Anal. Chem. 1989, 61, 441-446 (23) de Wit, J. S. M.; Parker, C. E.; Tomer, K. Chem. 1987, 59, 2400-2404.
(14) St. Claire. R. L., 111 Ph.D. Thesis, University of North Carolina at Chapel Hill, 1986. (15) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. AMI. Chem. 1984, 56, 479-482. (16) St. Claire, R. L.; Jorgenson, J. W. J . Chromatogr. Scl. 1985, 23, 186-191. (17) White. J. 0.; St. Cklre, R. L.; Jorgenson, J. W. Anal. Chem. 1988, 58, 293-296. (18) White, J. 0.;Jorgenson, J. W. AMI. Chem. 1986, 58, 2992-2995. (19) Saaverda, J. M.; Brownsteln, M. J.; Carpenter, D. 0.;Axelrod, J. Scl1974, 185, 364-365. (20) Brownsteln, M. J.; Saaverdra, J. M.; Axelrod, J.; Zeman, G. H.; Carpenter, D. 0. Roc. Meti. Acad. Scl. U . S . A . 1974, 7 1 , 4662-4665. (21) Kerkut, G. A.; Sedden, G. B.; Walker, R. J. Comp. Biochem. Physiol. 1967, 23, 159-162. (22) Emson, P. C.; Fonnum, F. J . Neurochem. 1974, 22, 1079-1088.
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RECEIVED for review July 12,1988. Accepted November 23, 1988. This work was supported by the donors to the Petroleum Research Fund, administered by the American Chemical Society, and the University Research Council of the University of North Carolina. R.T.K. received support from a North Carolina Governor's Board of Science and Technology Fellowship and from an American Chemical Society Analytical Fellowship sponsored by the Society for Analytical Chemistry of Pittsburgh.
Liquid Chromatographic Determination of Cyclophosphamide Enantiomers in Plasma by Precolumn Chiral Derivatization Joel M. Reid,' John F. Stobaugh,* and Larry A. Sternson2 The Department of Pharmaceutical Chemistry and the Center for Bioanalytical Research, The University of Kansas, Malott Hall, Lawrence, Kansas 66045
On the basis of reactlons described In the synthetic literature, a two-step chiral derlvatlzation sequence was developed for the antlcancer agent cyciophosphamlde (CP), The sequence Involves amidoalkylation of CP with anhydrous chloral containing 1 % dimethylformamlde followed by acylation of the resultlng secondary alcohol with a chiral carboxyllc acid chiorlde, (+)-6methoxy-a-methyl-2-naphthaleneacetyl chlorlde, to form a dlastereomerlc palr. Derlvatlred (-)-CP and (+)-CP exhlblted retention times of 17.2 and 20.7 mln, respectively, when chromatographed on Hypersil ODS with acetonltrlle/phosphate buffer (pH 6.8) as the mobile phase. Preparatlon of the indivlduai dlastereomers from enantlomerically pure CP enabled correlation of the chromatographlcally observed peaks with a partlcular enantiomer. Various aspects of the overall assay methodology have been systematlcally lnvestlgated (derlvatlzatlon solvents, temperatures, reactlon t h e , and work-up procedures) and optimized on the scale required for trace analysls In biological fluids. Calibration curves were establlshed for each enantlomer in spiked human plasma over the therapeutically relevant concentration range of 0.99-49.94 pg/mL.
INTRODUCTION Cyclophosphamide (CP, l), originally synthesized as a prodrug of nor-nitrogen mustard (I), is a widely used agent in cancer chemotherapy (2). CP requires an initial enzymatically mediated hydroxylation reaction (2) followed by a series of chemical transformations to produce the alkylating agent phosphoramide mustard, generally regarded as the species responsible for biological activity (3). Due to the presence of an asymmetrically substituted phosphorus atom, Present address: Mayo Clinic and Foundation, Division of Developmental Oncology Research, Department of Oncology, Rochesteri MN 55905. Present address: Eastman Pharmaceuticals, Product Development, 9 Great Valley Parkway, Great Valley, PA 19355. 0003-2700/89/0361-0441$01.50/0
CP exhibits optical isomerism. Previous investigations have shown the (+)-1 and (-)-1 enantiomers to possess R and S absolute configurations, respectively (4,5).Since introduction into clinical usage approximately 30 years ago, the drug has been utilized clinically as the racemic mixture. Numerous examples exist, in a variety of pharmacological categories, in which one stereoisomer of an enantiomeric pair possesses a superior therapeutic index compared with the other stereoisomer (6). Since CP requires enzymatic activation, the possibility exists for stereoselective metabolism and perhaps a distinct therapeutic advantage for one enantiomer relative to its optical antipode. Indeed, early testing in two murine tumor models, ADJ/PCGA plasma cell tumor and L1210 leukemia, revealed that (-)-CP exhibited a substantially greater therapeutic index compared to (+)-CP (7). Additionally, the initial enzymatically mediated hydroxylation step has been characterized by in vitro metabolism experiments with sevral animal species. Interestingly, with mice and rats the enantiomers were metabolized at essentially the same rate; however, the disposition of (-)-CP was substantially faster in rabbits (8). Only limited clinical studies addressing the issue of stereoselective metabolism have been reported for humans. In the most recent study, each enantiomer and the racemic mixture were sequentially administered to four patients by intravenous bolus injection (6).Stereoselectivity was determined by recovering the intact drug and two of the metabolites, 4-ketocyclophosphamide and carboxycyclophosphamide, from the urine of these patients. With this limited patient population, CP recovered from the urine revealed no enrichment of either enantiomer when administered as the racemate, but a statistically significant stereoselectivity was observed for the formation of 4-ketocyclophosphamide when (+)-CP was administered as compared to racemic CP. While these results are not suggestive that an enhanced therapeutic index will be realized by administration of CP as a single enantiomer, the aforementioned study was of a very limited nature involving only four patients in which significant interpatient variation of CP metabolism was observed. Ad@ 1989 American Chemical Society
