sample washed with 1 ml of 0.1 M HCL. The organic layer is next evaporated to dryness and redissolved in 20 ~1 of hexane for GCMS analysis. GCMS analysis is performed with a CPSlL-19 bonded fused-silica capillary interfaced directly with the mass spectrometer with helium as the carrier gas. The mass spectrometer is operated in the electron capture negative-ion mode with methane (0.2 Torr) as the moderator gas. The [M-PFB]- ions are monitored for the labeled and unlabeled forms of homovanillic acid at 0.5-sec intervals throughout the GC run. Peak areas are measured for the homovanillic acid forms using each of the selected ions and the ratios are calculated by comparison to standard curves constructed with mixtures of all forms with correction for ion overlap.

327 4. RESULTS

4.1. Systemic Levodopa Levels Our bolus infusion methodology produced levels of levodopa in blood as seen in Figure 3. These levels appeared maximal at t = 30 min and thereafter declined steadily to reach baseline concentrations at approximately t = 300 min. The mean (SD, range) concentration at t = 30 minutes for the nine subjects was 2.00 ~g/ml (0.43, 1.3-2.6). These results approximate serum levels of levodopa obtained after oral administration of 25/250 carbidopa/levodopa doses. Specifically, we have observed in other studies average systemic levodopa levels of 1.3 and 1.1 ~g/ml at 60 and 120 min, respectively, after a single oral 25/250 unlabeled carbidiopa/levodopa dose (3).

SERUM LEVODOPA LEVELS AFTER STABLE ISOTOPE LABELED LEVODOPA INFUSION 3

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Figure 3. The graph represents mean serum levodopa levels obtained over a 6-hr period following an intravenous bolus infusion of 150 mg of stable isotope labeled levodopa at time 0. Each point is the average of nine patients. Error bars indicate standard deviation.

328

SERUM HVA LEVELS AFTER INFUSION OF STABLE ISOTOPE LABELED LEVODOPA 400 Filled circle - +6 HVA Open circle +0 HVA 3OO

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Figure 4, The graph represents mean labeled (filled circles) and unlabeled (open circles) serum concentrations of homovanillic acid over a 6-hr period following an intravenous infusion of 150 mg of stable isotope labeled levodopa. Each point is the average of nine patients. Error bars indicate standard deviation.

4.2. Systemic Homovanillic Acid Levels It can be seen in Figure 4 that large amounts of the infused stable isotope labeled levodopa were decarboxylated in the periphery to homovanillic acid despite pretreatment with 50 mg of carbidopa (twice the carbidopa used in a standard oral carbidopa/levodopa tablet). Concentrations of the labeled homovanillic acid rose quickly and peaked at 60 min to a mean of 162 ng/ml accounting for approximately 85 percent of the total blood homovanillic acid. Unlabeled homovanillic acid serum concentrations, on the other hand, remained fairly stable and low throughout the 6-hr period with no suggestion of increased production.

4.3. Cerebrospinal Fluid Homovanillic Acid Levels We found that over 50 percent of homovanillic acid in lumbar cerebrospinal fluid was labeled 6 hr after a single bolus infusion of 150 mg of stable isotope

329

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Figure 5. The graph depicts the percent labeled homovanillic acid present in lumbar cerebrospinal fluid 6 hr after an intravenous bolus infusion of 150 mg of stable isotope labeled levodopa. The sample in tube #1 represents the l st-5th cc, and that of tube #5 the 21st-25th cc of cerebrospinal fluid, obtained from the lumbar puncture. The mean percent labeled homovanillic acid is given in parentheses for each of the two tubes. Each mean represents an average of eight patients.

labeled levodopa (Figure 5). For all patients there was evidence of a gradient for newly synthesized homovanillic acid in cerebrospinal fluid (Figure 6). Specifically, the mean (SD, range) absolute amount of labeled homovanillic acid in the initial cerebrospinal fluid collection tubes was 20 ng/ml (12, 4-43) as compared to 49 ng/ml (30, 19-107) in tubes #5. A correlation between area under the curve (AUC) for peripheral labeled homovanillic acid and labeled homovanillic acid in cerebrospinal fluid was not significant (R= -0.222). 4.4. Analysis of Blood-Cerebrospinal Fluid Transfer of Homovanillic Acid in a Primate Model

In order to confirm further the central origin of the labeled cerebrospinal fluid homovanillic acid present in our forementioned clinical studies, we have

330

LABELED HVA IN LUMBAR CSF 6 HOURS AFTER BOLUS INFUSION OF 150 MG LABELED LEVODOPA

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CSF TUBE NUMBER Figure 6. The graph demonstrates the absolute concentration of labeled homovanillic acid present in lumbar cerebrospinal fluid 6 hr after an intravenous bolus infusion of 150 mg of stable isotope labeled levodopa. The sample in tube #1 represents the l s t 5th cc, and that of tube #5 the 21st-25th cc of cerebrospinal fluid, obtained from the lumbar puncture. The mean concentration of labeled homovanillic acid is given in parentheses for each of the two tubes. Each mean represents an average of eight patients.

begun animal studies specifically designed to determine the ability of homovanillic acid in blood to transfer over into cerebrospinal fluid. We have examined one rhesus monkey which has been intravenously infused with labeled homovanillic acid (ring 1 ', 2', 3', 4', 5', 6' 13C) over a 3-hr period with 30-min blood and lumbar cerebrospinal fluid sampling. Levels of labeled homovanillic acid attained in blood averaged 147ng/ml (range 129167 ng/ml). The latter is a good approximation of labeled homovanillic acid blood levels in our human results. At these high labeled homovanillic acid blood concentrations, we found negligible penetration of labeled homovanillic acid into cerebrospinal fluid (Figure 7).

331

+6 HVA LEVELS DURING STEADY STATE LABELED HVA INFUSION IN PRIMATE

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Figure 7. The graph represents simultaneous serum and cerebrospinal fluid sampling in a single rhesus monkey during an intravenous steady state infusion of homovanillic acid (ring 1', 2', 3', 4', 5', 6' 13C) lasting 3 hr at a rate of 250 ~g/kg/hr. The filled circles represent cerebrospinal fluid concentrations of labeled homovanillic acid. The open circles represent serum concentrations of labeled homovanillic acid. Lumbar cerebrospinal fluid samples at the 150- and 180-min time period could not be obtained.

5. D I S C U S S I O N

The preliminary data presented in this chapter provide evidence that an in vivo methodology, using stable isotope labeled levodopa, can be successfully employed to examine important clinical pharmacologic problems in Parkinson disease (e.g. the role of central levodopa pharmacokinetics in limiting clinical response and the dose of carbidopa required to maximally inhibit peripheral levodopa metabolism). While the number of participating subjects is yet too small to infer conclusions regarding these specific problems; there are, nevertheless, several exciting relevant observations which deserve comment. First, we now strongly believe that the labeled lumbar cerebrospinal fluid homovanillic acid present in our studies is predominantly of central and not peripheral origin. It is known that stimulation of the substantia nigra results

332 in a stimulus dependent increase in striatal concentrations of homovanillic acid (28, 29) and a release of dopamine metabolites into ventricular cerebrospinal fluid (30). The presence of a steep gradient in cerebrospinal fluid homovanillic acid from ventricular to lumbar spaces is also argued as evidence that brain dopamine catabolism contributes almost exclusively to homovanillic acid levels in lumbar cerebrospinal fluid (26). The assumption of a primary central origin for cerebrospinal fluid homovanillic acid is crucial to the accurate interpretation of our levodopa metabolism studies. Most previous data in the literature, with the exception of one study (31), has tended to support such a central origin. However, criticism regarding interpretation of this literature could be raised. In the case radioactive [3H] homovanillic acid intravenous infusion data (17, 32), the finding of little penetration of blood homovanillic acid into cerebrospinal fluid could have been primarily related to the use of tracer doses of radioactive homovanillic acid. Another study (33) which found that less than 1 percent of ventricular or lumbar cerebrospinal fluid homovanillic acid was labeled following a constant rate intravenous infusion of deuterium tagged homovanillic acid might have been the result of "isotope effect" (i.e. deuterium labeled homovanillic acid as compared to unlabeled metabolite might have been less capable of crossing the blood-brain barrier). Finally, a number of studies using either unlabeled homovanillic acid or unlabeled levodopa which support a central origin (3437) could be criticized due to an inability to detect dynamic changes. Specifically, without using a label, large amounts of homovanillic acid entering and leaving the cerebrospinal fluid pool could be missed if total homovanillic acid concentrations remained relatively constant. The stable isotope methodology used in this study involves levodopa and homovanillic acid infused at full pharmacologic/physiologic concentrations (not tracer concentrations) which are both 100 percent labeled and devoid of "isotope effect". Our data support a central origin for cerebrospinal fluid homovanillic acid in three ways. First, in all patients there is evidence of a gradient for labeled cerebrospinal fluid homovanillic acid indicating a rostral origin for the newly synthesized metabolite rather than a diffuse blood-cerebrospinal fluid transfer of peripherally synthesized homovanillic acid. With levodopa (a substance known to cross the blood-brain barrier) no similar rostral/caudal gradient can be found (38). Second, there is no positive correlation between blood and cerebrospinal fluid labeled homovanillic acid levels. If blood levels of labeled homovanillic acid were contributing significantly to those in cerebrospinal fluid such a correlation would have been expected. For example, serum and cerebrospinal fluid levels of levodopa are very highly correlated (3). Finally,

333 in our animal study, high sustained levels of labeled homovanillic acid in blood averaging 147 ng/ml showed negligible penetration into cerebrospinal fluid. The next important observation relevant to our levodopa metabolism studies is the unexpectedly high percentage of labeled homovanillic acid in lumbar cerebrospinal fluid 6 hr after a bolus stable isotope labeled levodopa infusion. This is a remarkable finding which emphasizes a varied role for cerebrospinal fluid. Specifically, it supports one function of cerebrospinal fluid as a "sink" for dopamine metabolites to a much greater degree than had been previously recognized. We initiated our studies assuming that about 5 percent of homovanillic acid in lumbar cerebrospinal fluid would be labeled under our experimental conditions. These assumptions came from several sources. The first was previously cited ~80 data (22). The latter study employed an indirect methodology (labeled levodopa was not given). It demonstrated that after breathing ~80 for approximately 30 min, ~80-labeled homovanillic acid in lumbar cerebrospinal fluid peaked to about 5 percent of the total homovanillic lumbar cerebrospinal fluid concentration after 6 hr. The second source was a radioactive study (18) in which an oral tracer dose of levodopa was given to Parkinson disease patients. They reported that 2 hr after the dose, only "small" amounts of radioactivity in the form of labeled homovanillic acid could be seen in lumbar cerebrospinal fluid. Finally, two studies in Parkinson disease patients, who had Ommaya resevoirs post transplant, indicated little or no change in total ventricular cerebrospinal fluid concentrations of unlabeled homovanillic acid after unlabeled oral carbidopa/ levodopa (36, 37). In contrast, our results indicate that over 50 percent of homovanillic acid in lumbar cerebrospinal fluid is labeled after a single intravenous systemic levodopa dose. Our methodology, for the most part, reproduced normal physiologic conditions in treated Parkinson disease patients since we observed blood levodopa levels typical of patients receiving standard carbidopa/levodopa therapy (3). We believe our ability to demonstrate this high degree of labeling in the cerebrospinal fluid clearly demonstrates the advantages of stable isotope technology over other existing methods to examine levodopa pharmacokinetics. In the case of radioactive levodopa research (18), the most probable reason for the underestimation is that accurate quantitation of labeled metabolites is limited by use of only tracer amounts of labeled levodopa. As for the unlabeled levodopa studies (36, 37), it is likely that active cerebrospinal fluid dynamics are missed by an analysis of only unlabeled homovanillic acid (i.e. there may be very active transport of homovanillic acid into and out of a cerebrospinal fluid pool which cannot be seen

334 without labeling). Finally, in the 180 study (22) no labeled precursor was employed. This probably resulted in a wide distribution of 180 within the body and only a tiny incorporation of the label into dopamine. A third observation which deserves comment was the high degree of peripheral levodopa metabolism which persisted despite pretreating patients with 50 mg of carbidopa prior to the labeled levodopa infusion. This carbidopa dose represents twice that used in a standard carbidopa/levodopa preparation and it clearly did not prevent peripheral levodopa catabolism (approximately 85 percent of the blood homovanillic acid was labeled 1 hr after the infusion). The clinical implications of this data are significant. It demonstrates that substantially more carbidopa is required in carbidopa/levodopa tablets in order to successfully inhibit peripheral dopamine production. A more complete inhibition would allow more levodopa to enter brain since less would be catabolized in the blood. In addition, it is likely that greater carbidopa doses would also reduce the risk for cardiac arrhythmia. This side effect is presumed to be mediated by beta adrenergic stimulation of the heart by dopamine in the blood. Our results demonstrate that stable isotope technology will be a useful tool to explore in vivo pharmacologic issues in Parkinson disease and other diseases where metabolism of drugs by a specific tissue is important. The in vivo tracking of central and peripheral levodopa metabolism as a function of medication response is now possible since generated metabolites can be discerned from existing "background" levels.

REFERENCES 1. T.A. Hare, B.L. Beasley, R.A. Chambers, D.H. Boehme and W.H. Vogel, Clin. Chim. Acta., 45 (1973) 273. 2. U.K. Rinne, V. Sonninen and T. Siirtola, Eur. Neurol., 10 (1973) 301. 3. R. Durso, G. Szabo, H. Davoudi and R.G. Feldman, Clin. Neuropharmacol., 12 (1989) 384. 4. S. Algeri, S. Ruggieri, M. Miranda, M. Casacchia, PL Morselli and A. Agnoli, Eur. Neurol., 14 (1976) 219. 5. J.G. Pilling, J. Baker, L.L. Iverson, S.D. Iverson and T. Robbins, J. Neurol. Neurosurg. Psychiatry., 38 (1975) 129. 6. J.G. Nutt JG, Ann. Neurol., 22 (1987) 535. 7. J.G. Nutt and W.R. Woodward, Neurology, 36 (1986) 739. 8. K.G. Lloyd, L. Davidson and O. Hornykiewicz, J. Pharmacol. Exp. Ther., 195 (1975) 453. 9. S.E. Spencer and F.G. Wooten, Neurology, 34 (1984) 1105. 10. A. Goldberg, E. Melamed, J. Rosenthal, A. Reches and P. Tiqva, Neurology, 40[Suppl 1] (1990)291.

335 11. R.S.Burns, I. Kopinl, V. Weiss, M. Roznoski, M and M. Ebert, Neurology, 41[Suppl 1] (1991)378. 12. J.M. Cedarbaum and L.S. Schleifer, The Pharmacologic Basis of Therapeutics (Pergamon Press, New York, 1990), p. 463. 13. R.M. Pinder, R.N. Brogden, P.R. Sawyer, T.M. Speight and G.S. Avery, Drugs, 11 (1976) 329. 14. J.G. Nutt, W.R.Woodward and J.L. Anderson, Ann. Neurol,.18 (1985) 537. 15. L.T. Kremzner, S. Berl, M. Mendoza and M.D. Yahr, Adv. Neurol., 2 (1973) 79. 16. C.D. Ward, I.K. Trombley, D.B. Calne and I.J. Kopin, Neurology, 34 (1984) 198. 17. A. Pletscher, G. Bartholini and R. Tissot, Brain Res., 4 (1967) 106. 18. J.P. Morgan, J.R. Bianchine, H.E. Spiegel, L. Rivera-Calimlim and R.M. Hersey, Arch. Neurol., 25 (1971) 39. 19. E.S. Garnett, G. Firnau, C. Nahmias and R. Chirakal, Brain Res., 280 (1983) 169. 20. T.R. Browne, Clin. Pharmacokinet., 18 (1990) 423. 21. R.L. Wolen, J. Clin. Pharmacol., 26 (1986) 419. 22. T.N. Chase, A. Neophytides, D. Samuels, G. Svedvall and C.G. Swahn, Catecholamines: Basic and Clinical Frontiers, Vol. 2 (Pergamon Press, New York, 1979), p. 1569. 23. N.L. Foster, personal communication (1988). 24. K.G. Lloyd, Parkinson's Disease: Concepts and Prospects (Excerpta Medica, Amsterdam, 1977) p. 61. 25. Y. Agid, F. Javoy and J. Glowinski, Nature, 245 (1973) 150. 26. M.H. Ebert and M.J. Perlow. Structure and Function of Monoamine Enzymes (Marcel Dekker, New York, 1977) p. 963. 27. A.P.J.M. DeJong, R.M. Kok, C.A. Cramers and S.K. Wadman, J Chromatog., 382 (1986) 19. 28. R.H. Roth, L.C. Murrin and J.R. Waiters, Eur. J. Pharmacol., 36 (1976) 163. 29. J. Korf, L. Grasdijk and B.H.C. Westerink, J. Neurochem., 26 (1976) 579. 30. P.J. Portig and M. Vogt, J. Physio. [London] 204 (1969) 687. 31. L. Prockop, S. Fahn and P. Barbour, Brain Res., 80 (1974) 435. 32. G. Bartholini, A. Pletscher and R. Tissot, Experientia, 22 (1966) 609. 33. M.A. Elchisak, R.J. Polinsky, M.H. Ebert, K.J. Powers and I.J. Kopin, Life Science, 23 (1978) 2339. 34. L.M. Aizenstein and J. Korf, Brain Res., 149 (1978) 129. 35. H.C. Guldberg and C.M. Yates, Br. J. Pharmac. Chemother., 33 (1968) 457. 36. C.W. Olanow, L.L. Gauger and J.M. Cedarbaum, Ann. Neurol., 29 (1991) 556. 37. Z. Moussa, C. Raftopoulos, S. Przedborski and J. Hildebrand, Ann. Neurol., 31 (1992) 113. 38. R. Durso, unpublished observations (1995). 39. R. Durso, J.E. Evans, E. Josephs, G.K. Szabo, B.A. Evans, J. Handler, D. Jennings and T.R. Browne, Ann. Neurol., in press. 40. R. Durso, J.E. Evans, E. Josephs, G.K. Szabo, B.A. Evans, J. Handler, D. Jennings and R.G. Feldman, Neurology, 45(Suppl 4) (1995) A276. 41. R. Durso, J.E. Evans, E. Josephs, G.K. Szabo, B.A. Evans, J. Handler, D. Jennings and R.G. Feldman, Neurology, 48(Suppl)(1997) A184.

337

CHAPTER 18

DRUG INTERACTIONS

THOMAS R. BROWNE Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center

1. STABLE ISOTOPE LABELED (SlL) TRACER PROCEDURES 1.1. Standard Methods

Volume of distribution (Vd), clearance (CL) and elimination half-life (tl/2) are the parameters typically measured in pharmacokinetic drug interaction studies. A tracer dose of drug 1 is administered by the intravenous (i.v.) route before and after the addition of drug 2. After each infusion, plasma is sampled at multiple times, and plasma concentration of stable isotope labeled (SIL) and unlabeled drug is determined for each sample by gas chromatographicmass spectrometry (GC-MS). Vd, CL and t~/2 are calculated from plasma concentration versus time relationships by procedures described elsewhere (Figures 1 and 2) (1, 2). The values obtained, before and after adding drug 2, are compared by analysis of variance (see Browne et al. (1, 2) and Tables 1 and 2). Simultaneous determination of urinary excretion of drug 1 and its metabolites (not necessarily by GC-MS methods), before and after adding drug 2, is particularly desirable in drug interaction studies because the urine data can be utilized to detect altered routes of metabolism and exclude the possibility of noncompliance or altered fraction absorbed (Tables 1-3) (2, 3). If SIL tracer doses are administered orally, it is possible to measure directly only absorption half-life, t~/2, and tracer dose area under the plasma concentration versus time curve (AUC). CL and Vd cannot be measured. CL can be estimated using an assumed value for Vd (3). Note that combining data from SIL tracer studies, protein binding studies, and timed urinary excretion studies (not necessarily SIL), can determine all possible causes of observed decrease (or increase) in plasma concentration of drug 1 after adding drug 2 (Table 3).

338 -" - WEEK 0 x ...... x WEEK 4 o--- - --o WEEK 12

-

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TIME AFTER PHENYTOIN INFUSSION ( hours ) Figure 1. Serum concentration versus time relationships for 150 mg 13C15N2-phenytoin i.v. tracer doses for patient on chronic phenytoin therapy before (Week O) and 4 and 12 weeks after adding chronic phenobarbital therapy. From Browne et al. (1) with permission.

1.2. Assumptions SIL tracer m e t h o d s a s s u m e a b s e n c e of m e t a b o l i c i s o t o p e effect and a b s e n c e of " d e e p pool e f f e c t " on m e a s u r e d p h a r m a c o k i n e t i c p a r a m e t e r s , The a b s e n c e of t h e s e effects can be d e m o n s t r a t e d using s t r a i g h t f o r w a r d t e c h n i q u e s described in C h a p t e r s 2, 14 and 16 and Refs, 4, 5 and 6, Thus, SIL t r a c e r s t u d i e s are based u p o n t w o a s s u m p t i o n s w h i c h can be verified,

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TIME AFTER PHENYTOIN INFUSION ( hours ) Figure 2. Serum concentration versus time relationships for 150 mg 13ClSN2-phenytoin i.v. tracer doses for patient on chronic phenytoin therapy before (Week 0) and 4 and 12 weeks after adding chronic carbamazepine therapy. From Browne et al. (2) with permission.

340 TABLE 1.

Phenytoin Pharmacokinetic Values Before (Week 0) and 4 and 12 Weeks After Adding Phenobarbtal in Six Subjects Before

Week 4

Week 12

Significance a

Mean steady state plasma concentration (l~g/mL)

12.2 b _+ 5.4

13.2 _+ 5.7

15.0 +_ 5.6

0.51

Clearance (mL/min/kg)

0.29 +_ 0.18

0.31 _+ 0.19

0.29 _+ 0.20

0.43 c

Elimination half-life (hr) (L/kg)

34.3 _+ 16.4

29.6 _+ 12.5

32.5 _+ 17.2

0.53

Volume of distribution

0.69 -+ 0.06

0.64 _+ 0.09

0.60 +_ 0.05

0.10

aBy analysis of variance. bMean _ SD. Probability of missing a truly significant change of 20 percent or greater (Type II or beta error) <0.01. From Browne et al. (1) with permission.

TABLE 2.

Phenytoin Pharmacokinetic Values Before (Week 0) and 4 and 12 Weeks Afte Adding Carbamazepine in Six Subjects Before Mean steady state plasma concentration (l~g/mL) Clearance (mL/min/kg) Elimination half-life (hr) Volume of distribution (L/kg) 48 hr urinary excretion of phenytoin, p-pydroxy-phenylphenylhydantoin, and phenytoin dihydrodiol (mg)

13.2 b _ 6.6

Week 4

Week 12

Significance a

13.4 _+ 5.6

17.8 _+ 7.1

0.00025

0.271 _+ 0.148 37.7 _+ 19.3 0.65 -+ 0.08

0.246_+ 0.150 41.8 _+ 23.1 0.69 _+ 0.09

0.172_+ 0.074 50.3 _+ 27.3 0.63 -+ 0.08

0.025 0.0005 NS

580.2 _+ 146.0

ND

579.0_+ 79.0

NS

"'bSee Table 1. ND, not determined; NS, not significant. From Browne et al. (2)with permission.

341 TABLE 3. Evaluation of Data to Determine Which of Four Possible Mechanisms Explains Decrease (increase) in Drug 1 Plasma Concentration after Addition of Drug 2

Possible mechanism

.

Data to establish mechanism

Decrease (increase)in extent of absorption of drug 1 or decrease (increase) in compliance

Decrease (increase)in timed urinary excretion of drug 1 and its metabolites

Decrease (increase)in drug protein binding

Decrease (increase)in drug protein binding.

Increase (decrease) in drug volume of distribution

Increase (decrease) in volume of distribution of second drug 1 tracer dose

Induction (inhibition) of a metabolic pathway of drug 1

Increase (decrease)in clearance via production of products of metabolic pathway of drug 1

1.3. Racemic Drugs The effect of drug 2 on the pharmacokinetics of a racemic drug 1 can be determined using the "pseudoracemate" technique (7-9). Each enantiomer is labeled with a unique stable isotope and then the enantiomers are mixed to form a pseudoracemate. Tracer doses of the pseudoracemate of drug 1 are administered before and after adding drug 2. The plasma and urine concentration of each enantiomer can be quantitated by GC-MS. The pharmacokinetics of each enantiomer of drug 1 in the absence and presence of drug 2 can be computed. The effects of phenylbutazone on the enantiomers of warfarin are shown in Figure 3.

2. A L T E R N A T I V E M E T H O D S

2.1. Radioactive Tracer Studies The procedures described for SIL tracers can also be performed using radioactive labeled tracer doses of drug, Radioactive tracer methods offer advan-

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Figure 3. Serum concentration of warfarin and one-stage prothrombin times after single oral doses of (12C/1sC) pseudoracemic warfarin in six healthy subjects before and after multiple dose administration of phenylbutazone. From O'Reilly et al. (7) with permission.

tages of lower synthetic and analytic costs. Radioactive tracer methods have some disadvantages discussed below. 2.2. Measurement of Drug Plasma Concentration Steady state CL and tl/2 of drug 1, before and after adding drug 2, can be

343 estimated using measured mean steady state plasma drug concentration of drug 1 determined before and after adding drug 2, standard equations, dosing rate, and assumed values for compliance, fraction absorbed and Vd (3). Unfortunately, this method is dependent on seven assumptions, six of which are often untrue, seldom verified, and have the potential to introduce significant errors in estimated values of CL and t~/2. See Chapter 16 and Ref. 3 for complete discussion of this method.

2.3. Stopping Drug Administration Chronic administration of drug 1 can be stopped temporarily and serial plasma specimens for conventional analysis of plasma concentration obtained before and after adding drug 2. t~/2 during chronic administration of drug can be determined from plasma concentration versus time relationships. CL can be estimated from t~/2 and an assumed value for Vd (3). This method has several problems. First, patients may be exposed to risk because of being deprived of necessary medication (e.g. antiepileptic drugs). Second, elimination half-life data without Vd data may yield misleading information when utilized to make inferences regarding CL (3). Third, use of this method for drugs with nonlinear pharmacokinetics is problematic since elimination half-life at low plasma concentrations is less than at therapeutic plasma concentrations (10, 11).

2.4. Single Dose of Drug 2 Method A single dose of drug 2 is given (usually by the oral route) in the presence of therapeutic plasma concentration of drug 1. The tl/2 of drug 2 is calculated from plasma concentration versus time relationships. The tl/2 of drug 2 in the presence of drug 1 is compared with the t~/2 of drug 2 when administered alone (usually a literature value). This method is based upon three assumptions which may, or may not, be true for a given drug and usually are not validated in the performance of the study (Table 4) (3). If any of the assumptions are not met, the t~/2 data obtained may yield misleading results when used to predict drug 2 CL at steady state plasma concentration, or dosing rate necessary to achieve a given mean steady state plasma concentration. See Ref. 3 for a complete discussion of this method.

344 TABLE 4.

Assumptions of Single Dose of Drug 2 Method 1. .

.

Volume of distribution of drug 2 is the same alone or in thet presence of drug 1. Drug 2 does not possess concentration-dependent or time-dependent pharmacokinetics. Plasma concentration versus time relationships for drug 2 are followed for sufficient time to detect the terminal exponential phase of elimination (i.e. absence of deep pool effect as defined and discussed in Chapter 14 and Browne et al. (6)).

See Browne et al. (3, 6) for detailed discussion.

3. ADVANTAGES OF SlL TRACER METHODS 3.1. Safety Traditional pharmacokinetic methods of measuring pharmacokinetic drug interactions have involved temporarily stopping chronic administration of drug to measure elimination half-life or administration of a radio-labeled dose of tracer drug. The former technique is problematic (see above). Although the amount of radiation exposure risk to subjects in a study using a radio-labeled dose of tracer drug can often be demonstrated to be trivial, such studies are difficult to perform in many countries because of concerns of institutional review boards (especially with pediatric subjects) and difficulties with radioactive waste disposal. SIL tracer methods have none of these problems.

3.2. Assumptions Verifiable SlL tracer studies are based upon two assumptions that generally are true and can be verified (see above). Alternative methods are based upon assumptions which often are not true and seldom verified (see above) (3).

3.3. High Statistical Power Random "noise" is minimized in SIL tracer studies by: (1) the high precision and accuracy of GC-MS analytical methodology; (2) the ability to co-administer drugs and avoid cross-over errors; and (3) the absence of need to use assumed values for compliance, fraction absorbed, Vd, and mean steady state

345

plasma concentration (3). This results in high statistical power and the ability to produce statistically significant results with a small number of subjects. For example, using only six subjects per study, we were able to show in man with a high degree of statistical probability that carbamazepine decreases phenytoin clearance and phenobarbital has no effect upon phenytoin clearance (Tables 1 and 2) (1, 2). 3.4. Economic Savings

The high statistical power of SlL studies reduces the number of subjects and analytic specimens necessary to obtain definitive answers with adequate statistical power for regulatory agencies. Using SIL measurements of drug 1 CL before and after adding drug 2, it is often possible to prove the presence (difference with p value <0.05 by analysis of variance) or absence (difference insignificant, 0.80 power to detect a difference 0.20 or greater) using only six to eight subjects (1, 2). The commonly employed "measurement of drug plasma concentration method" has so many sources of error (Chapter 16, (3)) that studies with 30 or more subjects often fail to produce adequate statistical power for regulatory agencies, particularly when the drug interaction is small or nonexistent (1-3). In computing costs, $100.00 per GC-MS determination ($25.00-$50.00 per HPLC determination as described in Chapters 6 and 16), and $15,000.00 for SIL drug synthesis are typical values. When subject and time costs are factored in, the smaller number of subjects in SIL studies may make the SIL study more practical despite isotope and GC-MS costs (see Chapter 24 for detailed economic analyses). Substantial savings are possible by combining an SIL drug interaction study with other Phase I studies also employing SIL methods (see Chapter 24). 3.5. Urine Data can be Collected for Additional Information

Serial urine specimens can be obtained and analyzed for concentrations of drug and metabolites. Serial measurements of urinary excretion of drug and metabolites per unit time can be used to determine changes in: (1) routes of metabolism; (2) compliance; or (3) fraction absorbed (Table 3) (3).

4. DISADVANTAGES OF SIL TRACER METHODS

See discussion in Chapter 16.

346

ACKNOWLEDGMENT Supported by the United States Department of Veterans Affairs.

REFERENCES 1. T.R. Browne, G.K. Szabo and J.E. Evans et al., Neurology, 39 (1988) 639. 2. T.R. Browne, G.K. Szabo and J.E. Evans et al., Neurology, 38 (1988) 1146. 3. T.R. Browne, D.J. Greenblatt and G.E. Schumacher et al., in Antiepileptic Drug Interactions, W. Pitlick (ed) (Demos, New York, 1989), pp. 1-20. 4. T.R. Browne, A. Van Langenhove and C.E. Costello et al., Clin. Pharmacol. Ther., 29 (1981)511. 5. T.R. Browne, A. Van Langenhove and C.E. Costello et al., J. Clin. Pharmacol., 22 (1982) 309. 6. T.R. Browne, D.J. Greenblatt and G.E. Schumacher et al., J. Clin. Pharmacol., 30 (1990) 680. 7. R.A. O'Reilly, W.F. Trager and C.H. Motley et al., J. Clin. Invest., 65 (1988) 746. 8. R.A. O'Reilly, W.F. Trager and C.H. Motley et al., Clin. Pharmacol. Ther., 28 (1980) 187. 9. S.S. Murthy, W.L. Nelson and D.D. Shen et al., Drug Meta. Dispos. Biol. Fate Chem., 19 (1991) 1093. 10. T.R. Browne, J.E. Evans and G.K. Szabo et al., J. Clin. Pharmacol., 25 (1985) 43. 11. T.R. Browne, G.K. Szabo and G.E. Schumacher et al., J. Clin. Pharmacol., 32 (1992) 1141.

347

CHAPTER 19

APPLICATIONS TO PEDIATRIC PHARMACOLOGY

GERARD PONS and ELISABETH REY Service de Pharmacologie P~rinatale et P~diatrique, Universit~ Ren~ Descartes, Paris 5 H5pital Saint-Vincent de Paul, 82 Av. Denfert Rochereau, 75674 Paris C~dex 14, France

1. I N T R O D U C T I O N

Stable isotope labeling has not been used extensively in pediatric and perinatal clinical pharmacology. This method has, however, numerous advantages. It allows one to determine the concentration of drugs with great sensitivity, therefore allowing quantification in small volumes of biological samples. The method also allows quantification with great specificity, eliminating interference by drug metabolites found in some radioactive tracer methods. Stable isotope labeling is safe, since stable isotopes are not radioactive. The only possible toxicity may be related to an isotope effect, a slowing of biochemical reactions because of the greater mass of the stable isotope. Due to the large mass difference between deuterium and hydrogen, a significant isotope effect occurs only with deuterium labeling. However, significant toxicity related to the isotope effect of deuterium can only be produced by very high levels of deuteration (greater than 15 percent of body water) far greater than the amount of deuterium in a typical tracer dose of drug (1). Stable isotope labeling also allows noninvasive in vivo studies like the CO2 breath test. All these advantages explain the great potential interest of stable isotope labeling in pediatric pharmacology. Stable isotope labeling is useful in four different types of study (2): (i) drug metabolic pathways; (ii) pharmacokinetic studies; (iii) determination of compliance; and (iv) assessment of the therapeutic or unwanted effects of drugs.

348 2. STUDIES ON DRUG METABOLIC PATHWAYS

The isotope cluster technique is a powerful method of detection and structural identification of drug metabolites and a useful tool to trace the origin and sequence of addition of groups to a drug during biotransformation.

2.1. N7 Methylation of Theophylline in Neonates The N7 methylation of theophylline into caffeine (CAF) has been demonstrated in premature neonates by Brazier and colleagues using stable isotope labeling (3-4). CAF had already been detected in plasma of premature neonates receiving theophylline for treatment and prevention of apneas (5-8). Plasma concentration of CAF correlated with those of theophylline, variations occurring in parallel. Three hypothesis were raised in order to explain that CAF was present in plasma: (i) CAF either was transferred transplacentally; (ii) or transferred via human breast milk; or (iii) theophylline was transformed, in vivo, into caffeine. Indeed, CAF is a methylxanthine, like theophylline, but CAF has an additional methyl group on the N7-position. In order to verify the third hypothesis, Brazier and colleagues used theophylline labeled with two stable isotopes: nitrogen 15 on the 1 and 3 positions, and carbon 13 on the 2 position (3-4). Two premature twin babies received theophylline, 3 mg/kg every 8 hr, starting on day 2, for 8 days, as a mixture of 46 percent of the labeled form, and 54 percent of the unlabeled one. Using gas chromatographic-mass spectrometry, the authors showed that in plasma and urine caffeine molecules have the same labeling as the administered theophylline molecules. The mass spectrum of caffeine extracted from the urine showed the molecular cluster of caffeine, 194 and 197, that corresponds to unlabeled and trilabeled CAF, respectively. The other ion clusters produced by the fragmentation of caffeine and theophylline demonstrate that the caffeine molecules had the same labeling as the administered theophylline ones. The presence of labeled caffeine in plasma and urine of babies treated by labeled theophylline confirms biotransformation of theophylline into caffeine via N7 methylation. This metabolic pathway is virtually not detectable in adults (9).

2.2. C02 Breath Test The C02 breath test (CBT) allows one to study the demethylation and the decarboxylation of molecules. The principle of the C02 breath test is as

349 follows. One or several carbon atoms of an administered molecule is metabolized via a demethylation or a decarboxylation into formaldehyde, formic acid, formyltetrahydrofolate and C02. If the molecule is artificially enriched with C13 on the position to be metabolized, the expired gas is enriched with ~3CC02 proportionally to the drug metabolism. When the carbon atom belongs to a methyl group, the C02 breath test allows a noninvasive measurement of the demethylation of the molecule, as long as the transformation of formaldehyde produced into C02 is not a limiting step of the production of C02. It has been shown in rodents that the C02 breath test is a good method for measuring the activity of the isoenzymes depending on CYP 1A2 (10-11). Since it is not easy to measure directly the activity of the enzyme in human, a correlation between the rate of expired labeled C02 and an indirect measure of the enzyme activity, such as plasma clearance, has been looked for. Such a correlation has already been evidenced for aminopyrine (12) and caffeine (13-14) demethylations. This is consistent with the knowledge that demethylation explains most of aminopyrine and caffeine clearance.

2.2.1. Maturation of N-demethylation of caffeine in neonates and infants The C02 breath test has been used to study the maturation of N-demethylation of caffeine in neonates and infants (15). The study was performed in 12 children, 4 premature neonates and 8 infants 1-10 months old. They received oral caffeine citrate solution for treatment and prevention of apnea. The usual morning maintenance dose was substituted by 1, 3, 7-~3C trimethyl-xanthine. The expired gas was collected over a 1-min period using a face mask and a small dead volume two-way valve into a latex bag. The volume of expired gas was measured with a pneumotachograph connected to a flow transducer. Aliquots were transferred to vacuum-evacuated glass flasks. Five breath samples were collected between two consecutive doses; samples also were collected the day before the administration of labeled CAF for endogenous labeled C02 production, and during the test day. On the test day, samples were collected just before the labeled dose, 2, 4 and 6 hr after this dose and before the next scheduled dose. Blood samples were drawn by heelprick after each breath sample collection on the test day. Urines were collected on the test day. The ~3C-C02 enrichment arising from labeled CAF demethylation was determined by mass spectrometry and expressed as the A ~176176 increase from baseline. This instantaneous labeled C02 excretion rate was calculated as a percentage of administered labeled CAF at each sampling time. The cumulative labeled C02 excretion rate was calculated from the area under the curve of the instantaneous labeled C02 excretion rate as a function of time at 2, 4

350 and 6 hr after labeled CAF administration. CAF plasma concentration and CAF metabolites concentrations in urines were determined using HPLC (16). Caffeine plasma clearance has been calculated as the ratio of the administered dose, assuming 100 percent bioavailability, to the area under the curve of caffeine plasma concentration as a function of time between two consecutive doses. The demethylation process was estimated by the ratio of the number of methyl groups absent in the metabolites recovered in urines to the number of methyl groups contained in the molecules of the parent CAF (16). The cumulative labeled C02 excretion rate correlated with the CAF plasma clearance. CAF plasma clearance has been used as an indirect method of validating the C02 breath test as a monitor of CYP 1A2, assuming that CAF plasma clearance correlates with CYP 1A2-dependent CAF N-demethylation. This assumption should be valid since N-demethylation is the major route of CAF metabolism, and CAF plasma clearance is expected to be sensitive only to hepatic intrinsic clearance. As CAF is a drug with low extraction ratio and low protein binding, CAF plasma clearance is not expected to be influenced either by hepatic blood flow or protein binding. The correlation between cumulated ~3C-C02 excretion rate and CAF plasma clearance has already been found in smoking and nonsmoking adults (13). This method allowed us to describe the maturational profile of CAF Ndemethylation as a function of age: - No detectable change in expired ~3C-C02 from basal values was observed in neonates and young infants, whereas, changes were measurable in all infants older than 33 days postnatal age, and older than 45 weeks postconceptional age. - These changes parallel those of CAF plasma clearance and of demethylation ratio calculated from the urine data (16-17). CAF plasma clearance increases with post-natal age according to a single exponential curve. The plateau is reached during the second trimester of life (17). The demethylation ratio increases with post-natal age as a monoexponential curve. The plateau is reached during the second trimester of life and accounts for about 60 percent of all methyl groups. Demethylated metabolites are detectable as soon as 22 days postnatal age and 34 weeks post-conceptional age (16).

2.2.2. Maturation of N-demethylation of caffeine during puberty The use of the C02 breath test also has allowed study of the maturation of N-demethylation of caffeine during puberty. Lambert et al. (18) measured the

351 changes of 13C-C02 excretion rate after a 3 mg/kg administration of caffeine labeled on the N3 methyl group in 62 subjects 3-20 years old. The labeled C02 excretion rate was higher in children before puberty than in adults and decreased with increasing age. Using the five pubertal stages of Tanner, the authors showed that the labeled C02 excretion rate decreased with increasing pubertal maturation. The maturational profile during puberty differed according t o the gender. The excretion rate of adult patients was reached earlier in puberty in girls (early puberty) than in boys (mid-puberty). These changes in C02 excretion rate suggest decreasing enzyme function during puberty and a different maturational profile according to gender. 2.2.3. Effect of growth hormone therapy in growth hormone-deficient children on demethylation of caffeine.

The use of the C02 breath test allowed Levistsky et al. (19) to study the effect of growth hormone therapy in growth hormone-deficient children on cytochrome P450-dependent 3-N-demethylation of CAF. Six 4-15 year old GH-deficient children received caffeine labeled on the 3-N-methyl group as a single 3-mg/kg oral dose before and after a 4-week treatment with human growth hormone 0.1 IU/kg s . c . - three times a week. The 3-N-demethylation of CAF, as measured by the CBT, was significantly decreased following 1 month of growth hormone therapy suggesting a possible role of growth hormone on the expression of CYP 1A2. 2.3. Enantioselectivity

There is no available data in the literature on the use of stable isotope labeling in pediatric pharmacology for the study of enantioselectivity of drug metabolism. Using stable isotopes in adults, a highly stereoselective metabolism has been demonstrated for 4-hydroxylation of debrisoquine (20) and the metabolism of other enantiomers (see Chapter 22). In children, changes related to enzymatic maturation may significantly affect the relative rate of biotransformation of enantiomers as a function of age.

3. PHARMACOKINETIC STUDIES

Using stable isotope tracer methods, it is possible to perform pharmacokinetic studies that measure true steady state values and evaluate time-dependent and dose-dependent pharmacokinetic changes without exposing the subject

352 to radiation, without creating radioactive waste and without withholding necessary medication.

3.1. Time-Dependent Pharmacokinetic Studies Serial kinetic studies during maintenance therapy using stable isotope labeling have been used in children to study time-dependent kinetic changes at steady state without need to interrupt the ongoing treatment and without exposure to radioactivity. A unit dose is replaced, totally or in part, by an equal amount of the labeled drug. Mass spectrometry allows one to study separately the kinetics of the labeled and unlabeled compound, using the same blood samples.

3.1.1. Auto-induction of drug metabolism Carbamazepine (CBZ) is used in children for the treatment of partial seizures. The decrease in steady state plasma concentration of CBZ during long-term treatment suggests that CBZ induces its own metabolism. Bertilsson and colleagues (21) used tetradeuterium-labeled CBZ in three 10-13 year old children. A preliminary study in animals ruled out an isotope effect (22). Patients were given equimolecular amounts of labeled and unlabeled CBZ as an initial oral single dose. The plasma concentrations of the two forms of CBZ were determined simultaneously by mass spectrometry during the four subsequent days. The single oral dose kinetics of CBZ and CBZ-D4 were almost identical, indicating no isotope effect of deuterium labeling in these patients. Maintenance therapy with unlabeled CBZ was started on day 5. On three separate occasions during the first five months of maintenance therapy, a portion of the CBZ dose was replaced by an equivalent amount of labeled CBZ. The plasma kinetics were studied after each of these doses. The steady state CBZ total plasma concentrations during multiple dosing were lower after dose III (days 21 to 36) and IV (about 5 months) than after dose II (day 6). They were two times lower than the theorical steady state plasma concentrations predicted using the kinetic parameters obtained from the initial single-dose experiment. But they were close to those predicted using the kinetic parameters obtained after labeled CBZ administrations during multiple dosing. There was a considerable increase (around 0.7 to 1.9 times) in plasma clearance between dose I and III without subsequent increase. Since CBZ is primarily eliminated via metabolism, changes in kinetic parameters may be attributed to changes in metabolism. This study demonstrated

353 the auto-induction of CBZ metabolism and described its time course: occurring rapidly, and maximum within 2 to 4 weeks.

3.1.2. Drug interactions Other time-dependent kinetic changes may be studied in children using stable isotopes labeling: (i) induction of biotransformation; (ii)inhibition of biotransformation (23); and (iii) the effect of age during long term therapy.

3.2. Dose-Dependent Pharmacokinetic Changes Stable isotope labeled tracer methods also have been successfully employed for describing the pharmacokinetic properties of drugs with dose-dependent (nonlinear) pharmacokinetics in adults by Browne's group (see Chapter 16).

3.3. Other Pharmacokinetic Studies 3.3.1. Placental transfer of drugs Brazier et al. (24) developed a model using labeled theophylline in a pregnant ewe in order to investigate the placental transfer of drugs. This method allows one to carry out simultaneously in the same animal, infusions with the labeled drug to the fetus and with the unlabeled drug to the mother, and to study the kinetics of both labeled and unlabeled drug in both fetus and mother. This method is of more limited use in human since injection of drug in the umbilical vein of the fetus is possible, but serial sampling of the umbilical blood is not possible. The staggered stable isotope administration technique, therefore, may be of great potential interest to evaluate placental transfer and to calculate drug entry half-life. By infusing intravenously to the mother, a series of different stable isotope-labeled forms of the same drug at different times before a single collection of fetal blood via a puncture of the umbilical vein for diagnostic or therapeutic purposes, one can obtain the same information about the drug's distribution as would be obtained by infusing a single dose of the drug and performing serial collections of umbilical venous blood (25). However, this technique has several drawbacks. Synthesis of a series of stable isotopes of a drug is expensive. Presence of several stable isotopes of a drug may create analytic difficulties by increasing problems with overlap and interference by endogenous substances. One must have an approximate idea of the time required for the umbilical drug concentration to attain equilibrium in order to select proper times for administration of stable isotope-

354

labeled analogues and specimen collection. It would, however, also have to be known for serial sample collections using alternative techniques. The mother would be exposed to the potential morbidity of several intravenous infusions of the drug being investigated.

3.3.2. Other studies Other kinetic studies are of potential interest in pediatric pharmacology: (i) the study of enantioselectivity of drugs; and (ii) particularly the determination of absolute and relative bioavailability in early infancy.

3.3.2.1. Study of the enantioselectivity of kinetics of drugs Stable isotope-labeled isomers of a drug have been used to study stereospecific aspects of a drug's disposition in adults (26-28) but, to our knowledge, not in children (see Chapter 16). 3.3.2.2. Studies of drug bioavailability and bioequivalence Bioavailability is defined as the rate and extent of absorption of a drug from its dosage form into the systemic circulation. Absolute bioavailability is measured as the ratio of the availability of a pharmaceutic formulation of a drug to the availability of the same drug after intravenous administration. Relative bioavailability is measured as the ratio of the availability of two nonintravenous pharmacokinetic alternatives of the same drug. Bioequivalence is defined as the equivalent bioavailability of two or more pharmaceutic alternatives. The primary advantage of the isotopic method is that the drug can be administered concomitantly with by two routes (e.g. parenteral and oral) or in two formulations (e.g. solution and solid dosage). Thus, a single set of blood samples serves to describe the time course of the routes or formulations being compared. The concomitant administration reduces intrasubject variability inherent in dual administration of the conventional cross-over designs, considering that each subject serves as its own "real time" control. To achieve equivalent statistical certainty, the stable isotope technique can reduce the number of subjects by at least 50 percent when compared with the cross-over technique, resulting in reduced cost (29-31). The single assay for both forms further reduces variation. The stable isotope method minimizes both drug exposure and discomfort to the subject by reducing the time and number of samples collected, and there is no need for the individual to undergo the procedure more than once. Furthermore this technique is well suited to "pulse" administration, wherein the kinetics of a single dose during

355

multiple or chronic dosing regimens can be compared with single-dose kinetics. See Chapter 13 for more information.

4. STUDY OF COMPLIANCE

Stable isotope labeling could also be used in children as tracer to determine compliance (32). The main problem with the use of stable isotopes in compliance studies is the cost of the tracer, a biologically acceptable substance labeled with a rare stable isotope (~3C for example). To minimize the cost of such a study, Schwartz (32) proposed utilizing the cheapest enriched light isotope that is available, namely, deuterium. For drugs administered as liquids (e.g. insulin), D20 could be included in the drug as a diluent. Deuterated glucose could be used as a "filler" in solid drugs. Using this method, Schwartz could detect failure to comply with prescribed dose at the 10 percent deviation level in adult subjects. There is no available example of such a use in children.

5. STUDY OF THE EFFECT OF DRUGS

5.1. Treatment of Helicobacter Pylori Infection Active chronic gastritis is clearly related to the presence of Helicobacter pylori, both in children and adults. The prevalence of H. pylori infection in children diagnosed as having primary gastritis varies between 70 and 84 percent. Present methods for detecting H. pylori comprise techniques (bacterial cultures, urease tests, histological examination of antral biopsy specimens) that need endoscopy and, therefore, are invasive and difficult to use in children. The urea breath test is a noninvasive technique designed to identify the presence of urease activity in the gastro-intestinal tract. The principle of the urea breath test is that, in the presence of the enzyme urease, orally administered urea is hydrolyzed to C02 and ammonia. If the urea carbon is labeled with either the stable isotope 13C, or radioactive 14C, it can be detected in the breath as labeled C02 (33). H. pylori is the most common urease containing gastric pathogen and, therefore, a positive urea breath test can be generally equated with the presence of an H. pylori infection. The ~3C-urea breath test results compare well with the ~4C-urea breath test results (34). The great advantage of the ~3C-urea breath test in children is the absence of radioactive risk. Although the radioactive dose with ~4C urea is low (35), total avoidance

356 is better. Furthermore, repetition of the test that will be required at intervals, theoretically places the use of radioactive isotopes at a distinct disadvantage. The urea breath test has proven to be very robust. The degree of sensitivity (96 percent), specificity (93 percent), positive (83 percent) and negative predictive value (99 percent) of the ~3C-urea breath test compared with cultures as "gold standard" (36) in children corresponds to that reported in adults (37). Eradication of H. pylori infections has proven difficult. There was a need for simple, noninvasive tests that will rapidly identify and separate therapeutic modalities according to their effectiveness. Graham et al. (38) showed that the ~3C-urea breath test is fully the equal of any of the alternative invasive procedures in monitoring a clinical trial in which the treatment was tested for its ability to eradicate H. pylori infection. 5.2. Effect of Exogenous Pancreatic Enzymes on Fat Malabsorption in Cystic Fibrosis Patients The use of ~3C-labeled lipids to detect fat malabsorption in children is of particular advantage in a pediatric population because of the simplicity of tests based on ~3C-C02 measurements of respiratory C02: the collection of fecal samples is inconvenient for outpatients, expensive if hospitalization is required; and stool analysis is unpleasant to laboratory technicians (39). Fat malabsorption results from either an absence of bile acids or pancreatic lipase or from inadequate intestinal mucosa. Watkins et al. (40) administering 10 mg/kg 1-(~3C)-trioctanoin showed that children with normal fat absorption excreted a total of 25 _+ 2.5 percent of the dose as ~3C-C02 by 2 hr, while those with steatorrhea due to pancreatic insufficiency resulting from cystic fibrosis excreted 3.5_+ 2.5 percent of the dose in the same period of time. When exogenous pancreatic enzymes were fed, the children with cystic fibrosis excreted less fat, and the ~3C-C02 excretion increased 4-fold, showing the treatment decreases fat malabsorption (40). 5.3. Effect of Drug on Protein Metabolism and Turnover of Amino-acids Stable isotope labeling has been used to study the effects of steroids on protein metabolism in man (41). The mechanism of steroid related myopathy was not known: increase in protein catabolism? decrease in protein synthesis? or both? Being an essential amino-acid, during the post absorptive state, the flux of leucine equals the rate of disappearance. Leucine undergoes both irreversible oxidation into exhaled CO= and recycling into protein synthesis.

357 Protein catabolism and synthesis can be estimated by the measurement of leucine flow in vivo. This method was used to assess the effects of a short-term high-dose prednisone treatment (40 mg/m2/24 hr - max 60 mg/24 hr) in eight normal subjects in leucine metabolism from a primed continuous infusion of L-(113C)-Ieucine during both the post-absorptive and fed state. Prednisone increased the flux of leucine coming from protein (protein catabolism) but did not modify protein synthesis. Protein balance (synthesis minus catabolism) became more negative in the post-absorptive state and failed to become positive in the fed state. Although this study has not been performed in children, this type of study is of great potential interest in children on drug therapy.

5.4. Teratogenicity In pharmacokinetic studies, the occurrence of isotope effect can lead to grossly misleading biologic and analytic results and, therefore, should be avoided. In mechanistic studies, isotope effects can be used to advantage. It is possible to elucidate which drug metabolic pathway leads to a teratogenic metabolite by labeling the drug with deuterium at various metabolic sites and determining in animals the teratogencity of the different deuterated forms (42). Reduced teratogenicity of a deuterium-labeled analogue of the drug, in comparison with the unlabeled drug, suggests that the deuterium label was placed at a metabolic site important for the formation of the teratogenic metabolite and that formation of the metabolite was inhibited by kinetic isotope effect.

REFERENCES

1. P.D. Klein and E.R. Klein, J. Clin. Pharmacol., 26 (1986) 378. 2. T.R. Browne, A. Van Langenhove, C.E. Costello, K. Bieman and D.J. Greenblatt, Ther. Drug Monit., 6 (1984) 3. 3. J.L. Brazier, B. Ribon, M. Desage and S. Salle, Biomed. Mass. Spectrom, 7 (1980) 189. 4. J.L. Brazier, B. Salle, B. Ribon, M. Desage and H. Renaud, Dev. Pharmcol. Ther., 2 (1981) 137. 5. H.S. Bada, N.N. Khanna, S.M. Somani and A.A. Tin, J. Pediatr., 94 (1979) 993. 6. C. Bory, P. Bltassat, M. Porthault, M. Bethenod, A. Frederich and J.V. Aranda, J. Pediatr., 94 (1979) 988. 7. M.J. Boutroy, P. Vert, R.J. Royer, P. Monin and M.J. Royer-Morrot, J. Pediatr., 94 (1970) 996. 8. J.L. Brazier, H. Renaud, B. Ribon and B.L. Salle, Arch. Dis. Child., 54 (1979) 194.

358 9. D.D.S. Tang-Liu and S. Riegelman, Res. Com. Chem. Pathol. Pharmacol., 34 (1981) 371. 10. K. Biagas and A.N. Kotake, Fed. Proc., 42 (1983) 2872A. 11. G.J. Lambert, H. Leitz, D. Pang and A.N. Kotake, Pediatr. Res., 17 (1983) 150A. 12. R.J. Shulman, C.S. Irving, T.W. Boutton, W.N. Wong, B.L. Nichols and P.D. Klein, Pediatr. Res., 19 (1985) 441. 13. A.N. Kotake, D.A. Scholler, G.H. Lambergt, A.L. Baker D.D. Schaffer and H. Josephs, Clin. Pharmcol. Ther., 32 (1982) 261. 14. E. Renner, H. Weitholtz, R. Hugenin M.J. Arnaud and R. Presig, Hepatology, 4 (1984) 38. 15. G. Pons, J.C. Blais, E. Rey, N. Plissonnier, M.O. Richard, O. Carrier, P. D'Athis, C. Moran, J. Badoual and G. Olive, Pediatr. Res., 23 (1988) 632. 16. O. Carrier, G. Pons, E. Rey, M.O. Richard, C. Moran, J. Badoual and G. Olive, Clin. Pharmacol. Ther., 44 (1988) 145. 17. G. Pons, O. Carrier, M.O. Richard, E. Rey, P. D'Athis, C. Moran, J. Badoual and G. Olive, Dev. Pharmacol. Ther., 11 (1988) 258. 18. G.H. Lambert, D.A. Schoeller, A.N. Kotake, C. Flores and D. Hay, Dev. Pharmacol. Ther., 9 (1986) 375. 19. L.L. Levitsky, D.A. Schoeller, G.H. Lambert and D.V. Edidin, Dev. Pharmacol. Ther., 12 (1989)90. 20. M. Eichelbaum, L. Bertilsson, A. Kupfer, E. Steiner and C.O. Messe, Br. J. Clin. Pharmacol., 25 (1988) 505. 21. L. Bertilsson, B. Hojer, G. Tybring, J. Osterloh and A. Rane, Clin. Pharmacol. Ther., 27 (1980) 83. 22. J. Osterloh and L. Bertilsson, Life Science, 23 (1978) 83. 23. I.M. Kapetanovic, H.J. Kupferberg, R.J. Porter, W. Theodore, E. Schulman and J.K. Penry, Clin. Pharmacol. Ther., 29 (981) 480. 24. J.L. Brazier, B. Ribon, M. Desage, F. Comet, M. Lievre, M. Berland and B. Salle, Dev. Pharmacol. Ther., 7 (1984) 52. 25. T.R. Browne, J.E. Evans, D.L. Kasdon, G.K. Szabo, B.A. Evans and D.J. Greenblatt, J. Clin, Pharmacol., 26 (1986) 425. 26. T.A. Baillie and A.W. Rettenmeier, J. Clin. Pharmacol., 26 (1986) 448. 27. T.A. Baillie, A.W. Rettenmeier, L.A. Peterson and N.J. Castagnoli, Ann. Rep. Med. Chem., 19 (184) 273. 28. W.F. Trager, J. Clin. Pharmacol., 26 (1986) 443. 29. M. Eichelbaum, G.E. Von Unruh and A. Somogyi, Clin. Pharmacokinet., 7 (1982) 490. 30. M. Eichelbaum, H.J. Dengler, A. Somogyi and G.E. Von Unruh. Eur. J. Clin. Pharmacol., 19 (1981) 127. 31. H. d'A.Heck, S.E. Buttrill, N.W. Flynn, R.L. Dyer, M. Anbar, T. Cairns, S. Dighe and B.E. Cabana, Pharmacokinet. Biopharm., 7 (1979) 233. 32. H.P. Schwartz, Contr. Clin. Trials, 5 (1984) 573. 33. D.Y. Graham and P.D. Klein, Am. J. Gastroenterol., 86 (1991) 1118. 34. S. Dill, J.J. Payne-James, J.J. Misiewicz, G.K. Grimble, D. McSwiggan, K. Pathak, A.J. Wood, C.M. Scrimgeour and M.J. Rennie, Gut., 31 (1990) 1237. 35. B.J. Marshall, M.W. Planke, S.R. Hoffman, C.L. Boyd, K.R. Dye, H.F. Frieirson, R.L. Guerrant and R.W. McCallum, Am. J. Gastroenterol., 86 (1991) 438. 36. Y. Vandenplas, U. Blecker, T. Devreker, E. Keppens, J. Nijs, S. Cadranel, M. Pipeleers-Marichal, A. Goossens and S. Lauwers, Pediatrics, 90 (1992) 608. 37. D.Y. Graham, P.G. Klein, A.R. Opekun and T.W. Boutton, J. Infect. Dis., 157 (1988) 777.

359 38. D.Y. Graham, P.D. Klein, D.G. Evans, D.J. Evans, L.C. Albert, A. Opekun, G.R. Jerdack and D.R. Morgan, Am. J. Gastroenterol., 86 (1991) 1158. 39. P.D. Klein and E.R. Klein, J. Pediatr. Gastroenterol. Nutr., 4 (1985) 9. 40. J.B. Watkins, D.A. Schoeller, P.D. Klein, D.G. Ott, A.D. Newcomer and A.F. Hofmann, J. Lab. Clin. Med., 90 (1977) 422. 41. B. Beaufrere and H.W. Haymond, Ann. Endocrinol., 46 (1985) 347. 42. H. Spielman and H. Nau, J.Clin. Pharmacol., 26 (1986) 474.