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ditionally, CP is frequently administered as an oral dosage form to ambulatory patients and reports concerning the influence of route of administration on its disposition have not appeared. Thus, additional clinical trials of an expanded nature appeared to be warranted in order to define any relationship between stereoselective metabolism and efficacy for CP enantiomers. Unfortunately, to date, such studies have been substantially hindered by the lack of suitable analytical methodology. In the past, the stereoselective nature of C P metabolism was determined by recovering the intact drug from urine and obtaining 31PNMR spectra in the presence of a chiral shift reagent (9). While this approach provides the required selectivity, it is not suitable to support a large scale clinical investigation because of the need to isolate milligram quantities of CP and the limitation of only monitoring CP excreted in urine rather than directly determining the plasma levels of the individual enantiomers. Ideally, to support such a clinical investigation, a chromatographic based method should be available that allows for the determination of each enantiomer in plasma. Examples in which liquid chromatography has been used to achieve enantiomeric separations abound in the literature and have been the subject of several recent reviews and monographs (10-13). In general, enantiomeric separations are performed by utilizing chiral stationary phases and chiral mobile phase additives or via precolumn derivatization with chiral reagents to form diastereomeric products. Determination of CP enantiomers in a biological sample is an especially difficult problem as the physicochemical properties of the drug do not provide for the type of chiral interactions normally found in successful resolutions and the chemical reaction handles most commonly reported in chiral derivatizations are not available. In addition, detection at the required sensitivity can only be accomplished by absorbance measurement at 5210 nm, a region where numerous interferences are commonly encountered in biological fluid analyses. Presently, we wish to report the results of our research in which a two-step amidoalkylation/acylation sequence, which results in the formation of a diastereomeric pair, has been optimized and used in conjunction with liquid chromatography for the determination of C P enantiomers in plasma.
EXPERIMENTAL SECTION Apparatus. The chromatqraphic system consisted of an Altex Model llOA pump, Model 210 sample injection valve fitted with a 20-pL loop, and a Kratoe Model 783 variable-wavelength UV-vis detector. The high-performance liquid chromatography (HPLC) columns (15 cm X 4.6 mm i.d.; 3 and 5 pm particles) were packed with Hypersil ODS using an upward slurry technique (17). The smaller particle column was used in the determination of the enantiomers in plasma and the larger particle column was used for all other experiments. Chromatograms were obtained on a Houston Instruments Omniscribe strip chart recorder with quantitation accomplished by manual peak height measurements. The UV spectra were obtained by using a Perkin-Elmer 555 UV-visible spectrophotometer operated at ambient temperature. High-resolution 'H (300 MHz) NMR spectra were obtained on a Varian XL 300 high-resolution spectrometer. Routine mass spectra (MS) were obtained on a Nermag R-10-10 quadrupole mass spectrometer and high-resolution mass spectra (HRMS) were acquired on a VG Analytical ZAB magnetic deflection instrument. Melting points (uncorrected)were obtained on an Electrothermal melting point apparatus. Chemicals and Solvents. Cyclophosphamide monohydrate, (+)-6-methoxy-a-methyl-2-naphthaleneacetic acid [ (+)-3b-X, X = -OH (Scheme I)], 1-ethyl-3-[3-(dimethylamino)propy1]carbodiimide hydrochloride (EDCI), (N,N-dimethy1amino)pyridine (DMAP), and oxalylchloride were obtained from Aldrich. Anhydrous cyclophosphamide was prepared by exposing the monohydrate to anhydrous phosphorus pentaoxide in a sealed desiccator overnight (14). The optical isomers of cyclophosphamide
were a gift from INTERX Research Corp. Anhydrous Gold Label dichloromethane was dried by passage over activity I neutral alumina (Woelm). Solid-phase extractions (SPE) were performed on 3-mL, 500-mg disposable units (J. T. Baker). Column chromatography and TLC were accomplished on Silica Gel 60 (E. Merck). Reagent grade benzene was dried over anhydrous calcium chloride and redistilled prior to use. Water used throughout was deionized in mixed-bed ion-exchange columns followed by distillation from an all-glass still. All other chemicals were of the highest purity available and used as received. Reagent and Synthetic Preparations. Anhydrous Chloral. Chloral hydrate (25 g) was shaken with 15 mL of concentrated sulfuric acid in a Teflon-lined screw capped culture tube until the crystals disappeared. After centrifugation to aid in separation of the layers, the sequence was repeated. The upper chloral layer was recovered,transferred to a 50-mL round-bottomed flask, and distilled under Nz with a short-path distillation apparatus. The forerun, bp 2 as a function of reaction time determined on day l (0)and day 67 (0) when the amidoalkylation was conducted with the same batch of anhydrous chloral containing 1 % DMF which had been stored at -70 OC. The reaction was conducted at 30 OC.