361

CHAPTER 20

BREATH TESTS: DIAGNOSTIC STUDIES

BEAT MEYER-WYSS ~, PlUS HILDEBRAND = and CHRISTOPH BEGLINGER 2 1Department of Internal Medicine, St. Clara Hospital, CH-4058 Basel, Switzerland; 2Division of Gastroenterology, University Hospital, CH-4031 Basel, Switzerland

1. INTRODUCTION

Breath tests constitute a class of diagnostic tests used in clinical medicine since the 1960s when 1"C-labeled substrates became available. They have been developed with the intention of simplifying traditional tests of metabolic functions that often required intubation of the intestine or collection and analysis of feces or urine. Breath tests depend on the quantification of isotopically labeled C02 in expired air which is produced by a metabolic process within the body after administration of a labeled compound. Breath tests offer tremendous advantages in terms of patient comfort. If, however, the stable isotope 13C is used for labeling substrates, the analysis of ~3C02 requires expensive and sophisticated analytical devices in the laboratory. Furthermore, breath test results may be difficult to interpret because multiple steps are involved between administration of a labeled substrate and appearance of labeled C02 in breath. Thus, for developing new tests it must be made clear that generation of labeled C02 is the rate-limiting step. In this review we will survey the current breath tests based on stable isotopes which are available for clinical evaluation of gastrointestinal disorders. In the 1970s Schoeller and Klein (1) initiated programs to measure human nutrition and body metabolism using stable isotopes. They validated the methodology of ~3C-breath tests using isotope ratio mass spectrometry and prepared the way for a wider application. The potential of safe and simple procedures to study metabolic functions stimulated the development of a variety of ~3C-breath tests during the last decade. As a consequence, stable isotopes can be used to assess specific organ functions or to measure meta-

362 TABLE 1. Summary of Diagnostic Breath Tests Applied to Diagnose Gastrointestinal or Liver Dysfunction

Helicobacter pylori infection

Urea

Bacterial overgrowth

D-xylose (glycocholic acid)

Gastric emptying

Octanoic acid

Exocrine pancreatic function

Mixed triglyceride Trioctanoin Cholesteryl octanoate

Fat malabsorption

Triolein Hiolein Trioctanoin Palmitic acid

Liver function

Aminopyrine (methacetin) Ketoisocaproic acid

bolic processes. Based on this technology, tests have been developed to diagnose a variety of disorders such as fat malabsorption, exocrine pancreatic insufficiency, impaired liver function or infections of the gastrointestinal tract. A summary of the different tests used in clinical gastroenterology is given in Table 1. Monitoring the metabolism of a particular substrate by quantification of carbon dioxide generated by the metabolic process requires that the substrate be labeled with a carbon isotope. In principal, two isotopes can be used: the radioactive carbon isotope 14C, and the stable isotope 13C. The major drawback of ~4C-breath tests is the exposure of patients to ionizing radiation generated by radioactive ~4C. The radiation dose received during ~4C-breath tests is low and less than that received by having a chest X-ray; nevertheless, ~"C-breath tests should not be performed in children or young women who may become pregnant. Furthermore, ~4C-labeled tests are no longer permitted in many countries. As a consequence, in this review we will limit the discussion to breath tests using substrates labeled with the stable carbon isotope ~3C. Automated breath ~3C02 analysis by isotope ratio mass spectrometry is an accurate, precise and fast method nowadays. Taking breath samples is a simple procedure; the subject exhales through a straw of large diameter directly into a 20-ml glass tube which is immediately capped with a siliconcoated rubber stopper. The following information summarizes the current

363 knowledge on clinical applications of stable isotope breath tests in gastrointestinal disorders.

2. TESTS FOR DETECTION OF GASTROINTESTINAL INFECTIONS

2. 1. Urea Breath Test for the Detection of Helicobacter pylori Helicobacter pylori is a gram-negative bacterium that causes infection of the gastric mucosa of man. Significant evidence has accumulated in the last decade that shows that H. pylori causes chronic superficial gastritis (2) and there is a strong association between H. pylori infection and peptic ulcer disease (3). The evidence available has led a panel of the National Institutes of Health to conclude that all patients with gastric or duodenal ulcer who are infected with H. pylori should be treated with antimicrobial agents (4). A fundamental principal for specific antimicrobial therapy is the accurate diagnosis. There are several validated methods to diagnose H. pylori infection: they can be divided into invasive and noninvasive tests. The invasive tests include endoscopy with biopsy specimens taken for histologic demonstration of H. pylori, microbiological culture or direct detection of urease activity of H. pylori in the gastric tissue (Figure 1). Noninvasive tests for H. pylori infection include serology and a breath test. 13C-labeled urea is used to detect the presence of H. pylori infection by a breath test. The rationale is based on the fact that H. pylori exhibits an extraordinarily strong activity of the enzyme urease. Thus, orally administered, labeled urea will be rapidly hydrolyzed to ammonia and labeled C02 in the presence of H. pylori infection in the stomach. If the urea carbon is labeled by 13C, it can be detected as

Stomach 13C-Urea

,

,

13CO2

Helicobacter pylori Urease activity Figure 1. 13C-urea test principle for detection of urease activity of H. pylori in gastric biopsy specimens or by breath test.

364 30

25

0

20

H. pylori positive ~15 o

~) 10 o ~

a

H. pylori negative 0

10

20

30

40

Time (minutes) Figure 2. Typical 13C02 exhalation curves in H. pylori positive and negative subjects. Data are given as 13C change (%o) over basal values. Open circles represent H. pylori positive subjects, closed circles are H. pylori negative persons.

13C02 in the breath. Figure 2 shows typical breath test results of H. pylori positive and negative patients. Since the original description of the 13C-urea breath test (5), several modifications of the test procedure have been described. These include different test meals given together with the labeled urea, different doses of 13C urea, different breath collection techniques, and different ways of expressing the breath test results (6). In a recent multicenter trial involving centers from Switzerland, Germany and the UK, a standard protocol was followed (7). The results revealed that a single 30-min breath sample was sufficient to determine the H. pylori status of a patient. Infection was shown to be present by examination of gastric mucosa biopsy specimens submitted to histopathology, quick urease testing and microbiological culture. Comparing the breath test results with these data, we have shown that an increase of the delta ~3C02 value by 5%~ over the basal value separated healthy and H. pylori-

365 TABLE 2. Sensitivity and Specificity of Diagnostic Tests for H. pylori Detection

Sensitivity (%)

Specificity (%)

Invasive tests: Culture Histology Rapid urease test (CLOtestTM)

95 98 90

99 98 98

13C-urea breath test

98

98

Source: C. Beglinger, B. Meyer-Wyss and H. Merki, unpublished data.

infected subjects with a sensitivity of 98 percent and a specificity of 98 percent (Table 2) (8). The minimum dose of 13C urea administered is 1 mg/kg body weight or, for simplicity, as 100 mg per adult test person. As the substrate is becoming cheaper, the price of the labeled substrate is not a limiting factor anymore. It can be ingested together with a liquid meal or with orange juice; both of them delay gastric emptying, thus enabling the urease of the bacterium to react with the substrate. The 13C-urea breath test has been applied to thousands of patients and healthy volunteers. Excellent epidemiologic data have been aquired in children and adults (9); in addition, breath testing has been applied to monitor the success of antimicrobial treatment in eradication therapies for H. pylori. The ~3C-urea breath test represents, therefore, the best validated breath test introduced into daily clinical life. Its advantages are evident: (1) H. pylori infection can be detected noninvasively, circumventing the necessity of endoscopic biopsies; (2) the test procedure is quick and simple, it can be done in every private practice; and (3) the test can be repeated as often as required. In summary, the urea breath test is most useful for epidemiological studies of the prevalence of H. pylori and for evaluating success of antimicrobial therapy.

2.2. Xylose Breath Test for Detection of Bacterial Overgrowth The blind loop or stagnant loop syndrome is a condition where the small intestine becomes overgrown with a bacterial flora that resembles the bacterial flora of the normal colon (10). The condition, also known as small intestine bacterial overgrowth, is accompanied by secondary nutrient malab-

366 sorption and/or diarrhea. The traditional approach to making this diagnosis requires that the duodenum and upper jejunum be intubated and fluid aspirated for microbiological culture (11). As noted above, this technique is invasive since it requires intubation with a specific tube or an endoscope, and thus it is considered unpleasant by most patients. Several breath tests have been proposed to simplify identification of small bowel bacterial overgrowth. All these tests depend on the metabolism of an orally administered substrate by the abnormal bacterial population within the small intestine yielding labeled CO2 that is detected in the breath. A critical point in determining the utility of a breath test substrate is the ability of the test to distinguish between the normal colonic flora and pathologic bacterial overgrowth in the small intestine. Thus, the following characteristics are desirable (12): (1) bacteria in the small intestine should have a good contact with the substrate; (2) the substrate should not reach the colon to minimize contact with colonic flora (the test probe should be completely absorbed before the colon is reached); and (3) if the substrate is absorbed it should not be metabolized to avoid generation of labeled CO2 by human tissues. The 14C bile acid test was first developed as a clinical aid in detecting small bowel bacterial overgrowth (13). The sensitivity and specificity of the test were low, however, and prevented further validation. As an alternative, the xylose breath test was introduced by King and colleagues (14) and developed as a noninvasive, simple breath test. Eighty-five percent of patients with bacterial overgrowth have increased 13CO2 breath concentrations within 60 min of ingesting a small amount of labeled ~3C xylose. Basically, the bacteria in the small intestine metabolize 13C xylose to ~3C02 from where it is absorbed and excreted by the lungs. When small loads of xylose are given (usually 1 g xylose), most of the ~3Clabeled xylose is absorbed in the small intestine and only a fraction reaches the colon where it can be metabolized by the bacteria to ~3CO2. Although the test can be complicated by delayed gastric emptying, it has a good sensitivity and specificity (14). The 14C-xylose breath test has been extensively validated; the ~3C-labeled substrate has been tested, however, on only a very small number of patients only. Therefore, all information with regard to sensitivity and specificity of the ~3C-xylose breath test should be considered as preliminary. Nevertheless, the xylose breath test is a very promising test with excellent results obtainable within the limits outlined above: anaerobic bacteria metabolize xylose with high efficiency; small amounts of xylose are completely absorbed in the small intestine with minimal amounts reaching the colon; the ~3C xylose has an unlimited safety and the test can be repeated if necessary. The ~3C-xylose breath test is close to an ideal breath test for diagnosing

367 bacterial overgrowth provided further validation studies confirm these initial results in a larger group of patients.

3. TESTS FOR ASSESSMENT OF ORGAN FUNCTIONS

3.1. Octanoic Acid for the Measurement of Gastric Emptying Scintigraphic measurement of gastric emptying is generally accepted as the reference method to quantify gastric emptying rates (15). It requires complex and expensive equipment and is only available in specialized centers (16). Moreover, the use of radioactive markers makes the technique unsuitable for children or pregnant women. These considerations have led to the development of alternative methods which are easy to perform, noninvasive, and not unpleasant to the patient, but nevertheless correlate well with scintigraphy. Early developments were based on 13C-labeled acetate and 13C-labeled bicarbonate and have been validated for gastric emptying of liquid meals (17). Recently, ~3C octanoic acid was introduced and validated as a breath test for the measurement of gastric emptying of solids (18). The rationale is based on the fact that desintegration of the labeled solid phase of the test meal with subsequent absorption and oxidation of ~3C octanoic acid to ~3C02 takes place once the meal reaches the digestive environment of the duodenum. ~3C octanoic acid, a medium-chained fatty acid, is readily solubilized in egg yolk. The authors of the test therefore cooked the egg yolk with the labeled octanoic acid separately from the egg white into an omelette and served it with two slices of bread, which proved to be a good marker for the solid phase of a meal for gastric emptying studies. The 13C-octanoic acid breath test is an indirect method of measurement of gastric emptying involving several consecutive steps: gastric emptying followed by desintegration of the label in the duodenum, absorption in the duodenum followed by hepatic metabolization of the ~3C octanoic acid to ~3C02 with subsequent excretion by the lungs (Figure 3). Calculation of halfemptying times is complex and based on the assumptions that: (1) gastric emptying is the only variable; and (2) subsequent metabolic steps are constant. These assumptions are not totally valid. Nevertheless, the data so far published indicate that the pattern of gastric emptying of solids including its biphasic nature can be correctly determined by breath sample analysis (Figure 4). Preliminary studies indicate that the breath test is sensitive enough to detect pharmacological influences on gastric motor function. The group of Vantrappen (19) who developed and validated the test are currently evaluating

368 Absorption

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369 several clinical applications. In summary, the 13C-octanoic acid breath test is a promising noninvasive test which potentially allows one to reliably and safely assess gastric emptying rates of solids. Not enough data are available to determine its place as a clinical test.

3.2. ~3C Triglycerides for the Assessment of Pancreatic Insufficiency and Fat Malabsorption Triglycerides comprise the major part of fat ingested with food. They consist of glycerol esterified with three fatty acids of various chain length which can be either carboxyl- or randomly 13C-labeled. Upon gastric emptying, the triglycerides are hydrolyzed by pancreatic lipase, depending on the chain length of the resulting free fatty acids, they either diffuse rapidly through the mucosa or have to be solubilized by bile acids and are dependent on mucosal transport capacities (Figure 5). As a result, these biochemical properties of the fatty acids determine whether the breath test is either more specific for exocrine pancreatic function or for fat malabsorption. The 13C is then cleaved in the liver involving several metabolic steps and finally enters the carbon pool of the body or is partially exhaled as ~3C02. The appearance in the breath is slow, with peak concentrations observed after 4-6 hr after ingestion, which means that breath sampling has to take place in 30-rain intervals up to 6 hr. The results are either expressed as percent dose/hr at a certain time or cumulative dose over time. A variety of factors can influence the result, thus reducing sensitivity and specificity of the test. To give an example, pancreatic lipase has to be reduced by at least 60-80 percent to produce signs of maldigestion. Another assumption is the rate of endogenous C02 production

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370 which is usually taken as 9mmol/kg/hr; in reality this value may show a considerable variation. Finally, delayed gastric emptying, diabetes mellitus, hyperlipidemia, or liver diseases may alter the test result by different mechanisms. 3.2. I. Trioctanoin, mixed triglyceride or cholesteryl octanoate breath test for pancreatic insufficiency

Pancreatic function tests are usually performed if the diagnosis of pancreatic disease remains a possibility even when imaging tests are normal or inconclusive. They are also valuable to determine if exocrine insufficiency is contributing to malabsorption, to assess whether, or not, pancreatic enzyme replacement is sufficient, and to determine the extent of exocrine pancreatic insufficiency prior to pancreatic resection (20). Invasive tests of pancreatic function are the reference methods, but they are difficult to perform and require gastrointestinal intubation with subsequent quantification of enzyme capacity. Unfortunately, noninvasive tests are not as sensitive and specific as the intubation procedures. Different attempts have been made to develop breath tests for diagnosis of exocrine pancreatic insufficiency. These novel breath tests are based on the assumption that substrates have to be hydrolyzed by a specific pancreatic lipase and the cleaved octanoic acid is rapidly absorbed independently of duodenal solubilization (Figure 5). 1. Trioctanoin (1,2,3[carboxyl 13C] octanoyl glycerol) has to be hydrolyzed by pancreatic lipase, which seems to be the rate-limiting step. ~3CO2excretion is faster compared to other triglycerides with a peak observed after 2-3 hr (21). 2. Mixed triglyceride (1,3 distearyl 2[~3C] octanoyl glycerol) is based on the fact that the stearyl groups have to be split of the glycerol before the ~3Clabeled octanoate can be further metabolized (22). 3. Cholesteryl-[1-~3C] octanoate is hydrolyzed by the pancreatic carboxyl ester lipase, but not by lingual or gastric lipase. The pancreatic carboxyl ester lipase requires bile acids to achieve a maximal activity, thus, the test is not predominantly reflecting exocrine pancreatic function (23). As mentioned above, these tests not only reflect exocrine pancreatic function in form of lipase digestion, but can be influenced by various diseases. Vantrappen et al. (22) report an excellent correlation between intraduodenal lipase secretion and cumulative 13C02 excretion in the mixed triglyceride

371 br{:.,ath test. The published figures for sensitivity (0.89), and specificity (0.81), were only determined in patients with pancreatic disease and in normal volunteers. However, false positive tests were obtained in patients with nonpancreatic steatorrhea, liver disease and diabetes suggesting that the test is susceptible to errors in patients with bile acid deficiency, or in patients who have diseases of the small intestine or the liver. Similar limitations appear to be true with the trioctanoin and cholesteryl octanoate breath test. In summary, breath tests based on substrates requiring hydrolyzation by specific pancreatic enzymes are relatively simple noninvasive tests for assessment of pancreatic function. At present, it is not possible to define the sensitivity and specificity of these tests; they are probably most useful for monitoring the effect of pancreatic enzyme replacement therapy. Further studies are necessary in larger population groups to define their clinical value. 3.2.2. Triolem, hiolem, trioctanoin and palmitic acid breath tests for assessment of fat malabsorption

The conventional procedure for assessment of steatorrhea requires the measurement of fecal fat excretion for three days. This procedure is inconvenient, time consuming, unpleasant to perform for the laboratory technician and prone to inadequacy. Therefore, breath tests have been developed for quantification of fat malabsorption. As for pancreatic function tests, several steps are necessary in the pathway prior to exhalation of labeled CO2 in the breath: gastric emptying, hydrolysis by lipase, solubilization, mucosal absorption, hepatic metabolism and pulmonary excretion. Therefore, the same diseases as mentioned before may yield false pathologic results, thus reducing the specificity of the test. Breath analysis for 14CO2 following ingestion of 14C-labeled triglycerides was first proposed in the early 1960s and, thus, represents one of the earliest proposals for a diagnostic breath test (24). Concern regarding deposition of 14C in slowly metabolized fat pools prevented further development. A report from the Mayo Clinic in 1979 led to renewed interest in this test procedure. In this study, the investigators compared three different labeled triglyceride breath tests (14C triolein, 14C tripalmitin and 14C trioctanoin) to stool fat examinations (25). In this first report, sensitivity and specificity were excellent for 14C triolein, but less satisfying for 14C tripalmitin and 14C trioctanoin, respectively. Unfortunately, subsequent studies could not confirm these encouraging results. Furthermore, the concern with ~"C labels remained. Newer developments relied, therefore, on 13C-labeled substrates, but are based on the same principals. Breath tests with medium chain 13C-labeled

372 triglycerides have been described in the section on exocrine pancreatic function testing. Using triglycerides with long-chain fatty acids, hydrolization by lipases is not the only limiting step, bile salt solubilization and active absorbance by the small intestinal mucosa are also important factors for fat malabsorption (26). in general, the results have been controversial. First, the sensitivity and specificity of the different substrates is not very good in patients with diabetes mellitus, thyroid disease, liver disease or hyperlipidemia. Triolein is the substrate the absorption of which depends on the adequacy of all components, but the recognition of fat malabsorption does not indicate its etiology. Although trioctanoin was initially developed for the detection of fat malabsorption, it is more specific for exocrine pancreatic function. Tripalmitin and palmitic acid, comprising saturated long-chain fatty acids, were able to separate patients in earlier studies using 14C-labeled components, but the results with 13C palmitic acid were not as effective in identifying the cause of fat malabsorption (25). In fact, King et al. (27), were not able to distinguish between normal and abnormal fat absorption in their patients with the breath test in any of the measured parameters (peak labeled CO2 concentrations,

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373 cumulative 6- or 8-hr excretion or any hourly rate). Other groups have reported similar experiences (28). Recently, 13C hiolein was developed, a triglyceride with various long-chain fatty acids. In contrast to carboxyl ~3C-labeled substrates, 98 percent of all carbon atoms are randomly labeled, which offers the theoretical advantage of a higher ~3CO2 recovery rate and maybe a faster appearance of ~3CO2 in the breath. Initial experiments in Peter Klein's laboratory demonstrated that the randomly labeled fatty acids provided oxidative information equivalent to that of carboxyl-labeled substrates (29). Our own experience in a small group of volunteers showed, however, that ~3C hiolein is able to detect artificially induced fat malabsorption (30), but peak excretion rates occurred after 5 hr only and were most discriminative at 8 hr (Figure 6). Further developments (modification of meals, substrates) and additional validation experiments are necessary before an enhanced and expanded use of this test can be advocated in clinical gastroenterology.

4. LIVER FUNCTION BREATH TESTS

In clinical practice, the seriousness of liver disease is assessed on the combined information from clinical examination, routine biochemical tests and liver histology. During the past 20 years, several reports on quantitative liver function tests have been published in order to complement this traditional clinical assessment (31). Because the liver is the principal site of drug metabolism, most of these tests are based on the quantification of hepatic metabolism of a model compound. Pharmacological properties that qualify a drug for use as a test drug include: rapid and complete absorption from the gastrointestinal tract, distribution in total body water with negligible binding to plasma proteins, low hepatic extraction ratio and exclusive hepatic metabolism with negligible renal excretion (32). Thus, antipyrine clearance has been applied to identify and quantitate the impact of many factors and conditions on hepatic antipyrine metabolism. The determination of plasma clearance of a drug, however, is a rather cumbersone procedure, requiring repeated blood sampling and rather sophisticated chemical analysis. Bircher et al. (33), therefore, made an effort to develop a simple procedure for the measurement of at least one important hepatic metabolic step. Using an animal model, they were able to demonstrate that assessment of the exhalation rate of 14CO2 following intravenous administration of aminopyrine, specifically labeled at the two 4-N-methyl groups, yielded quantitative information on the hepatic demethylation of this compound which responded to experimentally induced pathologic changes of the liver in a predictable manner. Their studies formed

374 the logical basis for the numerous reports on liver function breath tests that have been published in the following years.

4. I. 73C-Aminopyrine Breath Test

Hepner and Vesell (34) applied a modification of the method published by Bircher et al. (33) to humans. They gave ~4C aminopyrine by mouth and measured the specific activity of ~4CO2 in breath after 2 hr. Based on their results, they proposed the procedure as a new quantitative liver function test. Lauterburg and Bircher (35) later confirmed the observations of Hepner and Vessell using a more complete analysis of the decay of specific activity of ~4CO2 in breath and demonstrated that the aminopyrine N-demethylation is affected to the same extent as other sensitive tests of liver function, such as galactose elimination capacity and BSP clearance (35). The introduction of the stable isotope 13C into aminopyrine represented a further extension of the use of ~3C-labeled substrates to assess hepatic drug metabolism. Schneider et al. (36) were able to show that after oral administration of 2 mg/kg of 13C-labeled aminopyrine resulted in rates of production of ~3CO2 that were nearly identical to those obtained with the ~4C-aminopyrine breath test. The diagnostic and prognostic implications of impaired aminopyrine breath test results have been investigated extensively. The findings can be summarized as follows: the aminopyrine breath test correlates with histologic features such as necroinflammatory activity and cirrhosis (37) and is able to distinguish cholestatic liver disease from chronic active hepatitis (38). It has also been shown that the aminopyrine breath test results can predict survival in alcoholic hepatitis and cirrhosis (39), in patients undergoing surgery (40) or in patients with acetaminophen intoxication (41). Despite the great number of encouraging publications, the aminopyrine breath test has not gained broad clinical importance. The reason is likely the fact that so far it has not been shown that the aminopyrine breath test is superior to the traditional clinical assessment using the simple Child-Turcot classification of hepatic functional impairment (42). Repeated testing of single patients, however, may overcome some of the problems associated with the interpretation of single aminopyrine breath test results. Reichen et al. (43) have been able to show that serial determination of galactose elimination capacity in patients with primary biliary cirrhosis may permit a very accurate prediction of death that was possible earlier than with the Mayo model. Thus, it is conceivable that repeated testing with the

375 aminopyrine breath test may also give additional important clinical information and prospective studies evaluating this concept are certainly needed.

4.2. Liver Function Breath Tests with Other ~3C-Labeled Substrates

A number of drugs have been evaluated in the attempt to develop quantitative liver function tests. The principles remained the same as outlined for the aminopyrine breath test. Labeling of methyl or ethyl side chains of drugs with ~3C will result in the formation of labeled ~3CO2 when the respective drug is oxidatively metabolized by the microsomal enzymes in the liver. Thus, 13C phenacetin (44), and its methyl analogue 13C methacetin (45-47), have been used as substrates for liver function breath tests and were found by the respective investigators to be at least as useful as the aminopyrine breath test. Similarly, a ~3C-caffeine breath test has also been published (48). However, the intrinsic conceptual problems remained the same, whatever substrate was used. First, there is a large interindividual variation of the activity of specific microsomal enzymes that is due to genetic and environmental factors. Second, many processes other than microsomal enzymatic activity may become rate-limiting in the generation of labeled ~3CO2 (absorption, distribution, protein binding, liver blood flow) and finally exhalation of labeled ~3CO2 may be affected by pulmonary disease. Third, the different breath tests each quantitate a very particular enzymatic reaction which may not be representative for the functional reserve of the entire liver (49).

4.3. Breath Tests for Detection of Alcoholic Liver Disease

Mitochondria are essential for cell function, and mitochondrial dysfunction could be a key determinant of alcohol-associated liver damage. Hepatic mitochondria of patients with alcoholic liver disease are pleomorphic and exhibit an increase in size, inclusion bodies and disorganization of cristae. Michaletz and coworkers have recently introduced a noninvasive probe of primarily hepatic mitochondrial function in experimental animals (50). (x-ketoanalogues of transchain amino acids are decarboxylated by enzymes that are mainly located in mitochondria and the decarboxylation of a-ketoisocapronic acid can be assessed in vivo by measuring the exahalation of labeled CO2 following the administration of labeled ketoisocapronic acid (KICA). In human studies, administration of 1 mg/kg 2-keto-l-~3C (isocaproic acid) resulted in peak exhalation of ~3CO2 that was significantly lower in alcoholics compared to healthy

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controls and patients with nonalcoholic liver disease (Figure 7) (51). Thus, the mitochondrial function a s r e f l e c t e d by the KICA breath test is impaired in chronic alcoholics. The functional impairment seems specific for ethanol abuse and not a reflection of decreased global hepatic function. The KICA breath test, therefore, could be a useful marker for excessive ethanol consumption.

REFERENCES 1. 2. 3. 4. 5.

D.A. Schoeller and P.D. Klein, Biomed. Mass Spectrom., 5 (1978) 29. S. Moss and J. Calam, Gut, 33 (1992) 289. E.A.J. Rauws and G.N.J. Tytgat, Lancet, 335 (1990) 1233. NIH Consensus Development Panel, J. Am. Med. Assoc., 272 (1994) 65. D.Y. Graham, P.D. Klein, D.J. Evans, D.G. Evans, L.C. Alpert, A.R. Opekun and T.W. Boutton, Lancet, I (1987) 1174. 6. D.Y. Graham and P.D. Klein, Am. J. Gastroenterol., 86 (1991) 1118. 7. R.P.H. Logan, S. Dill, F.E. Bauer, M.M. Walker, A.M. Hirschl, P.A. Gummett, D. Good and S. Mossi, Eur. J. Gastroenterol. Hepatol., 3 (1991) 915.

377 8. R.H. Eggers, A. Kulp, R. Tegeler, F.E. LQdtke, G. Lepsien, B. Meyer and F.E. Bauer, Eur. J. Gastroenterol. Hepatol., 2 (1990) 437. 9. P.D. Klein, Gastrointestinal Physiology Working Group, D.Y. Graham, A. Gaillour, A.R. Opekun and E. O'Brian Smith, Lancet, 337 (1991) 1503. 10. C.E. King and P.P. Toskes, Gastroenterology, 76 (1979) 1035. 11. P.K. Bardhan, K. Gyr, C. Beglinger, J. V6gtlin, R. Frey and W. Vischer, Scand. J. Gastroenterol., 27 (1992) 253. 12. P.P. Toskes, C.E. King, J.C. Spivey and E. Lorenz, Gastroenterology, 74 (1978) 691. 13. H. Fromm and A.F. Hofmann, Lancet, II (1971) 621. 14. C.E. King, P.P. Toskes, J.C. Spivey, E. Lorenz and S. Welkos, Gastroenterology, 77 (1979) 75. -'15. H. Minami and R.W. McCallum, Gastroenterology, 86 (1984) 1592. 16. R.C. Heading, P. Tothill, G.P. McLoughlin and D.J.C. Shearman, Gastroenterology, 71 (1976)45. 17. S. Mossi, B. Meyer-Wyss, C. Beglinger, W. Schwizer, M. Fried, A. Ajami and R. Brignoli, Dig. Dis. Sci., 39 (1994) S107. 18. Y.F. Ghoos, B.D. Maes, B.J. Geypens, G. Mys, M.I. Hiele, P.J. Rutgeerts and G. Vantrappen, Gastroenterology, 104 (1993) 1640. 19. B.D. Maes, M.I. Hiele, B.J. Geypens, P.J. Rutgeerts, Y.F. Ghoos and G. Vantrappen, Gut, 35 (1994) 333. 20. C.E. King and P.P. Toskes, Clin. Gastroenterol., 12 (1983) 591. 21. J.B. Watkins, P.D. Klein, D.A. Schoeller, B.S. Kirschner, R. Park and J.A. Perman, Gastroenterology, 82 (1982) 911. 22. G.R. Vantrappen, P.J. Rutgeerts, Y.F. Ghoos and M.I. Hiele, Gastroenterology, 96 (1989) 1126. 23. S.G. Cole, S. Rossi, A. Stern and A.F. Hofmann, Gastroenterology, 93 (1987) 1372. 24. A.F. Abt and S.L. Von Schuching, Bull. Johns Hopkins Hosp., 119 (1966) 316. 25. A.D. Newcomer, A.F. Hofmann, E.P. DiMagno, P.J. Thomas and G.L. Carlson, Gastroenterology, 76 (1979) 6. 26. C.E. King, P.P. Toskes, T.R. Guilarte, E. Lorenz and S.L. Welkos, Dig. Dis. Sci., 25 (1980) 53. 27. C.E. King, L.B. Snook and P.P. Toskes, Gastroenterology, 82 (1982) 1100. 28. W.F. Caspary, Clin. Gastroenterol., 7 (1978) 351. 29. P.D. Klein, D.L. Hachey, A.R. Opekun, P.E. Tacquard and D. Kyle, Gastroenterology, 100 (1991) A528. 30. H. Ashraf, P. Hildebrand, R. Meier, B. Meyer-Wyss, C. Beglinger, A. Christ and K. Gyr, Gastroenterology, 108 (1995) A715. 31. J. Bircher, Hepatology, 6 (1986) 1036. 32. E.S. Vesell, Clin. Pharmacol. Ther., 26 (1979) 275. 33. J. Bircher, A. KLipfer, I. Gikalov and R. Preisig, Clin. Pharmacol. Ther., 20 (1976) 484. 34. G.W. Hepner and E.S. Vesell, Ann. Intern. Med., 83 (1975) 632. 35. B.H. Lauterburg and J. Bircher, J. Pharmacol. Exp. Ther., 196 (1976) 501. 36. J.F. Schneider, D.A. Schoeller, B. Nemchausky, J.L. Boyer and P. Klein, Clin. Chim. Acta, 84 (1978) 153. 37. R. Carlisle, J.T. Galambos and W.D. Warren, Dig. Dis. Sci., 24 (1979) 358. 38. J. Galizzi, R.G. Long, B.H. Billing and S. Sherlock, Gut, 19 (1978) 40. 39. J.F. Schneider, A.L. Baker, N.W. Haines, G. Hatfield and J.L. Boyer, Gastroenterology, 79 (1980) 1145. 40. R.A. Gill, M.W. Goodman, G.R. Golfus, G.R. Onstad and M.P. Bubrick, Ann. Surg., 198 (1983) 701. 41. J.B. Saunders, N. Wright and K.O. Lewis, Br. Med. J., 280 (1980) 279.

378 42. J.P. Villeneuve, C. Infante-Rivard, M. Ampelas, G. Pomier-Layrargues, P.M. Huet and D. Marleau, Hepatology, 6 (1986) 928. 43. J. Reichen, T. Widmer and J. Cotting, Hepatology, 14 (1991) 504. 44. D.A. Schoeller, A.N. Kotake, G.H. Lambert, P.S. Krager and A.L. Baker, Hepatology, 5 (1985) 276. 45. P. Krumbiegel, K. G~nther, H. Faust, G. MSbius, K. Hirschberg and G. Schneider, Eur. J. Nucl. Med., 10 (1985) 129. 46. B. Meyer, A. Ajami, G.L. Davis and P. Toskes, Gastroenterology, 90 (1986) 1747. 47. K. Matsumoto, M. Suehiro, M. Iio, T. Kawabe, Y. Shiratori, K. Okano and T. Sugimoto, Dig. Dis. Sci., 32 (1987) 344. 48. A.N. Kotake, D.A. Schoeller, G.H. Lambert, A.L. Baker, D.D. Schaffer and H. Josephs, Clin. Pharmacol. Ther., 32 (1982) 261. 49. B. Meyer-Wyss, E. Renner, H. Luo and A. Scholer, J. Hepatol., 19 (1993) 133. 50. P.A. Michaletz, L. Cap, E. Alpert and B.H. Lauterburg, Hepatology, 10 (1989) 829. 51. A. Witschi, S. Mossi, B. Meyer, E. Junker and B.H. Lauterburg, Alcoholism, 18 (1994) 951.

379

CHAPTER 21

BREATH TEST: DRUG METABOLISM STUDIES

J.L. BRAZIER Facu/ty of Pharmacy, University of Montreal, CP 6128, Succursave Centre Vi//e, Montreal H3C 2J7, Quebec, Canada

1. I N T R O D U C T I O N

The increase in the availability of stable isotopes in diverse chemical forms, and the improvements in the instrumentation for their determination during the past decade, have resulted in their broad scientific application. Clinical pharmacology gained in power, with the use of stable isotopically labeled (SIL) molecules, in diverse human studies. With the rapid development of mass spectrometry coupled to gas chromatography, many human studies have been performed in various areas of drug research and development: metabolism, bioavailability and bioequivalence, drug follow up, as well as in studies of drug mechanisms. The major advantages of these SIL molecules is the absence of radioactivity and hazard generated by radiations. The only possible toxicity may be an "isotope effect" resulting from the substitution of the lighter isotope by its heavy counterpart leading to physicochemical characteristics modifications and metabolic switching (1). Nowadays, the trends are in developing noninvasive methods investigations as referred to by Loft and Poulsen (2). Among the noninvasive methods for studying or predicting xenobiotics metabolism, breath tests using first, 146 labeling and now 130, are increasingly used. Recent developments of continuous flow isotope ratio mass spectrometry (CFIRMS), and analytical systems devoted to the measurement of the 13002 content of breath, allowed the broadening of these applications especially in the area of metabolic studies.

380 2. BREATH TEST: PRINCIPLES AND MEASUREMENTS

2.1. Principles Historically, ~4C was the first isotope used to label drugs for metabolic studies. Exhaled CO2 from dealkylation of radiolabeled probes is collected by bubbling expired gas through a trapping agent (hyamine...). Trials were carried out by Rhodes and Houston (3) using ~"C-labeled antipyrine and rats placed in glass tubes with one-way flow for the collection of CO2. Pharmacokinetic parameters such as elimination rate constant were determined from the labeled probe. The method originally described for aminopyrine was extended to other drugs including caffeine, diazepam and erythromycin (4). The measurement of ~4CO2 is an absolute measurement of the amount of exhaled ~4CO2. There are some problems in the interpretation of the cumulated exhalation as a measure of the drug-probe clearance. Thus, only 50 percent of the labeled carbon generated by demethylation of aminopyrine is oxidized to bicarbonate accordingly to high variable kinetics and equilibrated into the HCO~ ~->CO2 pool before exhalation by lungs (5, 6). It is obvious that the use of radioactive compounds in man raises ethical and toxicological problems. So stable isotopes are preferred, not only in human studies, but also in preclinical animal studies. The breath test concept relies on the administration of a specific ~3C-labeled substrate whose metabolic utilization is monitored by measuring the elimination of ~3CO2 in the expired breath gases. This substrate acts as a metabolic probe whose target is the biological system which is implicated in the consumption of this substrate and, more specifically, of its labeled functional group(s). Informations on the integrity or changes of this particular biological system can be obtained from correlations existing between the kinetics of ~3CO2 elimination and the functional capacity of the system. Very low levels of stable isotopes and isotope ratios can now be measured accurately and with a high precision from micro-samples using continuous flow isotope ratio mass spectrometry coupled with gas chromatography. As an example, it is possible to describe the demethylation of ~3C aminopyrine. This substrate undergoes a cytochrome P450-dependent oxidative demethylation leading to its metabolites and [~3C]-formaldehyde which is further transformed into [~3C]-formate. The oxidation of [~3C]-formate occurs predominantly via the methionine-dependent folate pathway and generates ~3CO2, a part of which is eliminated by exhalation. Figure 1 shows the metabolic pathways allowing the production of CO2 after cytochrome P450 demethylation of N-methyl xenobiotics.

381 R-N-*CH 3

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H20 *CO 2

Figure 1. Production of carbon dioxide after cytochrome P450-dependent demethylations of xenobiotics. (*) labeled carbon.

2.2. Continuous Flow IRMS and Measurements

Isotope ratio mass spectrometers are designed for the continuous analysis of adjacent ion beams which necessitates a very high stability. These ions correspond to gas formed from the molecule to be analyzed. When measuring the isotopes of carbon, various isotopomers of CO2 are measured correspond-

382 ing to molecular weight of 44, 45 and 46, respectively. Consequently, three ion beams at m/z = 44, 45 and 46 are separated and collected in order to obtain the value of the 13C/12C ratio of the compound under analysis. The accelerated ions fly across a magnetic field which is kept constant. Then an optimized tuning of the accelerating voltage allows a continuous collection of the ion currents of interest. One of the original concepts in IRMS analysis is the use of a dual inlet for alternate admission of a sample and a reference gas and subsequent comparative isotope ratio measurements (7). This dual inlet can play the role of an interface between the IRMS analyser itself and various systems allowing, on-line, the preparation of the gaseous sample to be isotopically analyzed. Dynamic interfaces have been designed for such a purpose. This kind of dynamic interface consists of two parallel gas lines: a reference inlet line and a sample inlet line. These lines draw the sample and reference gas to a switching valve via two open splits. This valve ensures the admission of both gases into the ion source of the spectrometer. On the reference side of the dynamic interface, a pressure regulated flow of helium continuously sweeps the capillary where pulses of reference CO2 can be injected. The open splits completely isolate the mass spectrometer from any pressure transients which may occur during valve switching in the interface system and ensure a high stability in the ion source. A high efficiency cryogenic water trap ( - 1 0 0 _ 1~ is fitted along the sample inlet line. Figure 2

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383 displays the scheme of the dual inlet dynamic interface which allows the online preparation of the CO= to be isotopically analyzed. Obviously, water must be prevented from entering the mass spectrometer because the partial protonation of CO2 induces the formation of CO2H§ ions, thus interfering with the isotopic measurement. After the changing over valve, CO= enters the ion source and ions at m/z 44, 45 and 46 are continuously recorded (8). For the measurement of the isotopes of carbon, various devices or analytical systems allowing the preparation and purification of the CO=, to be isotopically analyzed, can be connected to the sample line of the dual dynamic interface.These devices can be: (i) a gas chromatograph and a combustion oven; (ii) an elemental analyser; or (iii) a gas chromatograph for the separation of gaseous mixtures containing CO=. When CO2 is present in a gaseous mixture it is possible to directly couple a gas chromatograph to the dynamic interface. The components of the mixture of gases can be separated on a column packed with Hayesep Q Porapack and detected using a thermal conductivity detector. A switching valve allows only CO2 to enter the isotope ratio mass spectrometer for isotopic measurement. Amounts of CO2 as small as 10 nanomoles are sufficient to ensure an accurate and precise determination of the 13C/12C ratio (9, 10). Various kinds of samples can be of interest in the area of drug metabolism. Among them are breath samples from small animals or humans, collected after administration of 13C-labeled substrates, atmospheres of in vitro, tissues, cells or culture media. It is also possible to associate CO2 micro-generators that can generate micro-volumes of CO2 using either chemical or enzymatic reaction from substrates to be analyzed for their isotopic content. The resulting CO2 produced by the reaction is purified on line by gas chromatography and immediately measured for its 13C content. The statistical combination of the isotopes of carbon (12C, 13C) and oxygen (160, 170, 180) to generate the CO2 molecules gives rise to the formation of various isotopomers whose molecular weights are 44, 45 and 46, respectively. In order to obtain a high precision and accuracy, reference gases of known isotopic composition are used and the dual inlet system allows an alternative admission of both sample gas and reference gas into the ionization source via a gas-switching valve. The measurement of the various ion beams allows the calculation of the 13C enrichment of the sample: ~13C (%o). The 13C abundance is generally expressed as (~13C (%o) according to the following relation:

813C (%o)= ([(13C/12C)sample/(13C/l=C)PDB]- 1)x 1000

384 This delta 813C (%o) value measures the variations in part per thousand of the carbon isotope ratio from the standard. For carbon, PDB was selected as the international reference. PDB is the Pee Dee Belemnitella (a fossil from the Pee Dee geological formation of South Carolina). The ~3C/~2C ratio from the calcium carbonate of this fossil is 0.011237. So compared to PDB, most of the natural compounds display a negative delta value, which is also true for CO2 values. For isotopic CO2 measurements, micro-breath samples are injected into the chromatographic column. A switching valve placed beyond the thermoconductibility detector (TCD) is activated between t~ and t2 (Figure 3), so that the CO2 separated form the breath sample is entirely transferred to the sample transfer line. Pulses of reference CO2 are admitted into the reference transfer line at preprogrammed times so that the peak of CO2 to be measured is framed by pulses of reference gas.

3. 13C0z AND DRUG METABOLISM 3.1. Action on Cyt P450-Mediated Drug Metabolism Enzymes The liver is the main organ involved in metabolism, especially in the transformations of endogenous as well as exogenous substances. The diversity and complexity of liver functions make its examination very difficult. Many mechanisms of biotransformation are based on oxidative reactions carried out by the cytochrome P450-dependent monooxygenase system. The reactivity of these enzymes can be modified by many chemical compounds, xenobiotics and drugs which react as inducers or inhibitors. Breath tests using ~3C-labeled substrates are used for diagnosis purpose and allow measurement of the metabolic activity of these mono-oxygenase systems in order to explore the liver function. As these enzymes can be induced or inhibited by drugs or chemicals, breath or micro-breath tests can be used to measure the changes in enzyme activities which can modify drug metabolism. During preclinical studies, 13CO2-breath tests can be used on small animals in order to detect inducing or inhibitory activities of drugs or metabolites as well as xenobiotics. Breath tests can also be utilized to measure modifications of the liver oxydative metabolism induced by drugs, or to monitor a potential hepatotoxicity.

385

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column switching and selective transfer of purified C02 to the IRMS source. (b) Record of the traces corresponding to ions at m/z = 44, 45 and 46 from the sample and the reference C02.

3.1.1. Induction

Enzyme induction by drugs and other xenobiotic chemicals, was discovered initially by virtue of the profound effects that induction can have on many pharmacologic responses as reported by Conney (11). Over the past decades,

386

many more situations have been uncovered in which induction of drugmetabolizing enzymes has been shown to significantly alter pharmacologic or toxic responses in vivo in laboratory animals and humans (12). It is of importance to rapidly detect inducing activities because there are two primary practical concerns related to P450 induction. First, induction may alter the. efficacy of therapeutic agents. Second, induction may create an undesirable umbalance between rates of "toxification" or "detoxification" in organisms exposed to drug or environmental chemicals. Some drugs that act as cytochrome P450 inducers are also substrates for the enzymes that they induce, they may stimulate their own metabolism, enhance their clearance and, thereby, reduce the duration or intensity of their pharmacodynamic effect. Many xenobiotic chemicals, drugs and/or metabolites can stimulate the metabolism and clearance of unrelated compounds. In the cases of drugs with low therapeutic index, induction may severely compromise efficacy. Since liver is the major site of xenobiotic metabolism, it is also frequently the prime target for toxicity from reactive metabolites. Studies of P450 induction in humans are intrinsically more difficult than in laboratory animals, however, the continued development of various technical approaches has led to considerable progress in this area. In vivo probes, among them micro-breath tests using 13C02, can be used in laboratory animals as well as in humans. The rapid screening of an inductive activity can be carried out on small animals as described by Guilluy et al. (9). Rats or mice are housed in tight all-glass metabolic cages swept by a continuous flow of air in order to ensure physiologic breathing conditions. Expired gases can be collected, either automatically or manually, in glass vacutainer at various times after 13C-labeled substrate administration. Small samples of breath air are collected because only 250 ~1 are needed for the measurement of the 13C/~2C ratio of the expired C02, by CFIRMS. Various substrates can be used to study the activity of phenobarbitalinducible P450 or other isoforms of cytochrome P450. Moreover, when using caffeine as metabolic probe the various N-CH3 groups at positions 1, 3 and 7 can be labeled separately allowing one to obtain informations on various isoforms of cytochrome P450. Figure 4 shows the variation of the ~3C enrichment of the C02 expired by a rat after administration of 1,3,7 [(13CH3)3]-caffeine as substrate in a twosequence trial. The first sequence corresponds to the measurement of the basal metabolism of the rat. Sequence II was performed after cytochrome P450 induction carried out by a single i.p. (30 mg/kg) of 3-methyl-cholanthrene, 24hr before the second administration of labeled caffeine. It can be observed that immediately after [~3C]-caffeine administration, the 13C02

387

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Minutes 0

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100 120 150 180

Figure 4. Kinetics of the expired 13002 by a rat after 1,3,7 [13C3]-caffeine administration. Basal kinetics and kinetic after one injection of 3 methylcholantrene (3 MC).

enrichment rises very rapidly up to +30.74 i~/ooPDB within 20 min. The maximum (~130po B difference versus basal value was +53.88(~%o, and was reached after only 30 min. This large change in C02 excretion clearly indicates the inductive effect of 3-methylcholanthrene on caffeine N-demethylase activity. Figure 5 shows a typical profile of the 13002 enrichment ((~130PDB versus time) from a rat after administration of [13C2]-aminopyrine (sequence I), and of the same substrate after a 6-day administration of phenobarbital (sequence II). In sequence II, immediately after [13C2]-aminopyrine injection, the 13002 enrichment of the expired air increases from -23.70(~%o (basal value) to --4.96(~130pDa, 20 min after administration. The maximum enrichment difference between sequence II (induced) and sequence I (noninduced)is +21.12 (~%o, 10 min after [13C2]-aminopyrine administration. Such an increase in the rate of excretion of 13002 derived from N-demethylation of isotopically labeled aminopyrine clearly indicates the inductive effect of phenobarbital. 3. 1.2. Inhibition

The activity of cytochrome P450 may be decreased by four main mechanisms. Thus, (i) a drug may compete with other substrates for reversibly binding to cytochrome P450; (ii) a reactive metabolite may covalently bind to a nitrogen

388

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Figure 5. Kinetics of 13CO2expired by a rat after [13C2]-aminopyrine before and after 6 days of treatment with phenobarbital (Pheno).

atom of the heme and destroy cytochrome P450 (13); (iii) reactive metabolites may also covalently bind to the apoprotein itself, with the formation of a hypoactive cytochrome P450; and (iv) either the drug itself, or one of its metabolites, may form complexes with the iron of cytochrome P450. Cimetidine, an H2-receptor antagonist, has the additional pharmacologic property of impairing hepatic microsomal drug oxidizing capacity (14). When co-administered with a number of drugs that are biotransformed by oxidative processes, cimetidine impairs metabolic clearance, leading to modified pharmacokinetic parameters: increased elimination half-life, increased area under plasma concentration versus time curve, and higher steady-state concentration during multiple dosing (15). This inhibition of drug oxidizing capacity is not an invariable component of the action of H2 antagonists, since ranitidine does not share this inhibition property. As a matter of fact, ranitidine does not impair the metabolism of several drugs including antipyrine, warfarin, propranolol, theophylline or phenytoin (16). Bell et al. (17), and Gerber et al. (18), have demonstrated that ranitidine does not inhibit the liver cytochrome P450 mixed function oxidase system because contrarily to cimetidine, ranitidine does not possess the imidazole ring thought to be the site of binding to cytochrome P450. Various studies of the pharmacokinetics of drugs associated with cimetidine and ranitidine have demonstrated inhibitory effect of cimetidine, whereas ranitidine had no effect.

389

APE/O C3BASAL

20

mCIMET IIDIFF

15 10

-5

Minutes

-10 0

5

10

15

20

30

40

50

60

70

80

Figure 6. Kinetics of 13CO2expired by a rat after [13C2]-aminopyrine in normal conditions (Basal) and 45 min after receiving a dose of cimetidine (Cimet). The difference between the two sequences corresponds to the (Diff) bars.

Micro-breath tests, using 13C-labeled aminopyrine and carried out on small laboratory animals, can provide fast and comprehensive information on these kinds of modifications of cytochrome P450. Figure 6 shows the kinetics of 13CO2 exhalation after [~3C]-aminopyrine intake in the basal conditions and the modified kinetics, when the same rat received cimetidine, 45 rain before aminopyrine administration. The figure also shows the difference between the two sequences expressed in atom percent excess versus the basal kinetics (APE/o). Figure 7 shows the results of the same trial carried out with ranitidine. The different behaviors of these two H= antagonists on the oxidizing activity is clearly shown using the rapid screening with ~3C-breath test on small animal. In 1991, Brady et al. (19) showed that hepatotoxicity of N-nitrosodimethylamine or CCI4 in rats was blocked by pretreatment with disulfiram. The authors demonstrated that cytochrome P450 II E1 was inhibited and inactived by disulfiram and/or its metabolites and that this mechanism could account for many of the reported inhibitory actions of disulfiram against chemically induced toxicity and carcinogenesis. P450 II El-dependent activities are decreased by treating animals with organic sulphur compounds such as diallyl sulfide, phenethyl isothiocyanide and dimethylsulfide. Disulfiram, a drug used

390

APE/O C3BASAL

25

mRANITIDINE INDIFFERENCE

20 15 10

Minutes

-5

0

5

10

15

20

30

40

50

60

70

80

Figure 7. Kinetics of 13C02 expired by a rat after [13C2]-aminopyrine in normal conditions (Basal) and 45 rain after ranitidine intake, There is no significant difference between the two sequences,

in avoidance therapy for alcohol abuse, and its metabolites, diethydihiocarbamate (DDTC) and CS2, exhibit cancer protective properties that resemble those described for diallyl sulfide. DDTC is rapidly formed from disulfiram in vivo. Disulfiram and its metabolites were tested by Brazier et al. (20) for their inhibitory effect using micro-[13C]-aminopyrine breath test (ABT) and [13C]caffeine breath test (CBT). With this technique, it is possible to design studies, in vivo, where the inhibitory substances to be tested can be administered at various time before administration of the ~3C-labeled probes in order to study where inhibition takes place and its duration. Figure 8 shows the inhibitory effect of a 164-mg/kg dose of DDTC administered 5 min only, before ~3C aminopyrine administration. A significant inhibitory effect of DDTC can be observed from this ABT result. As caffeine metabolism in rats gives rise to the production of ~3CO2 for a long time but with small ~3C enrichment, in normal conditions, CBT is not an ideal model to study cytochrome P450 inhibition. If rats are pretreated with 3MC, cytochrome P450 allows the metabolism to generate ~3CO2 giving a higher ~3C enrichment of the expired gas. Figure 9 shows the effect of a 164-mg/kg dose of DDTC administered only 5 minutes before [~3C]-caffeine intake in a rat pretreated by 3MC. The inhibi-

391

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30 20 ~. 10

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Minutes 0

5

10

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20

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40

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Figure 8. Kinetics of 13C02 exhaled by rats after [~3C2]-aminopyrine in normal conditions (Basal) and 5 rain after administration of a dose of 164 mg/kg of diethyldithiocarbamate. The (Inhib) bars shows the difference between the two sequences and the inhibition process.

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0

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Figure 9. Kinetics of ~3C2 expired by rats receiving [13C3] labeled caffeine after 3MC induction (Caf-3MC), by the same rats, 5 rain after a Na-diethylditihocarbamate administration of 164 mg/kg. The (Diff) bars clearly show the cytochrome P450 inhibition.

392

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3CS2: .4_ 5

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mmol/Kg 2.5 H before ABT 15

20

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100 120 150 180rain

Figure 10. Kinetics of 13CO2expired by rats after [13C2]-aminopyrine administration in normal conditions (Basal) and 2.5 hr after CS2 administration (3.4 mmol/kg). The (Diff) bars show the inhibitory effects of CS2 on cytochrome P450-dependent monooxygenases.

tory effect is extremely rapid and intense and the 13CO2 level is quite identical to that observed in a noninduced rat. These two examples clearly show that both ABT and CBT on small animals allow one to detect easily the inhibitory effect of DDTC on cytochrome P450. The same results can be obtained with the degradation product of DDTC, CS2. Figure 10 show its inhibitory effect on the ABT. It can be observed that 2.5 hr after CS2 administration, inhibition still persists in rats. Figure 11 presents the effect of a CS2 dose administered 20 min after [13C]caffeine intake in 3MC-pretreated rats. The ~3C enrichment is maximum in these rats. When CS2 is administered, the ~3C enrichment immediately falls down and reaches the basal level within 1 hr, whereas in rats not receiving CS2 this ~3C enrichment level is not reached after 3 hr. These results were obtained in vivo with each animal being its own standard, and without killing animals before enzyme activity measurement. These results are in total agreement with those previously obtained, in vitro, on microsomes. The inhibition of cytochrome P450 by disulfiram, DDTC and CS2 can be reflected well by the results of ABT and CBT which can be extended to many other drugs or xenobiotics.

393 CS2

APE/O/ mCS2 ................................ C3Induct 3MC

30 20 10

0

5

10

15

20

30

40

60

80

100 120 150 180

min

Figure 11. Kinetics of 13C02exhaled by rats receiving [13C3]-Iabeled caffeine after 3MC induction (induc- 3MC). Effect of 3.4 mmol/kg of CS2 administration, 20 min after labeled caffeine intake in rats induced by 3MC.

The previous examples on induction and inhibition of cytochrome P450 dependent drug metabolizing activities show that these noninvasive methods are reliable and have a number of methodological advantages. They do not raise the ethical problems caused by the sacrifice of animals (the administration of the test-labeled compounds is not considered as an invasive procedure). They permit the recurring use of the same animal, and the use of each animal as its own control; this drastically reducing the number of animals. Finally, they reflects the overall rate of metabolism.

3.2. Hepatotoxicity Breath tests can be used to evaluate in vivo the hepatotoxicity of drugs or chemicals. Mion et al. (21) conducted a study to evaluate in vivo the hepatotoxic effects of CCI4 administration to rats using breath tests. Carbon tetrachloride (CCI4) hepatotoxicity is mainly due to the formation of reactive metabolites through the cytochrome P450 system. In vitro incubation of liver microsomes in the presence of CCI4 produces a "suicidal" destruction of CCI4 bioactivating mechanism as a result of cytochrome P450 inhibition. In vivo,

394 a small dose of CCI4 decreases the toxicity of a second lethal CCI4dose: the first dose inhibits cytochrome P450, leading to a decreased bioactivation of the second dose. Mion's study was designed to evaluate in vivo the CCI4 autoprotection phenomenon and its temporal relationships with cytochrome P450 inhibition, based on 13C-breath tests: these noninvasive tests allow repeated enzymatic measurements on the same animal, each rat being its own control. Cytochrome P450 activities were measured by the [~3C]-aminopyrine breath test. The metabolic liver function was assessed by the [~3C]-galactose breath test. This test explores the hepatic clearance of galactose, of which the rate limiting step is the galactose kinase. This enzymatic process is not involved in CCI4 bioactivation, and a decrease of the test should reflect the CCI4-induced decrease of liver function. Some transaminase activities were measured in order to assess the severity of CCI4-induced liver damage. The effects of a single intra-gastric administration of CCI4 are an almost complete inhibition of N-demethylation on day 1, as assessed by the ABT (Aminopyrine Breath-Test). A slight, but significant, recovery was detectable on day 3. Seven days later, results of the ABT were identical to day 0. The galactose breath test (GBT) values also decreased significantly on day 3 and were not significantly different from day 0, or day 7 (Figure 12). A second CCI4 administration was performed either 3 or 7 days after the first one, to evaluate in vivo the duration of the CCI4 "autoprotection" duration. When CCI4 was given on day 3, the cytolytic effect was much less pronounced and the GBT values were not decreased. On the contrary, when administrated 7 days after the first CCI4 intake, the decrease of the GBT value was identical to the one induced by the first dose (Figure 13). These results clearly indicate an inhibition of aminopyrine N-demethylase lasting at least 3 days after CCI4 administration and a complete recovery 7 days later. These results are in accordance with in vitro microsomal studies carried out by Glend (22). This example demonstrate the facility and usefulness of noninvasive 13C-breath test to study in vivo, the evaluation of enzymatic activities. It is well known that methotrexate (MTX) exhibits hepatotoxicity during treatment (23). In order to assess the damage induced by this hepatotoxicity, liver biopsies regularly taken during MTX therapy have been proposed by Philips et al. (24). Guitton et al. (25) tried to use the ABT to detect the effects of MTX on P450-dependent mono-oxygenases during treatment and developed an animal model. In this study, three groups of rats were used: a control group, a group receiving a single dose 70 mg/kg of MTX by intraperitoneal route and a third group treated orally with 1 mg/kg of MTX every 2 days. ABT were performed throughout the study to monitor the metabolic activity. At

395 CCI4

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Figure 12. Effect of intragastric CCI4 administration on ABT. Successive ABT were performed before (0) and 1, 3 and 7 days after CCI4 administration (arrow) n = 10. The values of AUCs_3om i n were decreased by 92 percent at day 1, 78 percent at day 3, and return to pre-CCI4 values at day 7.

the end of the study (25 days), the rats were sacrificed and the cytochrome P450 concentration as well as microsomal enzymatic activities of aminopyrine N-demethylase, 7-ethoxyresorufin desalkylase, 7-ethoxycoumarin desalkylase and 7-pentoxyresorufin desalkylase were determined. A histological study of the liver was also performed as well as the measurement of haematological and biochemical parameters. All the rats receiving a single dose of MTX (70 mg/kg) showed discernible signs of acute intoxication. They also showed an inflammatory reaction. The evolution of 13C enrichment of the CO2 expired was quite different 7 days after MTX administration as compared to the basal kinetics (Figure 14). The profile of the [~3C02] versus time curve is modified with a lengthening of the time necessary to reach the maximum which increases form 5-40 min on day 7. The curve becomes progressively flatter as the metabolism slows down. The same features and profiles have been observed when inhibitors of the cytochrome P450-dependent monooxygenases are administered. That is due to the decrease in the input rate of the 13C02 into the HC03 <-~ C02 pool.

396

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MINUTES Figure 14. Results of the ABT in rats (Basal = normal conditions). Effect of a single 70-mg/kg dose of methotrexate measured by ABT, 7 days after MTX administration.

397

3.3. Methodologies Related to Micro-breath Test As micro-samples containing as low as 10 nmol of CO2 can be used for acurate measurements of 13C/12C ratios from gas mixtures containing CO2, it is possible to design protocols involving micro-generators of CO2. In metabolic studies, these micro-generators can be micro-organisms such as bacteria or parasites. The consumption of a 13C-labeled substrate spiking the culture medium can generate CO2 whose ~3C enrichment can be measured in the atmosphere of the culture or nutritive media. According to this principle, the in vivo, N-dealkylation of [~3C2]-aminopyrine was studied on the larvae of a helminth, Heligmosomo[des polygyrus by Kerboeuf et al. (26). Resistance to antihelmintics is an increasing problem throughout the world. Several mechanisms have been described. Nevertheless, detoxification processes can be involved such as metabolism by cytochromes P450. These enzymes have been discovered and characterized in a wide range of organisms including, bacteria, fungi, insects, molluscs and obviously vertebrates (27). However, the previous assays which were carried out to demonstrate the existence of cytochrome P450 and/or P450-1inked reactions in helminths were unsuccessful (28). To overcome these problems, the drug metabolizing enzyme activity was measured in the larvae of a helminth species, by the very sensitive in vivo method of GC-IRMS which avoids the homogenization process used in the in vitro methods. Heligmosomoi'des polygyrus was chosen as the model species because its larvae are commonly used for the study of the effects of anthelmintics. Figure 15 shows the effect of different conditions on the 813CpDBdifferences after 14 hr of incubation of the larvae in the presence of 0.5mM [~3C2]aminopyrine. There is no significant production of ~3CO2 in the tube containing larvae killed by boiling. The tube containing larvae and aminopyrine shows a production of ~3CO2 from the labeled probe corresponding to an enrichment of 30.23 (~13C%o. When 1-benzymidazole in DMSO solution is added, there is a strong decreased of the enrichment. 1-benzymidazole is a well-known inhibitor of cytochrome P450 activities as reported by Rodriguez et al. (29). The tube containing aminopyrine and DMSO only, (C) shows a slight decrease of the ~3CO2 production as compared with the tube (B) not containing the solvent. It is possible that in vitro methods fail to show a mono-oxygenase activity in helminths because of: (i) the effective absence of enzyme; (ii) a level too low to be detected by conventional methods; (iii) the destruction of the enzyme during homogenization; and (iv) the inhibition of the enzyme by some inhibitor liberated in the medium during homogenization. The use of

398

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B

C

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~T0 .

mT14(H)

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===~

-s0 Figure 15. Effect of different condition on ~13C/PDBdifferences after a 14 hr incubation of larvae of Heligosom6tdes polygyrus in presence of 0.5 mM [13C2]-aminopyrine. (A) boiled larvae; (B)larvae and aminopyrine; (C)larvae and aminopyrine and DMSO; and (D) larvae and aminopyrine plus the inhibitor 1-benzymidazole

GC-IRMS allows one to demonstrate the existence of the oxidative metabolism of aminopyrine by the larvae of H. polygyrus. It should be stressed that the activity of the enzyme is very low as judged by the very long time (14 hr) necessary for the cumulative formation of small amounts of 13CO2.

4. 13COz AND METABOLISM-FREE RADICALS Blood cells, especially polymorphonuclear leukocytes (PMNL), can act as ~3C02 micro-generator in the same manner as micro-organisms ( Section 3), and provide models for metabolic studies using CFIRMS, Lamrini et el, (30) used this new methodology to design metabolic studies on free radicals, After stimulation of polymorphonuclear leukocytes by various stimuli, glucose oxidation occurs by the hexose monophosphate shunt (HMPS) and generates NADPH and carbon dioxide, In the same conditions, PMNL give rise to the formation of superoxide ions, and other oxygen reactive species, including free radicals. Using [13C]-glucose labeled at various position C1, C2 or C6, it was possible using PMNL and CFIRMS to quantitatively evaluate

399 various metabolic pathways occurring during neutrophil activation: hexose monophosphate shunt (C1), glucose recycling (C2) and glycolysis (C6), PMNL (5.105 cells/1 ml) were incubated in the presence of the various D [~3C]-glucose isotopomers and stimulated by phorbol myristate acetate. ~3C02 released by the cells was displaced by acid treatment and the 13C/~2C ratios measured by CFIRMS. Quantitation of carbon dioxide produced by PMNL was performed by stable isotope dilution. Suppose an amount q (nmol) Of ~3C02 with an isotope abundance b (atom percent 13C) is produced by the C02 micro-generator. This amount q is diluted with a known amount Q of C02, and a known isotope abundance a. This C02 amount is present in the atmosphere of the tube. After reaction, the total amount of C02 is (Q + q) with a resulting isotope abundance A. The following equation can be derived: Qa + qb = (Q + q)A

The 13C02 amount produced by the micro-generator is very small, so q is quite negligible compared to Q, thus, (A - a ) = bq/Q

The knowledge of Q and a, and the measurement of A and q, allows one to determine b and hence the amount of 13C02 produced by the micro-generator. The determination of Q is performed by using a calibration curve obtained from [13C]-sodium hydrogen carbonate solutions. Figure 16 shows the variation of ~3C02 enrichment over a 60-min period corresponding to the C02 produced by 5.105 PMNL stimulated by PMA and incubated with 5 mM of D-[1-~3C]-, D-[2-~3C]- or D-[6-~3C]-glucose. Over a period of 30 min the amount of 13C02 produced from D-[1-~3C]-glucose was 6.91 + 3.84nm01 (n = 11 healthy subjects). The production from D-[2-~3C]glucose was only 4.08 + 2.81 nmol (n = 5 healthy subjects). No ~3C02 was produced from D-[6-~3C]-glucose. This example demonstrate that HMPS activities in stimulated leukocytes can be measured using various glucose isotopomers and CFIRMS. Each of these isotopomers provides precise informations on the metabolic mechanisms. [1-~3C]-glucose allows the quantification of HMPS activity glucose recycling is measured by the production of 13C02 from [2-~3C]-glucose and is shown to be dependent of the HMPS levels. Finally, the tricarboxylic cycle activity is measured by the ~3C02 production from [6-~3C]-glucose. As the cellular response at an oxidative stress is dependent of HMPS activity, the technology and methodology of ~3C/~2C ratio mea-

400 11111

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Figure 16. Production of 13C02 by stimulated polymorphonuclear leukocytes from [1-13C], [2-13C] and [6-13C]glucose.

surements by CFIRMS of C02 micro-generators is quite reliable to give informations on this cellular response. This technique can be potentially useful for pharmacological studies on HMPS, glucose recycling/or glycolysis activities in polymorphonuclear leukocytes or other types of cells. The detection of the production of hydroxyl radicals (OH~ by various biological systems has been carried out using various methods including the hydroxylation of various organic compounds: phenol, salicylate, phenylalanine or benzoate. Sagone et al. (31) reported a method based on the decarboxylation of [7-14C]-benzoic acid and giving rise to the production of 14C02. Lamrini et al. (32) adapted this principle to CFIRMS measurement of ~3C02 generated by hydroxyl radicals from [7-~3C]-benzoic acid. They used an acellular generator of hydroxyl radicals: iron meso-tetra-(4-sulfonatophenyl)-porphyrin and hydrogen peroxide. The production of hydroxyl radicals was proved by comparative studies with electron spin resonance (ESR) and LC analysis of the hydroxylation products of benzoic acid. It has been shown that the stable isotope technique permits on-line analysis of micro-quantities of carbon dioxide eluted from small samples. It displays considerable facility to perform repetitive measurements during the kinetic reactions because the complete analysis of one sample is achievable within 4 min. The highest sensitivity is obtainable with this technique: below 0.1 nmol of ~3C02 compared to 5-10 nmol of ~4C02. In this study, various hydroxyl radicals' scaven-

401 gers have been studied successfully: mannitol, methanol and demethylsufoxide. The production of 13C02 from [7-~3C]-benzoic acid was inhibited by these agents. The previous examples clearly show that the new technology of CFIRMS measurement of isotope enrichment of C02 from microcellular, as well as acellular micro-generators, provides a new, very useful tool for metabolic studies.

5. CONCLUSION The usefulness of breath tests has been demonstrated when using suitably radiolabeled drugs such as aminopyrine, phenacetin or caffeine. Aminopyrine and caffeine are demethylated in the liver. They are not excreted unchanged (or only at very low levels), so that their N-demethylation reflects their clearance characteristics. To study the modifications of drug-metabolizing liver activities, aminopyrine and caffeine are two useful substrates. Aminopyrine is a substrate of phenobarbital-inducible P450, and caffeine is a substrate of the 3-methylcholanthrene-inducible PI-450 isoenzyme. Hence, breath tests with 14C- or 13C-labeled aminopyrine and caffeine as substrates can be used, in vivo, as noninvasive probes of mixed function oxidase activity. Other labeled compounds can be used also as metabolic probes. An alternative to using radiolabeled compounds is the use of stable isotope-labeled molecules and, for breath-test purposes, tracers specifically labeled with ~3C. The limiting phase of breath tests is the C02 purification phase before isotope ratio measurement, because isotope ratio mass spectrometers have to work with pure CO2 only. The gas chromatograph-isotope ratio mass spectrometer system is able to effect automatically, on-line, the purification of C02 from small air samples, the determination of CO2 and the ~3C/~2C isotope ratio with a precision better than 0.2813CPDB and an overall run time of 3 min. Such a system allows noninvasive repetitive isotopic measurements on small animals in v/vo, It also allows investigations to be performed using small animals as their own standards in multi-sequence designed studies, The sole operation carried out on the animal is drug administration, It can be also used successfully with all kinds of C02 micro-generators used for metabolic studies purpose, The method described here can be used for studies designed to investigate the modifications induced in drug-metabolizing systems by other drugs or xenobiotics, The variations in the excretion rate of ~3C02 derived from N-

402 demethylation of the 13C-labeled substrates are significant markers of such modifications and activities.

ACKNOWLEDGEMENTS The author thanks Dr R. Guilluy who has made productive this area of research in the LEACM, and M. BUISSON for her skilful typographic assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

T. Browne, Clin. Pharmacokinet., 18 (1990) 423. S. Loft and H.E. Poulsen, Pharmacol. Toxicol., 67 (1990) 101. J.C. Rhodes and J.B. Houston, Biopharm. Drug Dispos., 4 (1983) 125. B. Lauterburg and J. Bitcher, Gastroenterology, 65 (1973) 556. C.S. Irving, A. Dale, N. Kanichi, L. Baker and P.D. Klein, Clin. Pharmacol. Ther., 42 (1982) 493. M. BrLich, L. Kling, W. Legrum and E. Maser, Arch. Toxicol., 60 (1987) 81. S.J. Prosser, S.T. Brookes, A.L. Linton and I. Preston, Biomed. Mass Spectrom., 20 (1981) 724. P.A. Freedman, E.C. Guillyon and J. Jumeau, I. Int. Lab., 2 (1988) 22. R. Guilluy, F. Billon-Rey and J.L. Brazier, J. Chromatogr., 562 (1991) 341. R. Guilluy, F. Billon-Rey, C. Pachiaudi, J. Jumeau and J.L. Brazier, Anal. Chim. Acta, 259 (1992) 193. A.M. Conney, Pharmacol. Rev., 19 (1967) 317. A.M. Conney, Cancer Res., 42 (1982) 4875. P.R. Ortiz de Montellano and M.A. Correira, Ann. Rev. Pharmacol., 23 (1983) 129. A. Somogyi and R. Gugler, Clin. Pharmacokinet., 7 (1982) 23. D.R. Abernethy, D.J. Greenblatt and M. Divoll, Pharmacol. Exp. Ther., 224 (1983) 508. W. Korch, M. Hoensch and M.D. Janish, Clin. Pharmacokinet., 9 (1984) 493. J.A. Bell, A.J. Gower, L.E. Martin, E.N. Mills and W.P. Smith, Biochem. Soc. Trans., 9 (1981) 113. M.C. Gerber, G.A. Tejwani, N. Gerber and S.R. Bianchine, Pharmacol. Ther., 27 (1985) 353. J.F. Brady, F. Xiao, M.H. Wang, Y. Li, S.M. Ning, J.M. Gapac and C.S. Yang, Toxicol. Appl. Pharmacol., 108 (1991)966. J.L. Brazier, C. Descours, F. Rispal, F. Billon-Rey and R. Guilluy, in Pharmacocinetique de la recherche la clinique, J. Bres and G. Panis (eds) (John Libbey Eurotext, 1992) pp. 193, 198. F. Mion, A. Gelo~n, M. Rousseau, J.L. Brazier and Y. Minaire, Life Sciences, 54 (1994) 2093. E.A. Glend, Biochem. Pharmacol., 21 (1972) 1697. D.J. Bjorkman, E.M. Memmond, R.G. Lee, D.O. Cleg and K.G. Tolman, Arth. Rhum., 12 (1988) 1465. C.A. Philips, P.J. Cera, T.F. Menge and E.D. Newman, J. Rhuma., 19 (1992) 229.

403 25. J. Guitton, G. Souillet, J.L. Riviere, F. Gerard, R. Guilluy and J.L. Brazier, Eur. J. Drug Metab. Pharmacokinet., 19 (1994) 119. 26. D. Kerboeuf, D. Soubieux, R. Guilluy, J.L. Brazier and J.L. Rivi~re, Parasitology Research, 81 (1994) 302. 27. D.W. Nebert, D.R. Nelson and R. Feyeresen, Xenobiotica, 19 (1989) 1149. 28. R. Precious and J. Barett, Biochem. Biophys. Acta, 992 (1989) 215. 29. A.D. Rodriguez, D.F.V. Lewis, C. lannides and D.V. Parke, Xenobiotica, 17 (1987) 1315. 30. R. Lamrini, P. Lacon, A. Francina, R. Guilluy and J.L. Brazier, Anal. Biochem. (1995) (submitted). 31. A.L. Sagone, M.A. Kecker, R.M. Wells and C. De Mollo, Biochem. Biophys. Acta, 628 (1980) 90. 32. R. Lamrini, J.M. Crouzet, A. Francina, R. Guilluy, J.P. Steghens and J.L. Brazier, Anal. Biochem., 220 (1994) 129.

405

CHAPTER 22

PROCUREMENT OF STABLE ISOTOPELABELED PHARMACEUTICALS

THOMAS R. BROWNE ~, GEORGE K. SZABO ~ and ALFRED M. AJAMI 2 1Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center; 2Mass Trace, Woburn, MA

1. INTRODUCTION

Pharmaceutical researchers generally ask three questionas regarding stable isotope-labeled drugs. First, how difficult are they to obtain? Second, how much do they cost? Third, how difficult is it to obtain FDA approval for human use of stable isotope-labeled drugs? This chapter will address questions one and two. Question three is reviewed in detail in Chapter 23 of this book.

2. PROCUREMENT OF STABLE ISOTOPE-LABELED DRUGS

Many stable isotope-labeled drugs may be purchased "off the shelf" from a catalog. Others must be custom synthesized. 2. I. Ordering from Catalogs

There are a number of companies offering stable isotope-labeled drugs (see Table 1). These companies generally have a catalog of "off the shelf" items and also offer custom synthesis. More than 100 "classical" drugs (e.g. acetaminophen, phenacetin, phenobarbital) are offered in these catalogs. In some cases, the drugs are literally "on the shelf" and can be delivered immediately. In other cases, the drug has been synthesized in the past by the company, and a new batch must be produced using a proven synthesis.

406 TABLE 1. Sources for Stable Isotope-Labeled Drugs

Aldrich, 1001 West Saint Paul Avenue, Milwaukee, Wl 53233, USA C/D/N Isotopes Inc., 88 Leacock Street, Pointe-Claire, Quebec, Canada Cambridge Isotope Laboratories, 50 Frontage Road, Andover, MA 01810-5413, USA EURISO-TOP, Route de I'Orme, Saint-Aubin, Cedex, France ICON Services Inc., 19 Ox Bow Lane, Summit, NJ 07901, USA Isotec Inc., 3858 Benner Road, Miamisburg, OH 45342-4304, USA Mass Trace, 3-G Gill Street, Woburn, MA 01801, USA

2.2. Custom Synthesis Most of the companies in Table 1 will custom synthesize stable isotopelabeled drugs. The purchaser supplies specifications (compound, chemical purity, isotopic purity, delivery date). The company will then explore the possible synthetic routes and provide the purchaser with a bid. Further details are given below.

3. COST

3.1. Ordering from Catalogs Most cataloged stable isotope-labeled drugs have a list price of $50-$1,000 per 100 mg (see Table 2). Price varies with the difficulty of synthesis and the amount and type of labeling. In general, deuterium-labeled analogues are less expensive than ~3C- or ~SN-labeled analogues, but also run greater risks of isotope effect (see Chapter 2). Often, substantial discounts for larger quantity ordered can be negotiated.

3.2. Custom Synthesis The major cost of custom synthesis of stable isotope-labeled drugs is the labor in synthesizing the drug. Stable isotope building blocks and small molecular weight precursors at 99 percent enrichment are relatively inexpensive. A representative selection, with approximate "street" prices, available

407 TABLE 2. Catalog Cost for Stable Isotope-Labeled Analogues of Common Drugs*

Drug

Cost per 100 mg ($US)

D,-acetaminophen D3-L-dopa ~3C6-L-dopa D5-phenobarbital 13C.phe nacet i n D~o-phenytoin 3C3-phe nytoi n ~3c,~sN2-phenytoin ~SN2-theophylline

45.00 60.00-140.00 950.00 160.00 35.00 360.00-950.00 445.00 760.00 460.00

*From current catalogs of companies listed in Table 1.

TABLE 3. Cost (Lowest Bidder) of Some Custom Syntheses Performed for Authors

Drug

Cost per 100 mg ($US)

D3-antipyrine (5-g order) 13C2,15N-acetaminophen (1-g order) 13C2,~SN-ammonium hippurate (30-g order) ~SN-carbamazepine (30-g order) ~3C2,15N-ethotoin (6-g order)

20.00 245.00 50.00 35.00 45.00

from all commercial suppliers includes: ammonia (15N), $120/L; carbon (13C, 99 percent) monoxide, $65/L; potassium cyanide (13C), $65/g; sodium acetate (1,2-~3C2), $100/g; and urea (15N2), $100/g. The cost of custom synthesis varies directly with difficulty of synthesis. If a company is synthesizing a compound for the first time, extra time and cost are figured for trial and error. If a company has synthesized a compound in the past, bids are usually lower. The cost (lowest bidder) for some compounds custom synthesized for our pharmacokinetic studies are shown in Table 3. When ordered in 5-30 g lots, the cost of custom synthesized drugs was similar to the cost of "off the shelf" drugs.

408 4. GUIDELINES FOR ORDERING STABLE ISOTOPE-LABELED DRUGS

4.1. Think Carefully About Labeled Sites and Possible Labels

Labeling is best accomplished by labeling sites which are unlikely to cause isotope effects (see Chapter 2) and are unlikely to be lost due to metabolic procedures (e.g. demethylation), or analytic procedures (e.g. acid hydrolysis). The number of labels is determined usually by resolution of mass spectrometric peaks; three or more labeled sites per molecule are often considered optimal. However, flexibility within these rules can save money by selection of the least expensive synthetic route and precursors.

4.2. Provide as Much Assistance to as Possible Synthesizer

Considerable cost savings may be obtained if the orderer provides the synthesizer with a written protocol for the desired synthesis and/or samples of key or rare intermediates.

4.3. Enantiospecific Syntheses

Enantiospecific syntheses, requiring the connection of several labeled synthons, are the most costly. Functional group transformation and site specific labeling options should be considered for maximal cost savings. Labeling by exchange, although in principle the least expensive approach for the preparation of tracers, should be reserved as an option of last resort, since positional stability and high labeled species abundance are difficult to obtain.

4.4. Always Obtain Multiple Bids

For both "off the shelf" and custom synthesized drugs, the quoted prices will vary greatly among companies. No one company consistently offers the lowest quotes. Quoted prices depend upon: (1) previous experience in synthesizing the drug or similar compounds; (2) cost of isotopes (companies preparing stable isotopes have lower costs than those purchasing stable isotopes); (3) how busy the company is; (4) whether or not the company believes the drug might have other potential purchasers; and (5) availability of documented synthetic route, including analytical protocols and standards.

409

4.5. Specify Chemical and Isotopic Purity Chemical purity is usually dictated by USP standards and FDA regulations. Isotopic purity is determined by mass spectrometric and nuclear magnetic resonance spectrometric analytic considerations. Chemical purity of drugs is usually 98 percent or greater. Isotopic purity is usually 99 percent at each labeled site. All parties to any custom synthetic work, especially with isotopes, should recognize the vagaries of analytical chemistry, where results are method, operator and equipment dependent. Ideally, there should be agreement on all protocols for establishing chemical identity, isomeric, enantiomeric and isotopic purity. There are definitive monographs for the analysis of many drugs. These are published in official compendia, such as the US Pharmacopoeia and the Food Chemicals Codex, or by government groups or professional societies concerned with analytical standardization, such as the National Institute of Standards and Technology (NIST), or the Association of Official Analytical Chemists. Compliance with the specifications of these types of organizations would constitute prima facie evidence of purity for any custom synthesized bioactive molecule. But note that many of the documented procedures may not be amenable to scaledown for evaluation of tracer-labeled materials, where the costs of destructive analysis, even of small amounts, often proves to be significant. This is a subject over which disputes may be inevitable, if the codification of expectations is not addressed in advance.

4.6. Check Chemical and Isotopic Purity Materials supplied by companies do not always meet the specified purity. Also, the FDA requires verification of chemical purity.

4.7. Specify a Defivery Date Custom synthesis requires time. Sometimes more than expected. Be certain the supplier has agreed to a firm delivery date.

ACKNOWLEDGMENT Supported by the United States Department of Veterans Affairs.

411

CHAPTER 23

REGULATORY ASPECTS

THOMAS R. BROWNE Departments of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston Department of Veterans Affairs Medical Center

1. INTRODUCTION The regulatory aspects of stable isotope-labeled (SIL) drug utilization in pharmaceutical research are regulated in the United States by the Food and Drug Administration (FDA) and can be divided into two areas. First, the use of a SIL analog of a drug (which will not be marketed in the labeled form) to perform one, or more, human research studies on the drug requires that the sponsor obtain an Investigational New Drug (IND) authorization for the SIL analog of the drug from the FDA. The second area of regulatory concern is the requirements for approval by the FDA of a new SIL drug to be marketed for use as a diagnostic or therapeutic agent, or as part of a device (e.g. ~3Clabeled urea used as part of a diagnostic test for Helicobacter pylori bacteria in the stomach).

2. INVESTIGATIONAL NEW DRUG (IND) APPLICATION An IND application is a request for FDA authorization to administer an investigational drug to humans (1-3). Such authorization must be secured prior to interstate shipment, or administration of the SIL analog of a drug. The requirement holds whether or not the unlabeled analogue of the drug is approved by the FDA. The FDA is committed to ensuring that investigational drug products are critically evaluated for good quality control. The FDA recognizes that the experimental nature of a drug product in an early stage of development may limit the amount of data available. At early stages of development, the

412 TABLE 1. Items Required for IND Application

1. 2. 3. 4. 5. 6.

7. .

9. 10.

Form FDA 1571 [21 CFR 312.23 (a)(1)] Table of contents [21 CFR 312.23 (a)(2)] Introductory statement [21 CFR 312.23 (a)(3)] General investigational plan [21 CFR 312.23 (a)(3)] Investigator's brochure [21 CFR 312.23 (a)(5)] Protocol(s) [21 CFR 312.23 (a)(6)] a. Study protocol(s) [21 CFR 312.23 (a)(6)] b. Investigator data [21 CFR 312.33 (a)(6)(iii)(b), or completed form(s) FDA 1572 c. Facilities data [21 CFR 312.23 (a) (6)(iii)(b)], or completed Form(s) FDA 1572 d. Institutional Review Board data [21 CFR 312.23 (a)(6)(iii)(b)], or completed form(s) FDA 1572 Chemistry, manufacturing and control data [21 CFR 312.23 (a)(7)] a. Environmental assessment or claim for exclusion [21 CFR 312.23 (a)(7)(iv)(e)] Pharmacology and toxicology data [21 CFR 312.23 (a)(8)] Previous human experience [21 CFR 312.23 (a)(9)] Additional information [21 CFR 312.23 (a)(10)]

acceptance/rejection criteria may not be as specific as in later stages. However, it is vital that such criteria be scientifically sound and based upon available scientific data. The IND approval process begins by contacting the FDA for the appropriate form (FDA 1571) and instructions for completing an IND application. The appropriate office is: FDA Center for Drug Evaluation and Research, Executive Secretariat Staff (HFD-8), 5600 Fishers Lane, Rockville, MD 20857; telephone (301) 594-1012; fax (301) 594-3302. The items required for the IND are listed in Table 1. Instructions for how to provide the required information for each item in Table 1 are provided by the FDA when form FDA 1571 is requested. Further details ofthe requirements for each item in Table 1 are contained in the Code of Federal Regulations (CFR) sections listed in Table 1. CFR's can be obtained by writing to: Superintendent of Documents, Attn: New Orders, P.O. Box 371954, Pittsburgh, PA 15240-7954. CFRs can be obtained also by using the Medical Library Electronic Reference Network (LERN) or Gopher systems (FDL, CFR or ORA GOLD DISC databases). CFRs are updated yearly. More recent regulations and notices by the FDA on CFRs are published weekly in the Federal Register. The Federal Register and the Federal Register Index (indices covering Federal Register contents) are available from the sources listed in the preceding paragraph.

413 TABLE 2. Divisions of FDA Center for Drug Evaluation and Research

Division

Telephone number (area code 301)

Cardio-Renal Drug Products Neuro Pharmaceutical Drug Products Medical Imaging, Surgical and Dental Drug Products Gastrointestinal and Coagulation Drug Products Anti-Infective Drug Products Anti-Viral Drug Products Topical Drug Products Generic Drugs

594-5300 594-5700 443-3560 443-0479 443-4310 443-4280 443-4280 594-0340

If the IND applicant is an independent clinical investigator, the IND application typically contains data on the drug's chemistry and formulation, results of preclinical studies (if any) and a description of the human studies to be performed. If the IND applicant is a drug company beginning work for a later request to market the drug, the entire proposed human research program must be described and additional information on drug manufacture, preclinical trials, investigator's credentials and institutional review boards must be provided. The IND application should be submitted to the FDA in triplicate at the following address: FDA, Center for Drug Evaluation and Research, Central Documents Room, Park Building, Room 2-14, 124020 Parklawn Drive, Rockville, MD 20852. All IND applications undergo a common initial review and then are sent to the appropriate FDA Division, depending on the type of drug (Table 2), for final review. The Division contacts the applicant with any questions and issues final approval of the IND. After submission of an IND application to the FDA, the Agency has 30 days to review it for safety issues. Clinical trials may begin when approval is received from the FDA or, in the absence of comment from the FDA, 30 days after submission. In cases where a previous investigator has obtained an approved IND for a specific SIL analog of a drug, another investigator may cite items in the previous IND. This eliminates the need to repeat certain items in the new IND application if the items in the new IND are the same as the old IND. Citing a previous investigator's IND requires written permission from the previous investigator to the Director of the FDA Division approving the initial IND.

414 The author has submitted several investigator initiated INDs to the FDA in recent years. The process has been prompt and fair. Listed below are a number of suggestions based in the author's experiences with previous IND submissions. (1) Verify the dose of labeled drug analog relative to nonlabeled drug analog. (2) Provide full details on the manufacture and control of the SIL drug substance. Alternatively, if a Drug Master File (DMF) for the SIL drug is on file at the FDA, a letter of authorization allowing the FDA to access this submission from the DMF holder on your behalf may be submitted. (3) A Certificate of Analysis should be provided by the contract manufacturer of the SIL drug substance with each shipment. (4) Report any identity testing of the SIL drug substance which has been performed. (5) A list of components, including all substances and in-process materials, used in producing the SIL drug product should be submitted. A quality designation (e.g. ACS, USP, NF) is required for each active and inactive component used in manufacture. (6) A quantitative statement of composition which specifies an appropriate range or definite weight of each ingredient is requested. (7) Provide a synthetic description of the manufacturing process which includes sufficient information to reproduce experimental results (e.g. reaction times, temperatures, scale, yields, etc). (8) Submit acceptance specifications and test methods for the starting materials and reagents used in synthesis. If acceptance is based on the supplier's certificate of analysis, provide recent examples to ensure proper testing is performed. (9) With regard to the drug substance specifications, include a gas liquid chromatography method for detection of residual solvents. (10) Full details on the assay method, including adequate acceptance specifications for the content of drug product, should be submitted. (11) Provide information on the container closure system used for shipping and storage of the drug substance. Provide storage conditions which will be enforced. (12) A stability protocol should be established for the drug substance and the drug product to ensure adequate stability throughout the storage period. (13) Data demonstrating that the drug product will remain within your specifications for the anticipated shelf-life should be proved.

415 Certain drugs and/or their SIL analogs may possess unique properties which are not covered in the standard IND application and instructions. In these situations the investigator may seek to have questions answered by the FDA prior to submitting the IND. Questions regarding the IND process may be directed to the FDA Center for Drug Evaluation and Research, Executive Secretariat Staff (address and telephone number above). Questions regarding the pharmacological properties of a drug may be directed to the appropriate Division of the Center for Drug Evaluation and Research (Table 2). Questions regarding SIL labeling may be addressed to the Medical Imaging, Surgical and Dental Drug Products Division of the Center for Drug Evaluation (Table 2).

4. APPROVAL OF A DRUG OR DEVICE FOR MARKETING

The approval of new drugs and devices in the USA for marketing falls under the authority of the FDA. The laws governing this authority are the Food, Drug, and Cosmetic Act of 1938, Revision of New Drug Application Regulations (1985), and Revision of Investigational New Drug Application Regulations (1987). These laws for drugs (1-3) and devices (4) have been reviewed elsewhere. These laws provide general guidelines for the types of information necessary for approval of a new drug or device. However, nowhere is there written an exact list of specific data required for approval of a new drug or device (specific study types, number of subjects, required statistical power). The specific studies performed for a new drug or device are determined by the following: (1) written FDA guidelines; (2) knowledge of past applications that have been accepted by the FDA; (3) knowledge of verbal statements made by the FDA at open hearings on new drugs or devices and their approval process; (4) knowledge of unique properties of the new drug or device being tested; and (5) a conference held by the sponsor with the FDA prior to initiation of each phase of testing. The specific research plan followed for a specific drug or device can be modified by: (1) unique properties of the drug or device; (2) specific concerns of the sponsor; and (3) specific concerns of the FDA conferees. Within the FDA, drugs are evaluated by the Center for Drug Evaluation and Research, while devices are evaluated by the Center for Devices and Radiological Health. There is an ongoing negotiation between the two Centers and potential marketers of SIL products attempting to define the jurisdiction and regulations for SIL products.

416

The current situation regarding approval of SlL drugs for marketing is ambiguous for several reasons. First, a set of FDA regulations specifically covering SIL drugs has not been finalized. Such a set of regulations is in the planning stage. Because the regulations are in the planning stage, neither the FDA or industry regulatory officials will discuss them publicly. Second, there are few SlL drugs or other products approved for marketing. Therefore, there is little information from previous approvals to provide guidance. Third, the first SlL product to seek FDA approval for large-scale marketing is SIL (13C) urea used in a breath test for detection of H. pylori gastrointestinal infections (see Chapter 20). The question has arisen as to whether ~3C urea is a drug (diagnostic agent) or is part of a diagnostic "device". At the present time, the FDA considers the 13C-urea breath test to be a drug-device combination, part diagnostic agent and part diagnostic device. Because of the above described situations, it is not possible at this time to list a clear set of regulations regarding FDA approval of SlL drugs or devices. The reader is advised to consult the specific Division of the FDA which will review the drug or device prior to initiating studies to be submitted as part of an application to market a SlL drug or device.

ACKNOWLEDGMENT

Supported by the United States Department of Veterans Affairs.

REFERENCES

1. T.R. Browne, in New Antiepileptic Drug Development: Preclinical and Clinical Aspects, J.A. French, M.A. Dichter and I.E. Leppik (eds) (Elsevier, Amsterdam, 1993) p. 31. 2. Drug Evaluations Annual 1995 (AMA, Chicago, 1995) p. 1. 3. D.A. Kessler, N. Engl. J. Med., 320 (1989) 357. 4. D.A. Kessler, S.M. Pope and D.N. Sundrall, N. Engl. J. Med., 317 (1987) 357.

417

CHAPTER 24

STABLE ISOTOPE TECHNIQUES IN DRUG DEVELOPMENT: AN ECONOMIC EVALUATION

THOMAS R. BROWNE Department of Neurology and Pharmacology, Boston University School of Medicine; Neurology Service, Boston; Department of Veterans Affairs Medical Center, Boston

1. INTRODUCTION

The studies usually required as part of early United States Food and Drug Administration (FDA) Phase I human testing of a new drug include: mass balance/metabolite identification, single-dose volunteer, multiple-dose volunteer, absolute bioavailability, single-dose patient, multiple-dose patient, and drug interaction studies (Table 1). Stable isotope labeled (SIL) tracer methods for performing each of these types of study have been published (see Chapters 11-13, 16 and 18). These publications have emphasized advantages of increased statistical power and reduction in number of subjects, subject risk, and study time. To date, the pharmaceutical industry has been slow in adopting these SIL methods, except in cases where standard methods have been tried and failed (1,2). This chapter will evaluate possible economic advantages of SIL methods. This evaluation will cover two areas: (1) use of SIL methods to perform single studies required for Phase I; and (2) use of SIL methods to perform multiple studies required for Phase I, including combining two or more studies into a single more efficient study. When used for a single study, SIL methods are least efficient economically. Whether one or several studies are performed, the same "start-up" costs are incurred: synthesis and formulation of SIL drug, validation of analytic method for quantitation of SIL drug and obtaining regulatory approval for use of SIL drug. By performing several studies with SIL tracers, start-up costs are spread over several studies, and the potential cost savings with SIL methods can be better exploited. Standard methods of performing the Phase I studies in Table

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2. METHODS

Table 1 lists the types of study typically performed in the Phase I evaluation of a new drug. Table 2 lists the specific items performed in each study along with the current costs for each item at Boston University Medical Center. Total final cost of the study to the sponsor (direct costs, regulatory filings, drug distribution, monitoring, etc.) is estimated by the standard pharmaceutical industry method of multiplying direct costs times four. The numbers obtained are representative of a typical study. Exact cost to the sponsor will vary with direct costs, overhead and sponsor activity.

3. MASS BALANCE/METABOLITE IDENTIFICATION STUDIES

Historically, these studies have been performed by: (1) administering radioactive (14C)-Iabeled drug; (2) measurement of radioactivity in urine and feces (to determine mass balance); (3) chromatography of urine to divide dissolved material into peaks; (4) recognition of peaks containing drug or metabolite by presence of radioactivity; and (5) identification of structure of drug or metabolite in "hot" peaks by mass spectrometry (MS). The analytic aspects of this method are simple and well established. However, the logistical aspects of this method can be problematic: (1) radioactive drug must be synthesized; (2) special institutional review board (IRB) procedures and permissions are required for administration of radioactive drug; (3) it is almost impossible to obtain IRB approval to perform a radioactive metabolic study in children in the US, even though children often have different patterns of drug metabolism than adults; (4) special procedures are required for storage and disposal of radioactive specimens; and (5) the sponsor may be assuming long-term liability risk (often causing pharmaceutical companies to seek outside or foreign contractors). These logistical considerations drive up costs and often result in delays in obtaining key information. Ideally, human mass balance/metabolite identification information should be obtained in early Phase I (Table 1). Delays can have serious consequences such as failing to know of poor absorption, or an active or toxic metabolite. Recently, techniques for performing mass balance/metabolite identification studies with SIL drug (~3C, ~SN) and isotope ratio mass spectrometry (IRMS)

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421 have been reported which generate information similar to that obtained with 14C-labeled drug and scintillation counting (see Chapters 6 and 11). The SIL/IRMS methods appear feasible for studies of low and medium (but not high) potency drugs. The overall cost of a mass balance/metabolite identification study performed with SlL- or 14C-labeling is approximately equal (see Table 2). However the SlL/IRMS method offers significant advantages: (1) the SlL/IRMS methods eliminates the five problems associated with ~4C-labeled studies listed above; and (2) the SIL/IRMS study can be combined with the singledose volunteer study, with a modest reduction in the overall cost of the two studies (see below).

4. SINGLE-DOSE VOLUNTEER AND SINGLE-DOSE PATIENT STUDIES

Single-dose studies typically collect information on the pharmacokinetics, safety and dose ranging of a new drug after administration of a single dose. A modest number of volunteers (e.g. 20) are studied to obtain information on dose ranging. Single-dose studies can be performed using SIL drug and MS detection (see Chapters 13, 16 and 18). However, this methodology usually is more expensive than standard methods (e.g. unlabeled drug, high performance liquid chromatography (HPLC) with ultra violet (UV) detection) because of the added costs of isotope synthesis and MS analytic methodology (Table 2). There are instances where quantitation of SIL-labeled drug with MS is more practical than quantitation of nonlabeled drug with conventional techniques, especially when inexpensive "table-top" mass spectrometers can be employed. Examples include high potency (i.e. low plasma concentration) drugs and drugs with low response to ultraviolet detection.

5. COMBINED MASS BALANCE/METABOLITE IDENTIFICATION AND SINGLE-DOSE VOLUNTEER STUDIES

Both mass balance/metabolite identification and single-dose volunteer studies involve administering a single dose of drug to healthy volunteers, collecting blood and urine for pharmacokinetic studies, and collecting safety data. The blood and urine specimens collected in a mass balance/metabolite identification study performed with ~4C-labeled drug cannot be used for later pharmacokinetic analysis because of problems with radioactive decay of sample, storage of radioactive specimens and radioactive contamination of analytic

422 instruments (e.g. HPLC) used to measure the concentration of drug in blood and urine for pharmacokinetic calculations. SIL-labeled drug is stable indefinitely in blood and urine samples and contains no radioactivity. Studies performed with single doses of SlL-labeled drug can have the SIL label "counted" by IRMS studies for mass balance determinations, and the blood and urine can then be stored for later analysis by HPLC or other methods for pharmacokinetic computations. Thus, the same subject and specimens can be used for both studies. Such a combination would result in a modest reduction in cost, time and number of volunteers (see Tables 1 and 2).

6. MULTIPLE-DOSE VOLUNTEER STUDY

Such studies are performed to determine the type of pharmacokinetic properties (linear, concentration-, or time-dependent) and the pharmacokinetic values (clearance, half-life dependent, volume of distribution) a new drug has during chronic administration and to obtain information on safety during long term administration. The standard method for performing such a study would be: (1) administer a single dose of drug and determine single-dose pharmacokinetic parameters from plasma concentration versus time relationships; (2) administer the drug chronically for a period of time; (3) stop the drug; (4) redetermine pharmacokinetic parameters from plasma concentration versus time relationships during the terminal "wash out" of drug; and (5) compare single dose and terminal pharmacokinetic values. A more satisfactory SIL method has been reported in which steps 3 to 5 are varied as follows: (3) administer a SlL-tracer dose of drug while the volunteer continues to take the drug; (4) determine tracer dose pharmacokinetic properties from tracer dose plasma and urine concentration versus time relationships; and (5) discontinue drug administration when tracer study is complete (see Chapter 16). This SIL technique will accurately determine the type of pharmacokinetic properties and the pharmacokinetic values (including Kr~ and Vva• for drugs with concentration-dependent pharmacokinetic properties). This SIL technique is superior to studying terminal plasma concentration versus time relationship because the SlL method measures true steady state pharmacokinetic values. Measurement of plasma concentration versus time relationships after stopping drug (i.e. when plasma concentration is constantly falling) does not permit direct determination of pharmacokinetic values for drugs with concentration-dependent pharmacokinetics. There are no economic benefits to utilizing SIL techniques to perform only

423 a multiple-dose volunteer study. Indeed, the SIL method is more costly because of the added costs of isotope synthesis and MS analysis (see Tables 1 and 2). The advantages of the SIL method are: (1) detailed and accurate information on changes in pharmacokinetic parameters during chronic administration for and drugs with concentration-dependent or time-dependent pharmacokinetics at steady state plasma concentration; and (2) improved statistical power to exclude concentration-dependent or time-dependent changes in drugs without such changes (see Chapter 16). Moreover, economic benefits can be obtained by combining a SIL multiple-dose volunteer study with other SIL studies (see below).

7. ABSOLUTE OR RELATIVE BIOAVAILABILITY/BIOEQUIVALENCE STUDIES

The classic technique for performing bioavailability/bioequivalence studies is a cross-over technique in which volunteers receive a dose of drug using the marketed preparation during one test session and a reference dose of drug as an i.v. infusion (absolute bioavailability), or an oral solution (relative bioavailability) during a second test session. Plasma concentration versus time relationships after each test dose of drug are used to compute area under the plasma concentration versus time curve (AUC) values. The ratio of AUC for marketed drug/AUC for i.v. solution (or oral solution) is a measure of absolute (or relative) bioavailability. Typically, this method requires study of approximately 24 volunteers in order to obtain adequate statistical power to demonstrate equivalence (i.e. adequate statistical power to detect a truly significant difference of 0.20 with a power of at least 0.80). The principal determinant of the number of subjects need is intraindividual variability in absorption and elimination between test sessions. Such variability produces random variation in AUC values, reduces statistical power, and increases the number of subjects required. An attractive alternative SIL methodology is available for performing bioavailability/bioequivalence studies. The marketed drug preparation is administered simultaneously with SIL reference preparation (i.v. solution or oral solution), and AUC values for each formulation are determined from plasma concentration versus time relationships for labeled and unlabeled drugs. This technique eliminates intraindividual variability and greatly improves statistical power. For a typical drug, 24 subjects are required to demonstrate equivalence of two preparations with adequate statistical power with the cross-over technique and only eight or less subjects with the SIL technique. Furthermore,

424 the subjects in an SIL study receive the test drug only once, reducing risk, hospital days and analytic specimens. See Wolen (4) and Chapter 13 for details on the methodologic and statistical aspects of SlL bioavailability/bioequivalence studies. The above considerations apply to "typical" drugs. Drugs with first pass metabolism have greater intraindividual variability in the AUC values obtained with the same formulation administered at different times than drugs without first pass metabolism. This increases the number of subjects necessary to obtain adequate statistical power. SlL techniques obviate these differences by simultaneous administration of both formulations and dramatically reduce the number of subjects needed to show equivalence with adequate statistical power. Drugs with saturatable first pass metabolism, nonlinear pharmacokinetics, prodrugs, and enantomers all present special problems for bioavailability/bioequivalence studies which can be solved using SIL methods (see Chapter 13).

8. COMBINED MULTIPLE-DOSE VOLUNTEER AND ABSOLUTE BIOAVAILABLITY STUDIES

Both of these types of study involve administering test drug to volunteers, collecting blood and urine for pharmacokinetic studies, and making safety observations (see Tables 1 and 2). By administering an SIL reference dose of drug (i.v. or oral solution) with the first chronic dose of drug (marketed drug preparation), it would be possible to determine simultaneously the absolute or relative bioavailability of the drug as well as the drug's single-dose pharmacokinetic parameters. This combined procedure would result in a dramatic reduction in subjects, hospital days, analytic specimens and cost (see Tables 1 and 2).

9. MULTIPLE-DOSE PATIENT STUDY

The objectives of this study are to obtain data on" (1) pharmacokinetic properties of the new drug during chronic administration (i.e. determine if a new drug has linear, concentration-dependent, or time-dependent pharmacokinetic properties); (2) drug interactions of new drug with medications with which the new drug will be co-administered (e.g. interactions of a new antiepileptic

425 drug with phenytoin or carbamazepine); and (3) preliminary data on safety and efficacy of the new drug. The standard methodology for obtaining multiple-dose pharmacokinetic data in patients is similar to the methodology employed in multiple-dose volunteer studies (see above). Usually 12 to 24 subjects are studied. Drug interactions of a new drug with an old drug usually are determined by measuring trough plasma concentration of old drug before and after adding new drug. Drug interactions of old drug with new drug usually are determined by administering a single dose of new drug to patients with a therapeutic plasma concentration of old drug and determining pharmacokinetic parameters of the new drug from plasma concentration versus time relationships. Pharmacokinetic parameters of new drug in the presence of old drug are compared with literature values of pharmacokinetic parameters of the new drug when taken alone. These two methods of studying drug interactions are highly suspect because they are dependent on multiple assumptions which often are untrue, almost never verified, and introduce variance into results. These multiple assumptions and their consequences have been reviewed in detail elsewhere (5). Drug interaction studies utilizing older methods typically involve 12 to 24 patients. Because of the multiple sources of variance in traditional drug interaction study methods, such methods lack statistical power. In particular, it is not uncommon for a study of 12, or even 24, subjects to lack adequate statistical power to prove the absence of difference in pharmacokinetic parameters of one drug in the presence of the other (i.e. power to detect a truly significant difference of 0.20 with a power of 0.80). Thus, the traditional methods employed on a multiple-dose patient study require 24 or more subjects and may yield suspect results. SIL tracer methods exist for determining presence or absence of pharmacokinetic changes during chronic administration (i.e. determining if a drug has linear, concentration-dependent, or time-dependent pharmacokinetic properties), including differential effects for enantiomers (see Chapter 16) and for determining presence or absence of pharmacokinetic drug interactions (see Chapter 18). These consist of administering a SIL-tracer dose of new drug before and during chronic administration and administering a tracer dose of old drug before and during the chronic administration of new drugs. These methods eliminate intraindividual variability and many other sources of variability (see Browne, et al. (5) for detailed discussion). There is increased statistical power because of reduced variance. Indeed the presence or absence of pharmacokinetic changes of new drug during chronic administration or of old drug after adding new drug can often be demonstrated using eight or

426 fewer subjects with 0.80 power to detect a truly significant difference of 0.20 (6, 7). Thus, two separate SlL studies to determine new drug pharmacokinetic properties during chronic administration and drug interactions between a new drug and an older drug would usually require 16 subjects. The cost of two SIL studies with eight subjects each would be approximately the same as studying 24 subjects with the traditional techniques (see Tables 1 and 2). The savings in subjects with SIL techniques would be offset by costs for SlL drug and MS determinations. In Chapter 7 it is suggested that deuterated analogs of many drugs can be separated from non-labeled drug by HPLC and quantitated by UV detection, an inexpensive and readily available technique. New antiepileptic drugs must be tested for drug interactions with the standard older antiepileptic drugs, carbamazepine and phenytoin. We have shown that deuterated analogs of carbamazepine and phenytoin can be synthesized, separated from nonlabeled analogs by HPLC, and quantitated by HPLC/UV (8, 9). It has been shown the deuterated analogs of carbamazepine and phenytoin do not exhibit a metabolic isotope effect in man (8, 10). This method was developed as a standard, inexpensive method of performing drug interaction studies of the effect of new antiepileptic drugs on older standard drugs. Other such deuterium/HPLC/UV techniques probably will be developed to study the interactions of new drugs with currently administered older drugs. It is possible to combine SlL studies of new drug pharmacokinetics during chronic administration and pharmacokinetic drug interactions of new and old drugs into one study utilizing eight subjects. Simultaneous administration of SlL-tracer doses of new and old drugs would be performed before and during chronic administration of new drugs to subjects continuously taking old drugs. Pharmacokinetic values obtained before and during chronic administration of new drug would determine if the new drug has time-dependent pharmacokinetic changes. Pharmacokinetic values of new drug obtained at low and therapeutic plasma concentration of new drug would determine if the new drug has concentration-dependent pharmacokinetic properties. Pharmacokinetic values of new drug in the presence of old drug would provide information on drug interactions of old drug with new drug when compared with pharmacokinetic values of new drug administered alone. Pharmacokinetic values of old drug obtained before and during chronic administration of new drug would determine presence or absence of drug interaction of new drug with old drug. The reduction in subjects from 24 subjects with traditional methods to eight subjects with combined SIL methods would reduce total costs for the study by more than 50 percent (see Tables 1 and 2).

427 10. MAXIMALLY EFFICIENT UTILIZATION OF SIL METHODS

Let us now investigate the possibilities of making maximal and optimal use of SIL and combined SlL techniques throughout a Phase I drug development program. Note first that the fixed development costs associated with utilization of SIL techniques (isotope synthesis and formulation, MS method development, regulatory approval) would be spread over several studies. The logic of this optimized SIL Phase I program can be followed by referring to Table 1. The six volunteers used in the SIL mass balance/metabolite identification study will yield excellent single-dose volunteer data. This will reduce the number of subjects in studies #1 and #2 of Table 1 from 26 to 20 and will reduce the cost by $80,000. SIL methods reduce the cost of the absolute bioavailability study alone from $960,000 to $222,000, a savings of $738,000. Combining the absolute bioavailability study with the multiple-dose volunteer study will reduce the total number of subjects required from 44 to 20 and will reduce the total cost by $860,000. Using traditional methods, obtaining information on multiple-dose patient pharmacokinetics and multiple-dose patient drug interactions would require at least 24 subjects compared with eight subjects using the combined SIL study method described above. Thus, the stable isotope method reduces multiple-dose patient subjects by 16 and costs by $1,000,000 (see Table 1). The combined savings over Phase I from combining studies and using stable isotope methods is 46 subjects and almost $2,000,000 (see Table 1). The time savings realized by eliminating 46 early Phase I study patients will vary from 12 to 24 months depending on company logistics. It needs to be emphasized that the data obtained using the proposed stable isotope combination methods are not "short cut" or second rate. In fact, the data obtained is equal or superior to data obtained by standard methods for several reasons. First, the bioavailability, pharmacokinetic, and drug interaction data obtained with the proposed stable isotope methods have been shown to have statistical power which is adequate for FDA purposes and is often superior to standard methods (see Chapters 11, 13, 16 and 18). This assures that the more long-term and expensive late Phase I and Phase II studies (safety, dose ranging, efficacy) will be carried out optimally. Second, the early availability of mass balance/metabolite identification data can prevent major problems discussed above. Third, the number of subjects required for safety and dose ranging studies (Studies 2, 3 and 5 in Table 1) is not reduced. To date, the combined SIL methods described in this paper have not been

428 utilized in drug development. However, the combined SlL methods represent a straightforward combination of published single study methods. We currently are performing a demonstration project to demonstrate the SlL combination studies described in this chapter. We hope others will utilize these combined SlL methods in the near future.

ACKNOWLEDGMENT Supported by the United States Department of Veterans Affairs.

REFERENCES 1. T.R. Browne, Clin. Pharmacokinet., 18 (1990) 423. 2. R. L. Wolen and W.A. Garland, in Synthesis and Applications of Isotopically Labeled Compounds, T.A. Baille and J.R. Jones (eds) (Elsevier, Amsterdam, 1989) p. 147. 3. T.R. Browne, in New Antiepileptic Drug Development: Preclinical and Clinical Aspects, J.A. French, M.A. Dichter and I.E. Leppik (eds) (Elsevier, Amsterdam, 1993) p. 31. 4. R.L. Wolen, J. Clin. Pharmacol., 26 (1986) 419. 5. T.R. Browne et al., in Antiepileptic Drug Interactions, W.H. Pitlick (ed) (Demos, New York, 1989) p. 1. 6. T.R. Browne et al., Neurology, 38 (1988) 639. 7. T.R. Browne et al., Neurology, 38 (1988) 1146. 8. G.K. Szabo et al., J. Chromotogr., 535 (1990) 271. 9. G.K. Szabo et al., J. Clin. Pharmacol., 34 (1994) 242. 10. T. R.Browne et al., Neurology, 44 (1994) 2410.

429

SUBJECT INDEX

Absolute bioavailability 246, 248, 250, 354, 417, 418, 420, 423, 427 Absorption 243, 309, 341,369, 419, 423 Absorption half-life 337 ABT 392 Accelerating voltage 86 Acceptance specifications 414 Accumulation 160 Accuracy 220, 221,226, 228 Acetaminophen 160, 223, 224, 225, 226, 227, 228, 231,231,374, 405, 407 Acetic anhydride-d6 234 Acetoacetate 148 Acetylcholine 158 AcetyI-CoA 158 Acetylcysteinyl 160 Acid hydrolysis 408 Adenosine triphosphate (ATP) 142, 147 Administration technique 261 ADP 147 Adsorption losses 93 Advantages of SlL tracer methods 312, 344 Advantages of the CF-IRMS method 228 AED 139, 195, 198, 200 AED parameters 186 AES 200 Albumen 8 Alcoholic liver disease 375 Aldehyde dehydrogenase inhibition 59 Aliphatic 14 Aliphatic hydroxylation 289 Alkali flame thermoionic detector 172 alI-E retinylidene 211 Alzheimer's disease 146, 149 Amine radical cation 290

Amino acid catabolism 94 Amino acid residues 281 Amino acid sequence 32 Amino acid 28, 97, 95,111 ~/-aminobutyric acid 142 Aminopyrine 349, 362, 373, 387, 391, 397, 401 Aminopyrine breath test 375, 390, 394 Aminopyrine N-demethylation 374 Ammonia 29, 30, 224, 407 Ammonium acetate 27, 33, 54 Ammonium carbonate 224, 225, 225 Ammonium hippurate 407 AMP 147 Amphiphilic 58 Analyte thermal stability required 69 Analyte 28, 36 Analyte-containing solvent 58, 63 Anaplerotic Flux 157 Anesthetics 161 Aniline 279 Anionic cationic functional groups 65 Anisotropy 206 Anthropods 203 Anticonvulsant drugs 99 Antiepileptic drug 128, 157, 158, 430 Antiepileptic properties 134 Anti-inflamatory drug 182 Antimicrobial agents 363 Antineoplastic agent 100 Antipyrine 407 Antipyrine clearance 373 Antitumor reactions of neocarzinostatin 104 APCI 26, 31, 50, 65, 66 APE 227 APEXTC 226 APEXTN 223, 225, 227 API 27, 38, 39, 65

430 Approval of a drug or device for marketing 415 Ar collision gas 94, 96 Arachidonic acid 108, 110 Area under the plasma concentration versus time curve (AUC) 198, 247, 264, 298, 310, 423 Argon 1, 32 Argon plasma 179 Aromatic 63 Aromaticity 40 Aromatic nitro groups 31 Array detector 21 Arteriovenous difference in drug plasma concentration 270 Aspartate 154 Assessment of exocrine pancreatic function 369 Assessment of fat malabsorption 371 Assessment of organ functions 367 Assessment of pancreatic insufficiency 369 Assessment of steatorrhea 371 Association of official analytical chemists 409 Assumptions in using CF-IRMS for MB/MI studies 220 Assumptions of SlL tracer methods 310 Atmospheric bands 2 Atmospheric pressure chemical ionization (APCI) 34, 36, 37, 48, 65 Atmospheric pressure interfaces 23, 25 Atmospheric pressure ion spray interfaces 27 Atmospheric pressure ionization (API) 20, 33, 61,239 Atmospheric pressure ionization interfaces (API) 50 Atmospheric pressure ionization-mass spectrometry (API/MS) 233 Atom percent excess (APE) 121 Atomic absorption (AAS) 177 Atomic emission line 184 Atomic emission spectroscopy (AES) 169, 170, 171,176, 179, 184, 189, 193, 201

Atomic fluorescence (AFS) 177 Atomic mass 2, 65 Atomic numbers 1 Atomic spectroscopy detection 177 Atomic table 2 Atomic weight 177 ATP hydrolysis 148 AUC 198, 424 Auto-induction 352 Aziridinium ion 101 B/E linked-scan 84, 85, 97, 103 Bacterial fixation 5 Bacterial overgrowth 365 Bacteriorhodopsin protein 203 Basal 390 Basal kinetics 387 Bathorhodopsin 203, 205 BE 76, 82, 87 BEB 79 BEEB 79, 80, 85 Beenakker type resonant cavity 179 Bell-shaped curve 289 Benoxaprofen 248 Benzoic acid 241,400, 41 Benzoxylcarbonyl methyl esters 100 Benzylic hydroxylation 235, 290 Benzylic radical 289 1-benzymidazole 397 BEqQ mass spectrometers 82 BEqQ 76, 85, 86 Bile acids 59 Bile 100 Bio-analytes 23 Bio-analytical chemistry 44 Bio-analytical problems 40 BioAnalytical systems 132 Bioavailability 9, 309, 318, 319, 354, 424 Bioavailability studies 195, 196, 245 Bioavailability/bioequivalency studies 250, 252, 257 Bioequivalence 243, 250, 252, 257, 354 Biofluid 160 Biologic fluid 193 Biological compounds 100, 112

431 Biological effects 171 Biological fluids 189 Biological macromolecules 38 Biological mass spectrometry 44 Biological materials 177 Biological matrices 100, 199, 229 Biological molecules 170 Biological polymers 100 Biological samples 108, 178 Biomedical analysis 187 Biomedical mass spectrometry 19 Biomolecules 32, 35, 64 Biopolymeric substances 44 Biopolymers 49, 62, 64, 81 Biotransformation 9, 275, 297, 315, 348, 353, 384 Blood 96, 220, 222 Blood (deproteinized) 222 Blood glucose concentration 149 Blood samples 99 Blood-brain barrier 315, 324, 332 Blood-cerebrospinal fluid barrier 324 Blood-cerebrospinal fluid transfer 329 BN50727 55 Boltzman distribution 143 Bolus infusion methodology 327 Bond angle 15 Bond length 15 Boron 1 Bovine retina 203 Bovine rhodopsin 203 Brain energy metabolism 147 Brain metabolites 153 Brain pathology 145 Brain slices 157 Brain tissue 97 Brainstem 98 Branched reaction pathway 286 Breath test 307, 361,363, 379, 380, 401,416 Bromine 55 BSA 234 BSP clearance 374 BSTFA 234 BSTFA-d18 234 Buspirone 124 Butylated carnitine levels 94

[13C 02] 121 CmD 14 D20 3, 6 12C 13, 319 12CO 173, 183 13C 2, 4, 6, 7, 9, 13, 15, 17, 41,104, 119, 121,123, 124, 130, 141,143, 170, 171,172, 174, 176, 178, 182, 188, 189, 191,194, 195, 199, 319 13CO 183,190 13CO2 6, 9, 123, 173, 176, 363 14C 419, 421 14N 13 14N/14N 121 15N 4, 7, 13, 15, 17, 41,119, 121,123, 130, 141, 170, 178, 183, 185, 199, 319 15N/14N 121 15N/15N 121 180 5, 7, 41, 170, 178 19F 141, 143 31p 141, 142, 143 34S 42 35CI 104 37CI 42, 104 81Br 42 1H 141, 143 2H 119, 178, 199 2H 7, 41 12C-13C intensities 172 13C atomic emission detection 194 13C bands 173 13C chromatogram 191, 192, 193 13C glucose 153 13C hiolein 373 13C isobutyl methyl xanthine 191,192 13C isotope 64, 206 13C isotopomer-based NMR method 159 13C labeled metabolite 145 13C labeling 7, 148, 151,173, 180, 185, 236, 380 13C MAS NMR 216 ~3C mass spectrometer 5 ~3C NMR spectra 155, 159 13C NMR 149, 151, 153 13C octanoic acid 367 ~3C signal 195

432 ~3C spectra 156 13C spectrum 145 ~3C subtraction 195 13C triglycerides 369 13C xylose 366 13C 150, 154, 155, 159, 169, 185 ~3C/12C isotope ratio 401 13C/12C ratio 199, 383 13C-CO2 enrichment 349 ~3C2-acetaminophen 226 ~3C6-acetaminophen 228 ~3C6-1evodopa 229 13C-Aminopyrine Breath Test 374 ~3Carbon 236 13C-breath tests 361 ~3C-containing molecules 7 13C-edited 150 ~3C-editing 153 ~3C-glycine 5 ~3CH3 isobutylmethylxanthine 195 ~3CH3 methyl ester 182 ~3C-labeled acetate 367 ~3C-labeled bicarbonate 367 13C-labeled caffeine molecules 196, 197 13C-labeled compounds 173 ~3C-labeled hiolein 372 13C-labeled isotopes 152 13C-labeled metabolites 153 13C-labeled mixed triglyceride breath test 369 13C-labeled polyunsaturated fatty acids 159 ~3C-labeled progesterone 200 ~3C-methane 5 13C-NAPA 246 13C-NMR 240, 241 13CO2 excretion 370, 372 ~3CO2 recovery rate 373 13CO2 151,384, 398 ~3C-octanoate 369 ~3C-octanoic acid breath test 367, 3 6 8 , 369 ~3C-octanoic breath test 365, 367, 368, ~3C-urea 363 ~3C-xylose breath test 366

14C bile acid test 366 14C trioctanoin 371 14C tripalmitin 371 ~"C-breath tests 362 14C-labeled triglycerides 371 35CI 103 l"C-xylose breath test 366 2-[~3C] caffeine 176 C/D/N Isotopes Inc. 406 C13H120~ 79 C15H~-o 79 C~e reversed phase columns 138 C-18 cartridge 59 C3-1abeled pyruvate 158 Ca=+ 162 [1,2,3-~3C3]acrylic acid 241 [1,2-13C]acetate 155 1,2,3[carboxyl ~3C] octanoyl glycerol 370 1,3,7-~3C trimethyl-xanthine 349 Caffeine 175, 180, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 195, 197 198, 348, 349, 401,307 Caffeine breath test (CBT) 390 Caffeine isotopomer 184, 188 Caffeine metabolites 189 Calcium 266 Calculation of the residual standard deviation 182 Cambridge Isotope Laboratories 321, 406 Capacity factor 135 Capillary column 179 Capillary electrophoresis 20, 57, 64, 68 Capillary gas chromatography 169, 187 Capillary glass GC columns 129 Capillary isotachophoresis 64 Capillary liquid chromatography 68 Capillary tube 25 Capillary 25, 28 Carbamazepine 98, 128, 130, 131, 132, 134, 136, 139, 262, 308, 339, 340, 345, 352, 407, 425, 426 Carbamazepine clearance 307 Carbamazepine-epoxide 98

433 Carbaryl 40 Carbidopa 317, 318, 334 Carbohydrate metabolism 148 Carbohydrates 38 Carbon dioxide 381,399 Carbon isotope 172, 175, 185, 189, 191 Carbon radical 287 Carbon 407, 55 Carbon-deuterium bonds 135 Carbon-based radical 290 Carbon-hydrogen bond cleavage reaction 275, 287 Carbon-hydrogen bond oxidation 285 Carboxy terminal group 109 Carboxyl oxygen 110 Carboxyl-labeled substrates 373 Cardiac arrhythmia 316, 317, 334 Carnitine 95 Carnitine deficiency 94 Carnitines in plasma 94 Carnitines in urine 94 Catalog cost for stable isotope-labeled analogues of common drugs 407 Catalytic hydrogenation 244 Cationic functional groups 65 CBT 392 CBZ 99 CBZ/deutero-analogue 132 CE-MS 35 Cellular Ca2+ 163 Cellular phospholipid synthesis 147 Center for devices and radiological health 415 Center for drug evaluation and research 415 Central dopamine metabolism 320 Central levodopa metabolism 319 Central levodopa/dopamine metabolism 319 Central nervous system 315, 317 Cerebellum 98 Cerebral lipid metabolism 159 Cerebral metabolism 146 Cerebrospinal fluid 262, 263, 324 Cerebrospinal fluid homovanillic acid levels 328

Certificate of analysis 414 Cesium 58 CF/FAB 26, 33, 49, 50, 58 CF/FAB interfaces 48, 57 CF-GC-IRMS (continuous flow gas chromatography mass spectrometry) 199 CF-IRMS 122, 220, 221,222, 228, 229, 230, 232, 386, 398, 399, 400 CFR 412 [1-13C]glucose 151, 153, 154, 156, 157 [2-13C]glucose 151 [6-~3C]glucose 151 [1,2-13C]glucose 155 Charge delocalization 214 Charge dispersal mechanism 63 Charge transfer 66 Charged analyte ions 35 Charged analyte molecule or parent ion 20, 21 Charged ions 58 Charged molecules 19 Charged radical-cation 29 Charge-exchange 26 Chemical and isotopic purity 409 Chemical Ionization (CI) 20, 25, 29, 37 Chemical ionization processes 60 Chemical purity 409 Chemical reaction interface mass spectrometry (CRIMS) 52, 119, 122, 220, 230 Chemical shift 145, 147, 207, 240 Chemotherapeutic drugs 162 3-13CH3 labeled caffeine 192 Chloramphenicol 55 Chlorine 1, 55 Cholestatic liver disease 374 Cholesterol 4 Cholesteryl octanoate 362 Cholesteryl octanoate breath test 371 Cholesteryl-[1-~3C] octanoate 370 Chromatographic efficiency 58 Chromatographic isotopic separation 128 Chromatography 177, 178, 200, 221 Chromophore 203, 208, 211,215 Ch ro mopho re-protei n interactions 208

434 Chronic active hepatitis 374 Chronic alcoholism 97 11-cis retinylidene 211 CI mass spectra 49 CI 31, 38, 52, 54 Cibenzoline 309 CID 76, 82, 86, 87, 88, 89, 98, 99 Cimetidine 388 Cirrhosis 374 CK activity 148 CK expression 148 CK flux 148 CK iso-enzyme 148 CK kinetics 148 CL 297, 298, 311,342, 343, 345 [1-13C]-Iabeled glucose 149 Clearance via production 313, 341 Clearance 264, 267, 268, 269, 270, 271,272, 273, 297, 298, 300, 301, 305, 306, 308, 310, 337, 340, 345, 422 Clinical gastroenterology 373 Clinical studies 323 [13C6]-L-kynurenine 55 Clobazam 234 Cluster ion formation 35 [~3C6]-L-tryptophan 55 Cmax (mg/I) 198 CO2 breath test (CBT) 348, 349, 350, 351 CO2 excretion rate 351 CO2 380 Coaxial delivery system 58 Code of federal regulations 412 Coefficient of regression 197 Coefficient of variation 221 Coefficients 196 Collision region 75 Collision 63 Collisional activation dissociation (CAD) 64, 75 Collision-induced dissociation 81 Collision-induced dissociation (CID) 75 Collisions 22 Colors 208 Column efficiency (N) 135 Combined mass balance/metabolite

identification and single-dose volunteer studies 421 Combined multiple-dose volunteer 424 Comparative isotope ratio measurements 382 Competitive or non-competitive inhibition 306 Complementary techniques 35 Complex mixtures 22 Compliance 312, 313, 341,343, 344, 345, 355 Concentration 343 Concentration-dependent (nonlinear) pharmacokinetics 297, 298, 299, 301,303, 305, 311,312, 344, 422, 423, 426 Conjugated aromatic systems 31 Conjugated compounds 63 Container closure system 414 Continuous flow fast atom bombardment (CF/FAB) 48, 56, 57 Continuous flow fast atom bombardment interface (CFFAB) 23, 24 Continuous flow gas chromatographyisotope ratio mass spectrometry (CFGC-IRMS) 170 Continuous flow-isotope ratio mass spectrometry (CF-IRMS) 119, 120, 220, 379, 381,382 Continuous-flow 27 Cortex 98 Cortisol 231 Cost of an MB/MI study 229 Cost 406, 407, 422, 426 Coupling constant 211 Covalent bonds 29 CP/MAS spectra 213 [1,2,313C3]propionic acid 241 Creatine kinase 147 Creutzfeldt-Jakob disease 146 [10,20-13C2]-rhodopsin 212 CRIMS (chemical reaction interface mass spectrometry) 122, 199, 232 CRIMS 122, 232 Cross-over study designs 245, 246, 251,254, 257

435 Cross-over technique 423 CSF 160, 263 Custom synthesis 406, 409 CVP 304, 305 Cyclic aziridinium intermediate 101, 103, 104 Cyclophosphamide 101, 103, 104 CYP 1A2 349, 350 CYP2B1 286, 292 CYP2D6 294 Cystic fibrosis patients 356 Cytochrome P450 275, 276, 277, 287, 288, 289, 290, 291,292, 380, 384, 388, 389, 393, 394, 397 Cytochrome P4502B1 287 Cytosolic proteins 146 D2-imipramine 247 D2-nicotine 249 D20 160 D3-methadone 246 D2-1abeled analogues 108 D2-1abeled fragment 104 D20 105 D3-carnitine 94 D3-GSH-NMF 100, 101, 102 D3-NMF 100, 101, 102 D4-5-hydroxytrypta mine 98 D4-CBZ 99 D4-1abeled analogue 98, 104 D4-1abeled indolethylamine 97 D4-tryptamine 98 D4-Tyr 96 Do-ME 96, 97 D6-5-HMTLN 98 D8-HTLN 98 Dg-TLN 98 Daltons (Da) 77 Data acquisition 40 Data analysis 40 Daughter fragment ions 39 N-dealkylation 289, 291 O-dealkylation reactions 290 Debrisoquine 351 Decadeuterated compounds 130 Decadeuterated ethotoin 137 Decadeutero phenytoin 134

Decadeuterocarbamazepine 130 Decadeuterophenytoin 130 Decarboxylation 348 Deep peripheral compartment 264 Deep pool effect 261,264, 265, 266, 267, 268, 269, 270, 272, 272, 273, 310, 338, 344 Demethylation of caffeine 351 Demethylation 188, 348, 349, 350, 408 N-demethylation 279, 289, 349, 350, 401 Demethylsufoxide 401 3D emission spectrum 175 Deproteinization 221 Deprotonated [M + H] + molecular ion 74 Deprotonation 290 Derivatives 22 Derivatization reagents 178 Desorption ionization method 38 Detection limits 53 Detection of neurotransmitters in vivo 158 Detection of the NMR signal 144 Determination of absorption 9 Determination of stable isotopes 177 Detoxification 386, 397 Deuterated caffeine 180 Deuterated compounds 106, 128, 134, 135, 139, 170, 426 Deuterated ethotoin 136 Deuterated internal standard 130 Deuterated isotopomers 133, 187 Deuterium [2H] substitution 131 Deuterium atoms 185, 187 Deuterium concentration 184 Deuterium ion cluster techniques 238 Deuterium isotope effect 275, 276, 277, 278, 280, 286, 290, 294, 295 Deuterium labeling 4, 15, 16, 123, 180, 244, 347 Deuterium substitution reaction 134 Deuterium 2, 3, 4, 5, 14, 15, 17, 40, 129, 134, 170, 171,180, 184, 185, 188, 234, 235 Dextrose injection 323 Diagnostic breath tests 362

436 Diarrhea 366 1,2-di b ro m e-3-ch Io ropropa ne (DBCP) 239 2,3-didehydrosparteine 294, 295 5,6-didehydrosparteine 294 Dideuteromethyl isotopomers 187 Dietary ammonium citrate 4 Diethydihiocarbamate (DDTC) 390, 391 Diethylstilbesterol (DES)isomers 59 Di-GSH-derivative 101 10,11-dihydrocarbamazepine 132, 133 Dihydroxyphenylacetic acid 319 Dimethylanilines 289, 291 Dimethylsulfide 389 Dipole moment 15 Direct chemical ionization (DCI) 49 Direct current (dc) 76 Direct insertion probe 19 Direct liquid introduction interface (DLI) 23, 25, 26, 48, 49 Direct-current plasma 178 Disadvantages of HPLC-CRIMS 231 Disadvantages of SlL tracer methods 313, 345 Disadvantages of the CF-IRMS methods 230 Dissociative mechanism 293, 294 Distribution 9, 273 Disulfiram 59, 389 Divisions of FDA Center for Drug Evaluation and Research 413, 415 D-labeling method 106 DLI interfaces 23, 25, 26, 48, 49 1D MAS NMR 210 2D MAS NMR dipolar correlation spectroscopy 210 DMSO 397 DNA 105, 106, 107 DNA damage 104 Dopa-decarboxylase activity 317 Dopa-decarboxylase inhibitor 315 Dopa-decarboxylase 317, 322 Dopamine 158, 317, 322, 334 Dose ranging 418, 421,427 Dose-dependent pharmacokinetic changes 353

Dosing interval 303 Dosing rate 303, 304, 305, 343 Double-focussing 77 Double-focussing mass spectrometers 78 Double-focussing sector mass spectrometers 78 Drug conjugates 56 Drug disposition 65 Drug distribution 261 Drug interaction studies 337, 417 Drug interactions 261,297, 298, 309, 337, 344, 353, 418, 424, 425, 426, 427 Drug master file (DMF) 414 Drug metabolic pathways 348 Drug metabolism 20, 352, 384 Drug metabolites 89 Drug monitoring 20 Drug protein binding 341 Drug-conjugate metabolites 34 Drug-conjugates 24 Drug-free bile sample 101 Drugs in biological fluids 9, 160 Drug-tissue entry rate constant 262, 264 Dual inlet dynamic interface 382 Dual inlet IRMS system 119 Dual selective detection of lSN label in N2 gas and subsequent 13C label in CO2 gas 225 Dumas combustion techniques 122 D-xylose 362 Dynamic fast-atom bombardment interface (CF-FAB) 27 Dynamic interfaces 382 Dynamic measurement AES 178 Dynamic processes 9 Elab 96 EB 76, 82, 85, 87 EB double-focussing 96 EBE 79 EBEB 79, 80, 85 EBEB mass spectrometer 103 EBqQ 76, 80 EBqQ hybrid mass spectrometer Economic evaluation 417

100

437 Economic savings 313, 345 Effect of drugs 355 Efficacy 418, 425, 427 El 24, 25, 38, 52, 54 El mass spectrum 42 Electric field strengths 84 Electric sector 75, 76, 78, 80, 87 Electrospray (ES) 48 Electrolysis 3 Electrolyte-mediated chemical ionization 33 Electrolytic dissociations 2 Electron capture (EC) 66, 97 Electron donation 15 Electron impact (El) 49 Electron ionization 20, 29, 340, 37 Electron multiplier 21 Electron spin resonance (ESR) 400 Electron transfer mechanism 290, 291 Electronegativity 292 Electronic and chemical environment 145 Electronic effects 289 Electronic transition system 175 Electronic vibrational spectrum 175 Electrospray and ionspray 62 Electrospray (ES) 23, 25, 26, 27 Electrospray (ES)ionization 34 Electrospray/ionspray ionization 37 Electrospray/ionspray LC-MS interface 62 Elemental analysis 44 Elemental chromatogram 180, 191 Elemental labels 178 Elimination 9, 344 Elimination half-life 264, 297, 298, 299, 300, 301,305, 309, 310, 337, 340, 343 Elimination rate constant 299, 300, 301,303 Emission spectrometer 200 Enantiomer 255, 256, 308, 341,351, 424, 425 Enantiomeric interconversion 255 Enantiomeric purity 409 Enantiomeric racemization 255 Enantioselectivity of drugs 351,354

Enantiospecific syntheses 408 Endogenous 276, 384 Endogenous levels 97 Endoscopy 363 Energy of activation 13 Energy-to-charge ratio 84 Enflurane 162 Enkephalins 96 Enrichment 2 Entry half-life 262, 264 Enzymatic maturation 351 Enzyme binding kinetics 15 Enzyme induction 272, 298, 385 Enzyme structure 288 Enzyme substrate complex (ES) 293 Enzyme urease 363 Enzymeoxene 293 Enzyme-substrate complex 283 EOS 293 Epidemiological studies 365 Equilibrium 9, 144 ES ionization 63 ES 38, 50 ES/IS 65, 66 ES-MS 63, 64 ES-active 63 ESD 279, 281,282, 283 ESH 279, 281,282, 283 Essential amino acids 7, 8 Ethane 176 Ethotoin 131, 137, 407 EURISO-TOP 406 Excretion 9, 160, 275, 297, 315 Exocrine insufficiency 370 Exocrine pancreatic function 372 Exogenous 276 Exogenous pancreatic enzymes 356 Expired labeled C02 349 Extraction 221 Extraction efficiency 93 19F NMR measurements 162 19F NMR spectroscopy 162 ~9FNMR 152,161,161,163 ~9F nucleus 160 ~9F spectrum 161, 162 ~9F indicators 162

438 19F NMR detection of fluorinated drugs 160 2-19F 152 FAB 28, 31, 39, 52, 57, 96 FAB ionization 94, 108 FAB ionization source 97, 100, 104 FAB mass spectra 102 FAB probe tip 95 FAB/MS spectrum 101, 106 FAB/MS 74, 104, 105, 107 FAB/MS/MS 104 Facilities data 412 Factor of bioavailability 198 Faraday cup collectors 121,382 Fast atom bombardment (FAB) 20, 25, 31, 32, 37, 74, 93, 112 Fast atom bombardment ionization 112 fast atom bombardment mass spectrometry (FAB-MS) 56, 239 Fat malabsorption 356, 362, 369 Fatty acid catabolism 94 Fatty acid metabolism 159 FDA Center for Drug Evaluation and Research 412, 413, 415 FDA Division 413 FDAPhasel 229,418 FDA 229, 405, 409, 412, 413, 414, 415, 416, 417 FDG-6-P 152 FDL 412 Feces 219,220 Feces (extracted) 222 Feces (whole) 222 Fenoprofen 182 [FeO]3+-substrate complex 288 [18F] fluorodopa 319 FFR1 85, 86, 87 FFR2 85, 86 First field-free region (FFR1) 78 First pass metabolism 424 First quadrupole mass filters 77 First-order 193 FK506 55 Flame emission (FES) 177 Flame photometric detectors 172 Fluorinated dopamine 319

Fluoro-BAPTA 162, 163 Fluoro-deoxy-6phosphogluconate 152 Fluoro-deoxyfructose 152 Fluoro-deoxyglucose 151 FIuo ro-d eo xy g Iu cose-6- p h osph ate (FDG-6-P) 151 Fluoro-deoxysorbital 152 5-fluoro-deoxyuridine 162 5-fluorouracil 162 Fluoromethyl alanines 162 Fluorometric assays 96 e-fluoro-/3-alanine 162 Fluoxetine + norfluoxetine 161 Fluphenazine 161 Fluvoxamine 161 Food and Drug Administration (FDA) 411 Food Chemicals Codex 409 Food Drug and Cosmetic Act of 1938 415 Forensic analysis 59 Form FDA1571 412 Form(s) FDA 1572 412 Formaldehyde 349 Formic acid 5, 349 Formyltetrahyd rofolate 349 Fourier transform analysis 144, 240 Four-sector mass spectrometers 79 Fraction absorbed 312, 313, 337, 343, 344, 345 Fragment 64, 110, 433 Fragment ion 28, 106, 22, 29, 30, 33, 41, 194 Fragment ions (F+) 81 Fragment or daughter ions 21 Fragmentation of angiotensin III 110 Fragmentation 69 Free-induction-decay 144 Frequency domain MAS 207 Frequency spectrum 144 Full-scan MS 82 GABA 155, 156 GABA metabolism 158 GABA spectra 158 GABA synthesis 149

439 GABA-T inhibition 158 GABA-T inhibitor 159 GABA-transaminase 158 Galactose breath test 394 Galactose elimination 374 Gas chromatograph (GC) 122 Gas chromatographic combustionisotope ratio mass spectrometric (GCC-IRMS) 121 Gas chromatographic-mass spectrometry (GC-MS) 73, 170, 237, 319, 326, 337 Gas chromatography 175, 185, 383 Gas chromatography-atomic emission detection (GC-AED) 169, 189 Gas chromatography-isotope ratio mass spectrometry (GC-IRMS) 120 Gas or liquid chromatograph 19 Gastric emptying 371 Gastric lipase 370 Gastrointestinal disorders 361,363 Gastrointestinal infections 363 Gastrointestinal or liver dysfunction 362 Gastrointestinal tract 355, 373 GCAED 185,189,200 (GC-AES GC-AED) 171 GC-IRMS 200, 385, 398 GC/MS 97, 234, 250 GC-MS 20, 25, 28, 48, 51, 62, 67, 97, 234, 241,250, 341,344, 345 GC-MS Inlet 24 GC/MS/MS 97, 98 GC/MS-SlM quantitation 98 GC-pyrolizer-MS system 121 GC-AED 174, 176, 177, 179, 184, 186, 191,194, 195, 197, 199 GC-AED isotope detection 186 GC-MIP coupling 179 GC-MS 194, 195, 200, 238 Gentamicin 264, 266 Glucose 141,157 [13C]glucose 157 Glucose metabolism 151,157, 158 Glucose oxidation 398 Glucose signal 149 Glucose transport 149

Glucose utilization 150 Glucuronic acid 59 Glucuronide 160, 238 3-glucuronides 65 6-glucuronides 65 Glutamate 141,152, 154, 156, 157 Glutamate enrichment time courses 157 Glutamine cycle 155 Glutamine synthesis 149, 156, 157 Glutamine synthetase 155 Glutamine 7, 154, 155, 156, 156, 157 Glutathione 151, 59 Glutathione conjugates 66, 100 Glutathione conjugation 101 Glutathione (GSH) 100 Glycerin 32 Glycerol 58, 369 1,2-glyceryl dinitrate 253, 254 1,3-glyceryl dinitrate 253, 254 Glycine 4, 5 Glycogen 5,150, 151 Glycolysis 150 Gopher systems 412 G-protein transducin 206 G-protein 203 Gram-negative bacterium 363 Growth and development 307 Growth hormone therapy 351 Growth hormone-deficient children 351 GSH 105 GSH conjugation 104 GSH-NMF 100, 101, 102 GSH-cyclophosphamide 104 Guidelines for ordering stable isotopelabeled drugs 408 Gylcerol 113 2H-labeled internal standards 56 1H NMR detection 150, 160 1H NMR spectrum 141, 145, 146 1H NMR 153 2Hp2C ratios 186 2H/~2C values 187 Halogens 31 [2Hlo]-CBZ 132

440 Heat labile biomolecules 23 Heated inlet chamber 33 Helicobacter pylori 355, 363, 411, 416 Helium carrier gas 122 Helium 52, 53, 54, 179, 220 Heme-oxene-substrate complex (EOS) 293 Hepatic antipyrine metabolism 373 Hepatic encephalopathy 146 Hepatic functional impairment 374 Hepatic metabolism 315, 371,373 Hepatocytes 235 Hepatotoxicity 384, 389, 393 Heptafluorobutyryl derivatives (HFBTEN) 97 Heteronuclear 13C-1H 145 Hexapole collision cell 76, 77 Hexokinase 151 Hexose monophosphate shunt (HMPS) 151,398 High-energy CID 78 High molecular weight biomolecules 20 High molecular weight biosubstances 24 High performance liquid chromatography (HPLC) 47, 220, 236, 355, 421 High temperature surface ionization 52 High-energy CID 80, 81, 85, 111 High-resolution capabilities of EB and BE mass spectrometers 79 Hiolein 362, 371 Hippocampus 98 Hippuric acid 241 Histamine 30 Histology 365 Histopathology 364 HMPS 399 5-HMTLN 98 Homogenization process 397 Homovanillic acid 315, 318, 319, 320, 321,324, 325, 326, 328, 329, 332, 333 HPLC columns 57, 58 HPLC flow 69 HPLC methods 138

HPLC technology 23 HPLC 24, 36, 51, 57, 64, 69, 100, 103, 160, 178, 222, 230, 232, 422, 426 HPLC-TSP-MS 61 HPLC-CRIMS 230, 231,232 HPLC-CRIMS aplications to mass balance studies 231 HPLC-CRIMS instrument 123 HPLC-CRIMS: advantages 231 5-HTLN 98 Human pituitary 96 Human plasma 65 Hybrid EBqQ 82 Hybrid instruments 87 Hybrid mass spectrometers 76, 81, 85 Hybrid sector 76, 77 Hybrid sector mass spectrometers 80 Hydoxylation 234 Hydrochloric acid 325 Hydrogen 55 Hydrogen atom abstracting species tert-butoxy radical 291 Hydrogen atom abstraction 288 Hydrogen atom abstraction mechanisms 291 Hydrogen atom radical recombination mechanism 288 Hydrogenated linseed oil 4 Hydrolization 372 Hydrophilic compounds 66 Hydrophilic loops 203 Hydrophobic c~-helices 203 Hydrophobic moiety 59 /3-hydroxybutyrate 148 6-hydroxydopamine 317 e-hydroxy fragmentation 108, 109 Hydroxy functional group 107 Hydroxyl radicals (OH~ 290, 400 Hydroxylation 279, 287, 400 o~-hydroxylation 285, 286 5-hydroxymethyltryptoline (5HMTLN) 97 5-hydroxytryptoline (5-HTLN) 97 Hyperactivity 320 Hyperlipidemia 372 Hypothalamus 98 Ibuprophen

160, 309

441 ICON Services Inc. 406 ICR 76 Identification purposes 33 Identity testing 414 Immunoaffinity column 59 Increased column efficiencies 136 IND 411,412, 413, 414, 415 Indoleamines 97 Indolethylamine internal controls 98 Indolethylamines 97 Inductibility coupled plasma 178 Induction 385 Induction (inhibition) 341 Inert target gas 81 Inflammation 146 Inflammatory reaction 395 Inhibition 387 Initial electron transfer mechanism 290 Initial hydrogen atom abstraction mechanism 290 Injection volumes 93 Inlet system 19 Institutional review board (IRB) 219, 412, 419 Inter-element ratios 169 Interface 53, 67 Interferon-~/ 55, 56 Intermolecular deuterium isotope effect 293 Intermolecular isotope effect 292 Internal standard 22, 40, 54, 98, 132, 195 Intersubject variability 150 Intra-ligand interactions 210 Intra-ligand internuclear distances 210 Intramolecular competition 277 Intramolecular competitive design 277 Intramolecular deuterium isotope effect 280, 282, 283, 285 Intramolecular isotope effect 277, 282, 291 Intrinsic isotope effect 275, 284 Intrinsic primary isotope effect 291, 292

Invasive tests 363, 365 Investigational new drug application 411 Investigator data 412 in vitro models 146 in vivo 250, 315, 316, 318, 334, 347, 375 in vivo isotope effects 129, 134 in vivo microdialysis 57 in vivo NMR spectrum 145 Ion accelerating voltage 79 Ion chromatography 68 Ion cluster 233 Ion cyclotron resonance (ICR) 75 Ion fragments 19 Ion molecule reactions 66 Ion sensitive ligands 160 Ion source 20, 28, 38 Ion source interfaces 60 Ion trap mass analyzer 21 Ion trap 37 Ion-cluster 194 Ionic and polar 19 Ionic substances 32 Ionic surfactants 59 Ionization mass spectrometry (APIMS) 238 Ionization modes 69 Ionization process 34 Ionization techniques 32 Ionization-MS 54 Ion-molecule interactions 32 Ionspray (IS) 26, 48 Ionspray (IS)interface 64 Ionspray (IS) ionization 34, 35 Ionspray API-MS detection 237 Ion-trap mass analyzer 37 Ion-trapping mass spectrometers 38, 76 IRMS 121, 122, 421,422 Irreversible inhibitors 159 IS 50 IS ionization 63 Isolation valve 382 Isomeric purity 409 Isotachophoresis 68 Isotec Inc. 406

442 Isotope atomic emission 172, 176 Isotope cluster technique 233, 348 Isotope dilution MS/MS 93, 94, 95, 97 Isotope effect 129, 134, 224, 225, 270, 275, 277, 280, 288, 290, 293, 310, 320, 332, 347, 352, 357, 379, 406, 408 Isotope enrichment 200 Isotope labeling 206, 215 Isotope labeling (SIL) 220 Isotope measurement 199 Isotope peak shift technique 234 Isotope ratio mass spectrometry (IRMS) 119, 123, 169, 188, 236, 238, 361,362, 419 Isotope synthesis 427 Isotope tracers 100 Isotopes detection 181 Isotopes of oxygen and hydrogen 175 Isotopes ratios 199 Isotopic dilution 156 Isotopic effect 171 Isotopic enrichment 155 Isotopic labeling 15, 16, 64, 93, 100, 150, 156 Isotopic purity 129, 409 Isotopic separation 129, 130, 133, 134, 138 Isotopic shifts 171 Isotopically labeled internal standard 94 Isotopically labeled internal standard D3-carnitine 95 Isotopically labeled species 98 Isotopomer distributions 155, 156 Isotopomer/analogue pairs 132 Isotopomers 129, 135, 136, 185, 186, 399 IUPAC (pg) 183, 184 IUPAC method 181 J-coupling 145 Jet separator 28

Kco 187 e-ketoanalogues 375 Keto-enol tautomerism 236 (x-ketoglutarate 152, 153, 157

(x-ketog Iuta rate/g Iuta m ate exchange 149 Ketoisocaproic acid 362 KICA breath test 376 Kinetic energies 81 Kinetic energy filter 87 Kinetic isotope effect 13, 14, 16, 17 Kinetic-energy-to-charge ratios 78 Kinetics of drugs 354 Kinetics of the expired 13CO2 387 Kr. 298, 299, 303, 304, 422 K,-nNmax 305 k! retention factor 135 Krypton 1 Kynurenine 56 Labeled caffeine 195, 197 Labeled compounds 195 Labeled drugs 15 Labeled homovanillic 330 Labeled isotopomer 195 Labeled lactate 151 Labeled levodapa 330 Labeled parent drugs 194 Labeling isotopes 187 Labile compounds 56 Lactate dehydrogenase 152 Lactate 141, 150 Lactic acid 150 Larmor condition 144 Larmor equation 143 Larvae 397 Laser desorption 52 Last-in first-out phenomenon 271, 272 LC technology 24 LC-APCI-MS 66, 67 LC-CF/FAB-MS 59 LC-CI-MS/MS 40 LC-CRIMS 53 LC-ES/IS-MS 65 LC-ES-MS 35 LC-FAB-MS 57 LC-MS 20, 23, 24, 35, 39, 47, 48, 51, 65, 243 LC-MS detector 63 LC-MS inlets 25

443 LC-MS interface 26, 48, 49, 51, 52, 62, 66, 69 LC-MS-MS 40, 243 LC-PB-CRIMS 55 LC-PB-EI/MS 55 LC-PB-MS 53 LC-PB-NCI/MS 55, 56 LC-TSP-MS 61 L-Carnitine 94 LC-CRIMS 124 LC-MS with atmospheric pressure ionization interface 239 LC-MS with fast atom bombardment interface 239 LC-MS with thermospray interface 238 L-dopa 407 Least square linear regression analysis 225 Less-abundant isotopes 43 Leucine 4, 356 Levodopa central and peripheral metabolism 318 Levodopa-induced clinical response 319 Levodopa metabolism studies 333 Levodopa 226, 315, 316, 322, 323, 325, 332 Liability risk 419 Ligand-protein interactions 203, 206, 208, 210 Limit of detection of 13C 181 Limit of detection of deuterium 183 Limit of detection of nitrogen lSN 183 Limit of detection 181, 184 Limited dynamic range 53 Limits of detection 183 Linear calibration curves 54 Linear dynamic range 184 Linear pharmacokinetics 267, 271, 272, 298, 299, 301 Linear regression analysis 222 Linked-scan 87 Linoleic acid 108 Linolenic acid 108 Lipid resonances 151 Lipophilic interactions 139

Lipophilicity 15 Liquid chromatography/mass spectrometry (LC/MS) 73, 238 Liquid inlet stream 178 Liquid matrix 32 Liquid secondary ion emissionMS 56, 96 Liquid/vacuum interface 113 7Li spectroscopic imaging 163 Lithium 1 Liver biopsy 263 Liver function breath tests 373, 375 L-kynurenine 56 LLQ 224 Long-chain fatty acids 372 Low or high molecular-weight compounds 19 Low thermal stability 54 Low volatility compounds 54 Low-energy CID 76, 80, 81, 85, 110 Lower limit of quantitation (LLQ) 222, 230 Low-resolution structure 210 Lumbar puncture 263, 264, 320, 330 Luminal lipase 369 Lumirhodopsin 205 Lysine 4 m/z ratio 21, 22, 35, 36, 37, 38, 40, 64, 76, 77, 79, 84, 86, 88 Magic Angle Spinning (MAS) NMR 206 Magnetic moment 142 Magnetic nuclear resonance 170 Magnetic sector analyzer 21, 36 Magnetic sector scanning 37 Magnetic sector 75, 78, 79, 80, 122 Magnetization transfer 147 Magnitude of kH/ko 287 Magnitude of transverse magnetization 144 Major fragments 42 Make-up gas flow 35 Malabsorption 370, 372 MALDI, 38 Mannitol 401 Manufacture 414

444 Maprotiline 247 Margin of safety 7 MAS NMR investigations of rhodopsin 208 MAS NMR 206, 212, 214, 215, 216 Mass analyzer 21, 32, 33, 34, 35, 36, 37, 38, 39, 75 Mass balance 124, 219, 232, 422 Mass balance/metabolite identification study 219, 417, 418, 421,427 Mass isotopomer patterns 8 Mass measurement 44, 104 Mass number 1 Mass-selection 74 Mass shifts 106 Mass spectra 43 Mass spectral identification and quantitation 128 Mass spectrometer (MS) 1, 19, 54, 58, 63, 64, 74, 75, 81, 86, 87, 221 Mass spectrometric detectors 127 Mass spectrometric fragmentation pattern 15 Mass spectrometry (MS) 20, 22, 29, 47, 65, 127, 128, 134, 139, 188, 219, 233, 352, 419 Mass spectrum 22, 29, 31, 36, 40, 41, 90, 348 Mass to charge ratio 19 Mass Trace 406 Mass-analysis 75 Mass-analyzed-ion-kinetic-energy spectrometry 85 Mass-analyzer 84s 87 Mass-analyzing 110 Mass-selected 86 Mass-selection 84 Mass-selective detection 127 Mass-to-charge (m/z) ratio 21, 74, 121 Matrix ions 58 Matrix-assisted laser desorption ionization (MALDI) 20, 32, 37, 54 Maturation of N-demethylation of caffeine 350 Maximum plasma concentration 198, 309 Maximum residence time 174

Maximum velocity 298 MB 49 MB interfaces 52 MB/MI 219, 220, 221,228, 229, 232 MB/MI study (14C) 420 ME 97 Mean steady state plasma drug concentration 343 Measurement of distribution half-life and volume of distribution 261 Measurement of drug plasma concentration 342 Measurement of drug plasma concentration method 345 Measurement of gastric emptying 367, 368 Measurement of rate of entry of drug into tissues 261 Mechanisms of drug metabolism 9 Medical imaging surgical and dental drug products division of the center for drug evaluation 415 Medical Library Electronic Reference Network (LERN) 412 Mefloquine 248 Membrane integrity 146 Membrane protein receptors 206 Membrane receptors 206 Mercury 1 Metabolic compartmentation 154 Metabolic disposition 42 Metabolic isotope effect 266, 310, 338, 426 Metabolic pathway 15, 134, 146, 187, 194, 303, 304 Metabolic process 193 Metabolism-free radicals 398 Metabolite 92, 132, 188, 190, 191,194 Metabolite detection 232 Metabolite formation 305 Metabolite identification 9, 59, 231, 235 Metal activity 138 Metaproterenol 248 Metarhodopsin I 205, 213 Metarhodopsin II 205 Metastable ion 82

445 Metastable ion decomposition 81 Methadone 256 Methane 30, 39, 176 Methanol 401 Methionine-dependent folate pathway 380 Method of Quimby and Sullivan 182 Methods to test for deep pool effect 266 Methotrexate (MTX) 394 Methoxsalen 248 3-methylcholanthrene 387 Methyl ester 43 Methylene chloride 325 a-methyltestosterone 248 Methyltryptoline (MTLN) 97 4-methylumbelliferyl glucuronide 34 Methyluric acids 189 Methylxanthine 189, 348 Metronidazole 160 Mg 2§ 147 MH § ion 90, 97, 99, 100, 101,102, 103, 104, 106, 107, 110, 111 MH + ions of butylated Phe and DsPhe 96 (M+H) + 63, 109 [M + H] § carboxylate anions 108 [M-HF] § ions 98 MHz range 143 Michaelis constant 298 Michaelis-Menten formulation 149 Microbore/nanobore LC 35 Micro-breath test 389, 397 Micro-HPLC-negative ion 59 Microsomal enzymes 375 Microsomial mono-oxygenases 188 Microwave-induced plasma 179, 230 MIKES 85 MIKES spectra 85 Mild ionization technique 30 Mitochondria 375 Mitochondrial/3-oxidation of fatty acids 159 Mitochondrial dysfunction 375 Mitochondrial inner membrane 94 Mixed triglyceride (1,3 distearyl 2113C] octanoyl glycerol) 370

Mixed triglyceride or cholesteryl octanoate breath test 370 MK-434 67 Mobile phase 28, 53 Molar volume 15 Molecular emission spectroscopy 174 Molecular ion 29 Molecular modeling 211 Molecular structure 41 Mollusks 203 Momentum separator 28, 52, 53, 54 Momentum-to-charge 78 Monoamine oxidase 322 Monodeuteromethyl isotopomers 187 Monohydroxy metabolites 92 Monohydroxy unsaturated fatty acids 108 Monoxide 407 Morphine 65 Moving belt 26 Moving belt (MB)interface 48 Moving-belt interface 25 MPTP 317 MS 20, 101,220, 426, 427 MS/MS 39, 65, 74, 77, 78, 79, 89, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 104, 108 MS/MS (collision activated decomposition) 37 MS/MS analyses 81, 82, 93 MS/MS analysis scheme for identifying drug metabolites 88 MS/MS instruments 88 MS/MS Phe/Tyr ratios 96 MS/MS scan methods 82 MS/MS scan modes 83 MS/MS scans 93 MS/MS spectrum 39 MS/MS/MS analyses 86 MS/MS/MS 110, 111, 112 MTLN 98 Mucosal absorption 371 Multi-collector mass spectrometer 119 Multicompartment metabolic models 156 Multiple ionization modes 69

446

Multiple sector 77 Multiple sector mass spectrometers 85 Multiple-charge 64 Multiple-dose patient study 417, 418, 420, 427 Multiple-dose volunteer study 417, 418, 420, 422, 427 Multiply-charged 64 Multiply-charged ions 69 Multiply-charged molecular ion 64 Multiply-labeled octanoate 159 Multi-step pathways 41 Myelin breakdown 146 Myo-inositol as a Glial Marker 146 14N/15N ratios 174, 175, 199 lSN NMR spectra 157 15N 13C2-acetaminophen 226 lSN 155 lSN2-PHT 130 15N-acetaminophen 222, 224, 225 lSN-enrichment 7 lSNitrogen 236 lSN-labeled 157 15N-nitroglycerin 251,253 15NO 123 N,N-diethyl-13C-benzamide 241 N7 methylation of theophylline 348 NaBD4 106 NaBH4/H20/CH3OH 106, 107 N-acetylaspartate 145 N-acetylprocainamide (NAPA) 246 NADPH 151 Nano-scale chromatography 57 Naproxen 160 National Institute of Standards and Technology (NIST) 409 Natural abundance 227 [lSN 13C]-carbamazepine 61 NCS-chrom 104, 105, 106, 108 NCS-chrom A 107 N-demethylation of caffeine 349 Nebulizer 28, 53 Necroinflammatory activity 374 Negative charge 31 Negative chemical ionization (NCI) 31

Negative ion chemical ionization (NCl) 55 Negative molecular anion 31 Negatively charged alkene 109 Neon 1 Net magnetization 143 Net spin angular momentum 142 Neurochemical detection 145 Neuroleptics 161 Neuronal and glial metabolism 156 Neuronal density 146 Neuronal-axonal marker 145 Neutral aldehyde fragment 109 Neutral loss 88, 92, 93 Neutral loss scan 82, 83, 87, 100 Neutral loss spectrum 88, 91 Neutral loss-precursor scans 87 Neutral particles 22 Neutrals (Ni) 81 [5-1SN] glutamine 157 Nicotine 248, 249 NIH shift 236 Nimodipine 309 Nitrobenzyl alcohol 113 Nitrodisc | 252, 253 Nitro-Dur II| 252, 253 Nitrogen isotope 174, 175 Nitroglycerin 250, 252, 253, 254 N-methylformamide 100 N-methylformamide (NMF) 239 N-methyI-N-(trideuteromethyl) aniline 279 NMF 102 NMF conjugates 100 NMR 143, 144, 206 NMR analysis 240 NMR measures 148 NMR phenomenon 142 NMR shifts 209 NMR spectra 154 NMR spectroscopy 141,144, 149, 154, 240 NMR-active isotopes 141 NMR-active nuclei 141 N,N-bis(dideuteriomethyl) aniline 279, 280 N,N-bis(dideuteriomethyl) aniline analogs 280

447 n-octanes 284 Nonalcoholic liver disease 376 Noncompliance 337 Nondissociative 294 Nondissociative kinetic mechanism 294 Nondissociative mechanism 293 Nonessential amino acid 8 Noninvasive methods 379 Noninvasive tests 363, 370, 394 Nonlinear and time-dependent pharmacokinetics 313 Nonlinear calibration curves 53, 54 Nonlinear pharmacokinetics 267, 270, 271,272, 343, 424 Non-linear response 69 Nonpolar compounds 19 Nonradioactive tracers 188 Nonrenal clearance 309 Nonreproducibility 136 Nonvolatile 23, 26, 44, 49 Nonvolatile buffers 27 Nonvolatile polar and ionic molecules 33 Nonvolatile/thermolabile compounds 123 Normal column HPLC 68 Normal (full-scan) MS 74 Normal phase 26 Nuclear spin 142 160 isotope 319 180 chromatograms 175 1802-labeled analogue 111 180-labeled internal standards 56 O-acetyl carboxypentafluorobenzyl ester 326 Occipital cortex 154 Octane-1,2,3-2H7 285 Octane-l-2H 3 285 Octanoic acid 362, 367 1-octanol 286 2-octanol 286 Octapole collision cell 76, 77 O-demethylation 278 Oleic acid 108 Oligonucleotides 24, 27, 34, 35, 38

Oligosaccharides 24, 27, 48, 56, 59, 62 3-O-methyldopa 319 Opioid peptides 96 Oppenheimer (pg) 183, 184 Oppenheimer's method 181, 182 Opsin + all-E-retinal 205 ORA gold disc databases 412 Oral route 197 Ordering from catalogs 405, 406 Organic molecules 170, 178 Orifice 52 Out-of-plane deformation 210 Oxene 293 Oxene-heme complex 288 Oxidation 369 Oxidative pathways 150 Oxidative stress 399 Oxygen consumption 152 Oxygen isotope 2 Oxygen 31, 55 Oxypentifylline 160 31p NMR 142, 147, 152, 163 31p spectrum 148 P450 catalyzed oxidative reactions 289 P4502B1 286, 288 P4502B4 288 P450b 286 P451A1 288 Palmitic acid 362, 372 Palmitic acid breath tests 371 Pancreatic carboxyl ester lipase 370 Pancreatic function tests 371 Pancreatic insufficiency 370 Pancreatic lipase 370 Pancreatic resection 370 Paracetamol 160 Para-ditrideuteromethoxybenzene 278 Para-hydroxylation 134 Parallel pathway mechanism 293, 294 Para-methoxyanisole 278 Para-trideuteromethoxyanisole 278 Parent drug 188 Parkinson disease 315, 316, 317, 318, 319, 320, 321,322, 331,333, 334

448 Particle beam (PB) 26, 48 Particle beam LC-MS 51 Particle beam LC-MS interface 51 Particle charging 53 Particle size 53 Particle-beam (PB)interface 28 Particles 52 Pattern of fragment ions 21 PB 49 PB interface 28, 48, 52, 53, 54, 55 PB-massive particle 54 Peak retention time (k! as a unit of time t) 136 Pediatric pharmacology 347, 351 Pediatrics 307 Pee Dee Belemnitella 384 Penicillins 160 Pentachlorophenol 42 Pentafluorobenzyl derivatives 56 Pentapeptide methionine enkephalin (ME) 96 Pentapeptides 97 Pentose phosphate cycle 151 Pentylfluroroaryl 238 Peptide 27, 28, 32, 35, 56, 59, 62, 64 Perferryloxy heme oxidizing species 276 Peripheral decarboxylation ,317 Peripheral dopa-decarboxylase 315 Peripheral levodopa metabolism 315, 316, 317, 318, 321,334 Peripheral levodopa toxicity 318 Pesticide 40 PET scanning 319 Pharmaceuticals 66 Pharmacokinetic applications 244 Pharmacokinetic parameters 198 Pharmacokinetic properties (linear, concentration-, or timedependent) 422 Pharmacokinetic studies 22, 351,353 Pharmacokinetics 20, 65, 98, 219 Pharmacologic 316 Phasel 419,427 Phase I human testing 417 Phase II studies 427 Phenacetin 405, 407

Phenethyl isothiocyanide 389 Phenobarbital 221,261,262, 263, 299, 300, 338, 340, 345, 387, 405, 407 Phenylacetic acid derivatives 43 Phenylalanine 4, 7, 95 Phenylbutazone 341,342 Phenylketonuria (PKU) 95, 96 Phenytoin 128, 130, 131,132, 134, 136, 139, 248, 261,262, 268, 269, 270, 271,301,302, 304, 305, 307, 312, 338, 339, 340, 345, 407, 425, 426 Phenytoin dihydrodiol 340 Phorbol myristate acetate 399 Phosphatase 256 Phosphate 27, 256 Phosphate metabolism 147 Phosphocreatine (PCr) 142, 147 Phosphodiesters 147 Phosphohexose isomerase 152 Phospholipids 59 Phosphomonoesters 147 Phosphoramide 101, 103, 104 Phosphorous 55 Phosphorylated compounds 142 Photochemical isomerization 214 Photodiode array 176, 179, 189 Photointermediates 214 Photoisomerization 205, 215 Photon 203 Photoreceptor 203 Photosynthesis 5 p-HPPH 302, 305 PHT/deutero-analogue pair 132 Pilocarpine 66 Pilot bioavailability/bioequivalency study 250 PKU 96 Placental transfer of drugs 353 Plasma 98, 99, 160, 175, 176 Plasma clearnace 309 Plasma levels 197 Plasma sources 178 Platinum 54 Pneumatic nebulization 35 Pneumatic nebulizer 53, 64 Polar and ionic 23 Polarity 15

449 Polycyclic aromatic hydrocarbons 55 Polymorphonuclear leukocytes (PMNL) 398 Polynucleotides 48, 62 Polypeptides 43 Polytherapy 98 Positive- or negative-ion 36 Positively charged ions 36 Post capillary 64 Post column 58 Post-column derivatization 138 Post-column flow-split ratios 35 Post-column jet separator 24 Post-mortem pituitaries 96 Post-natal age 350 Post-separation derivatization 63 Potassium cyanide 407 Potential toxicity 160 p-pydroxy-phenylphenylhydantoin 340 Precision 220, 221,228 Pre-column 58 Precursor 93 Precursor ion scan 82, 83, 86, 88, 91, 92 Precursor (or parent) ion 74 Prednisone 357 Primary isotope effect 286 Principal lines 2 Probe 57, 58 Procainamide 264, 266 Procurement of stable isotope-labeled pharmaceuticals 405 Pro-drug formulations 256, 424 Product inhibition 303, 306, 307 Product ion scans 82, 83, 84 Product ion spectrum 84 Product (or daughter)ions 75 Progesterone 200 Propoxyphene 256 Propranolol 256 Protein binding 15, 337 Protein metabolism 356 Proteins 24, 27, 32, 35, 38, 62, 64, 146 Protocol 412 Proton exchange 236 Protonated hexapeptide 111

Protonated MH + 74 Protonated Schiff base 208 (Pseudo)electroch romatog raphy 68 Pseudo-first-order unidirectional rate constants 148 Pseudomolecular ions 56 Pseudoracemate technique 235, 255, 256, 257, 308, 309, 341,342 Psychiatric disorders 96, 161 Pulmonary disease 375 Pulmonary excretion 371 Pyruvate carboxylase 155, 157 Pyruvate dehydrogenase 157 Pyruvate 5, 150, 152, 157 Q/E linked-scan 85 Q1 94, 96 QlqQ2 86, 88 QlqQ2 mass spectrometer 94, 96, 97, 99 Q2 89, 94, 96 QITMS 76 Quadrupole 38, 62, 77 Quadrupole ion-trap mass spectrometers (QITMS) 37, 75 Quadrupole mass filter 37, 75, 76, 79, 80, 84, 87 Quadrupole mass filter analyzer 21, 34, 37 Quadrupole mass spectrometer 122 Qualitative and quantitative determinations 169 Quantify tryptoline (TLN) 97 Quantitation of drug transformation 9 Quantitative analysis 22, 40, 53, 58, 66 Quantitative capabilities 61 Quantitative capabilities for CF/FAB 59 Quantitative MS/MS methods 99 Quantitative statement of composition 414 Quantum mechanics 171 Quasi-molecular ion 29, 30, 32, 33, 39, 40 Quasi-molecular ion [M + H] + 35 Quick urease testing 364

450 Quimby (pg/s)

183, 184

R-(+)-D6-propranolol 256 R-(+)-propranolol 255 Racemates 308 Radiation 231 Radiation-absorbing matrix 33 Radio frequency 37 Radioactive 219, 228, 229, 344, 419, 421,422 Radioactive (14C) 219 Radioactive flow detectors 236 Radioactive isotopes 236, 243, 356 Radioactive label 88, 229 Radioactive labeled levodopa 318 Radioactive tracer studies 341,342, 343 Radioenzymatic assay (REA) 94 Radio-frequency controllers 127 Radio-frequency (rf) 76, 144 Radioimmunoassay (RIA) 96 Radionucleotides 319 Radioreceptor assay (RRA) 96 Raman spectror 214 Ranitidine 388, 390 Rapid urease test (CLOtestTM) 365 Rate-limiting step 13, 14, 361,370 REA 95 Reaction mechanism 42 Reactive metabolites 388 5(x-reductase inhibitor 67 Regression lines parameters 197 Regulatory approval 417, 427 Regulatory aspects 411 Relative bioavailability 197, 245, 246, 247, 354, 423, 424 Relative peak separation 136 Relaxation 144, 210 Reliable instrumentation 220 Renal biopsy 264 Renal clearance 309 Renal impairment 309 Reproducible LC method 139 Reservoir inlet 19 Residual solvents 414 Resolution 104, 221 Resolution equation 135

Resonance Raman experiments 212 Respiratory carbon dioxide 5 Retention mechanisms 138 Retention time 135 Retinals 208 Reversed phase 26 Reversed phase chromatography 138 Reversed phase column 136 Reversed phase separations 138 Revision of investigational new drug application regulations (1987) 415 Revision of new drug application regulations (1985) 415 Rf-only quadrupole 77 Rf-only quadrupole collision cells 76, 80, 82 Rhodopsin 203, 205, 206, 211,212, 215 RIA 97 Rotational resonance 207 Rotational resonance dipolar recoupling 211 Routes of metabolism 337, 345 RRA 97 (R)-1,1,1 -tride ute ro-2phenylpropane 282

Safety 312, 344, 418, 421,424, 425, 427 Safety of stable isotope labeling 307, 310 Saturatable first pass metabolism 424 Scan rate 22 Scavengers 400 Schiff base 203, 206, 214, 215, 216 Scintillation counting 421 SDS/PAGE gel electrophoresis 65 Secondary isotope 13, 15 Second field-free region (FFR2) 78 Second linear pathway 306, 307 Second quadrupole mass filters 77 Secondary isotope effects 129, 286 Second-generation product ions 110 Selected ion monitoring (SIM) 74, 122 Selected ion monitoring mass spectrometry (SIM-MS) 119

451 Selected-reaction monitoring (SRM) 82, 83, 88 Selectivity (a) 135 Selectivity 184 Selenium 55 Sensitivity 74, 220, 221,228 Sensitivity and specificity of diagnostic tests 365 Sequence-specific fragment 110 Serology 363 Serotonin 158, 161 Serum 65, 220, 226, 324 Serum (deproteinized) 222 Serum (whole) 222 Serum concentration versus time relationships 16 Serum whole matrix 228, 229 Serum whole matrix diluted 228, 229 Shape selectivity 138 Shelf-life 414 Signal processor/data system 21, 22 Signal-averaging times 147 Signal-to-noise (S/N) ratio 74, 98 SIL labeling 220, 229, 415 SIL molecules 170 SIL tracer dose 299 Silica capillary 58 Single- and multiple-ion scanning 37 Single dose of drug 2 method 343 Single dose volunteer safety/pharmacokinetic study 229 Single labeled analogue 22 Single-dose experiment 352 Single-dose kinetics 355 Single-dose patient 417, 418 Single-dose volunteer and single-dose patient studies 418, 420, 421,427 Site-directed mutagenesis studies 208 Skin metabolism 248 Snapshot 181 Sodium acetate 407 Solid phase extraction 160 Solid-liquid extraction 55 Solids 19 Soliton 215 Solubilization 371 Solute 138

Solvent-mediated chemical ionization 25 Solvent-mediated CI, 26 [34S]omeprazaole 240 [34S]omeprazole 237 Sorbinil 152 Source temperature 22 Sources for stable isotope-labeled drugs 406 Spatial ligand structure 210 Spectral characteristics 69 Spin coupling 145 Spin-lattice relaxation time 161,144 Spiramycin 55 SRM 88, 93 SRM mass spectra 97 Stability protocol 414 Stable fragment ions 29 Stable isotope 1, 2, 7, 9, 14, 22, 42, 43, 47, 48, 50, 55, 59, 73, 74, 93, 98, 99, 100, 101, 112, 127, 128, 129, 170, 171,177, 180, 189, 193,201,229, 255, 257, 316, 319, 320, 379 Stable isotope administration technique 353 Stable isotope breath tests 363 Stable isotope labeled (SIL) tracer techniques 3, 93, 261,263, 265, 297, 337, 353 Stable isotope labeled derivatizing agent 234 Stable isotope labeled levodopa 321, 324, 330, 330 Stable isotope labeling 47, 347, 351, 356 Stable isotope methodology 5, 232, 233, 246 Stable isotope synthesis 257 Stable isotope technology 316, 333 Stable isotope utilization 244 Stable isotope-labeled DES internal standards 59 Stable isotopically labeled (SIL) 5, 169, 188, 379 Stable or labile compounds 19 Staggered stable isotope administration technique 262, 263, 264

452 Standard curve 93 Standard deviation 181 Standard fluorometric methods 95 Start-up costs 417 Stationary phase 138 Statistical power 345, 423, 424, 425 Steady state clearance 267, 268, 269, 270 Stearate 369 Stereoselective hydroxylation 281 Stereoselective metabolism 351 Stereoselective pharmacokinetic studies 255 Stereoselectivity 281 Steroids 59 Stopping drug administration 343 Storage and disposal of radioactive specimens 419 Storage conditions 414 Striatum 98, 317 (S)- 1,1,1-t rid eute ro-2phenylpropane 281,282 Structural analysis 29 Structure elucidation 31, 52, 100 Structure-independent detection 199 Study costs 420 Substrate saturation 305 Succinic 4 Sulfate 59, 238 Sulfonamide 88, 92 3"Sulfur 237 Sulfur 1, 55 Sulfur conjugated paracetamols 160 Superoxide ions 398 Suprofen 309 Surface wave plasma 179 Synchronously scan 87 Synthesis and formulation of SII drug 134, 417 Systemic homovanillic acid levels 328 Systemic levodopa levels 327 2S-[2H]-5,6-didehydrosparteine 295 2S-[2H]-sparteine 295

tl/2 297, 311,342, 343, 304, 312 tmax (h-l) 198 T2 relaxation time 144

Tamoxifen-ds 234 Tamoxifen-do 234 Tandem-in-space mass spectrometers 75, 76, 77 Tandem-in-time 75 Tandem-in-time ion-trapping mass spectrometers 86 Tandem mass spectra 81 Tandem mass spectrometry (MS/MS) 39, 40, 73, 74, 75, 128 Tandem scan methods 73 Target pharmacokinetic parameters 197 TCA cycle 152, 156, 157 TCA cycle flux 149, 152, 153 TCD 385 Telazol 324 Teratogenicity 357 Terminal carboxy oxygens 112 Terodiline 248 Terrestrial natural abundance of various stable isotopes 177 Tetradeuterium-labeled CBZ 352 Tetradeutero 129 Tetrahydrofuran 130 Theophylline 248, 348, 353, 407 Theoretical computations 221 Thermal conductivity detector 383 Thermal spray TS, 20 Thermal stability 54 Thermally unstable compounds 31 Thermoconductibility detector (TCD) 384 Thermolabile analytes 23 Thermolabile substances 32 Thermolability 44 Thermospray (TS) 25, 26, 27, 48, 60, 61 Thermospray interface (TS) 23, 27, 50 Thermospray ionization (TSI) 33, 37 Thermospray mass spectrometry 239 Thermospray spectrum 34 Thermospray vaporizer 53 Thermostability 44 THF 136 Thin layer chromatography 68 Thioglycerol 113

453 Three-dimensional displays 181 Three-sector mass spectrometers 79 Thymidine 108 Thyroid disease 372 Time (t) to reach a given serum concentration 304 Time for plasma peak 198 Time to maximum plasma concentration 309 Time to reach steady state plasma concentration 303, 305 Time-dependent pharmacokinetic properties 297, 298, 299, 301,312, 313, 344, 352, 423, 426 Time-of-flight 37 Time-of-flight mass spectrometers 38 Tin 1 Tissue metabolism 315 TLNs 97, 98 TMS derivatives 235 Tobramycin 264, 266 Torsional angles 212 Toxicity studies 5 Toxification 386 Tracer dose 9, 264, 266, 267, 268, 269, 270, 271,272, 273, 297, 299, 300, 301,303, 337, 338, 339, 341, 422, 425 Tracer dose area under the plasma concentration versus time curve (AUC) 337 Tracer dose elimination rate constant (k) 298 Transchain amino acids 375 Transdermal delivery systems 248, 251 Transdermal nicotine systems 249 Transderm-Nitro 10| 250 Transderm-Nitro | 252, 253 Transfer time 385 Transmission 144 Transport interfaces 51 Transport kinetic parameters 150 Trazodone 248 Trideuteromethylcaffei ne 183 Trifluoperazine 161 Trifluoroacetamido 238

Triglyceride breath tests 371 Triglycerides 372 Trimethylsilyl (TMS) 43, 238 Trimethylsilyl ester 43 Trioctanoin 362, 370, 371 Triolein 362, 371 Tripalmitin 372 Triple quadrupole mass spectrometer 76, 77 Tryptolines 97, 98 Tryptophan 56 TS 36, 38 TSI 33, 39 TSP interface 66 TSP LC-MS interface 60 TSP 50 Tungsten 54 Turnover of amino-acids 356 Two double-focussing instruments 80 Tylosin 55 Tyr-Gly-Gly-Phe-Met 96 Tyrosine (Tyr) 4, 95, 96 Tyrosi ne-g lyci n e-g lyci ne-phe nyla la ninemethionine 96 [U-13C] glucose 155 [U-13C]glutamate 155 Ultra-high field MAS NMR 210 Ultraviolet detection 421 Uncharged nonvolatile material 35 Uncompetitive inhibition 306 Unimolecular 82 United States Food and Drug Administration 417 Unlabeled caffeine 185, 195, 197 Unlabeled compound 200 Unlabeled internal standard 98 Unsaturated fatty acids 110 Uracil 188 Urea breath test 363 Urea 224, 407, 411,416 Urease 224 Uric acids 188 Urinary bile 59 Urinary excretion 188, 313, 337, 340, 341,345 Urine (diluted) 224, 225

454 Urine (urease treated) 224, 225 Urine and plasma extracts 94 Urine diluted with water 224 Urine whole matrix 226 Urine 100, 160, 189, 191,192, 219, 220, 222, 223, 224, 225 US Pharmacopoeia 409 USP 409 UV detection 426

Vinylic fragmentation 109 Visual signal transduction 206 Volatile analytes 28 Volatile buffer 27 Volatile electrolyte 33 Volatility 26, 44 Voltages 22 Volume of distribution (Vd) 262, 271, 272, 297, 300, 301,309, 311,337, 340, 341,344, 422

Vmax 298, 299, 303, 304, 422 1Nmax 307 Vacuum pumps 25 Vacuum system 22 Validation of analytic method 417 Valproic acid 99, 128, 134 Van der Walls forces 15 Vaporized analyte molecules 25 Vd 298, 311,312, 343, 344 Verapamil 246, 256 Vertebrate 203 Vestec universal interface 54 ~/-vinyl 159

Warfarin 309, 341,342 Washout of drug 266, 422 Wavelength 176 Weighted (1/X2) least square linear regression 224, 225, 226, 228, 229 Whole urine 222 Xanomeline 65 Xanthines 180 Xenon 1, 32 Xylose breath test Zonisamide

365, 366

88, 89, 90