occur with complete randomness a t the newly created asymmetric center, thus forming a diastereomeric pair from each enantiomer. Obviously, if the first result were obtained, enantiomeric (*)-2 would not be separable by conventional chromatography but would provide a handle for acylation with a chiral carboxylic acid to form diastereomers. If the second result occurred, diastereomeric (*)-2 should be amenable to separation by conventional chromatography, but determination of the CP enantiomeric composition at this stage would not be possible. This results because (R)-and (S)-CP would each react with chloral to form diastereomeric pairs, e.g., (R)-CP forms (R,R)-2and (R,S)-2while (S)-CP forms (S,S)-2 and (S,R)-2(the second configurational assignment refers to the newly created asymmetric center), which are themselves enantiomerically related. When &)-2 was synthesized on the preparative scale from ( i ) - l ,(+)-l,and (-1-1, the products obtained exhibited identical retention values when characterized by normal phase silica-gel or reversed-phase bondedsilica chromatography. These results are suggestive that the reaction of CP with chloral proceeds in a highly stereospecific fashion. Alternatively, if formed, diastereomeric (*)-2 may not be separable by the chromatographic methods investigated. Experiments are currently in progress which should allow for the unambiguous characterization of this reaction. Selection of the Acylation Reagent. Previously, Bodor et al. (16) have reported the acylation of racemic 2 with (+)-3a-X (X = -OH, Scheme I) to form diastereomerically related esters that were chromatographically separable. For the present application, this carboxylic acid was deemed unsuitable due to the relatively weak molar extinction coefficient 7000). Also, the observed, even at lower wavelengths corresponding carboxylic acid chloride is a viscous oil at room temperature, making handling and purification difficult, which is of substantial concern due to a report that racemization may occur during distillation (23). However, the chiral carboxylic acid shown in Scheme I, (+)-6-methoxy-a-methyl-2naphthaleneacetic acid [(+)-3b-X, X = -OH, €232 700001, is commercially available in the enantiomerically pure form and the corresponding carboxylic acid chloride is a solid amenable to purification by recrystallization. Therefore, based on these aspects, the acid chloride form of (+)-3b was selected for evaluation as a chiral reagent. Optimization of the Acylation Reaction. The use of (+)-3b-X (acid chloride form) as a chiral derivatization reagent in the proposed sequence is contingent on the formation of chromatographically separable diastereomers. On the preparative scale, using a carbodiimide coupling agent, (*)-2, (-)-2, and (+)-2 were reacted with (+)-3b (free acid) to form (f)(+)-4b, (-)-(+)-4b, and (+)-(+)-4b,respectively. These diastereomers were easily separable by normal-phase chromatography on silica gel or by reversed-phase chromatography
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Flgure 3. Chromatograms of the diastereomeric products resulting from the derivatiration of (f)-1 extracted from human plasma. The R and S labels refer to the configurationof each enantiomer according to conventional stereochemicalnomenclature and correspond to the products (+)-(+)-4b and (-)-(+)-4b, respectively. Chromatogram l a was a blank while l b was from the determination of plasma spiked with (*)-1 at a concentration of 1.0 pg/mL with respect to each enantiomer.
on bonded-phase silica with acetonitrile/water mobile phases. Synthesis of (-)-(+)-4b and (+)-(+)-4bfrom (-)-1 and (+)-l, respectively, allowed for the correlation of a chromatographically observed peak with a particular enantiomer. To enable investigation of the acylation step in a more complicated system, CP was spiked into water and recovered by extraction with ethyl acetate, and the amidoalkylation reaction was conducted. After completion of the f i s t reaction, excess chloral, ( f ) - 2 and several unknown substances were present in the reaction mixture. The acylation reaction would not proceed in this environment, thus necessitating the development of the following cleanup methodology. After completion of the amidoalkylation step, the reaction mixture was diluted with dichloromethane and the organic layer was recovered, washed with sodium carbonate solution, and loaded onto a solid phase extraction (SPE) cartridge. The cartridge was washed, with no loss of (f)-2, with dichloromethane (10 mL), dichloromethane/acetone (9/1,6 mL) and dichloromethane/methanol (49/1,2 mL). Elution of (f)-2 was accomplished with a n additional quantity of dichloromethane/methanol (49/1,3mL). After solvent evaporation, the resulting residue was dissolved in purified anhydrous dichloromethane and the (f)-2 acylated with (+)-3b-X(X = -C1, Scheme I). In the presence of the nucleophilic catalyst 4-(dimethy1amino)pyridineformation of (f)-(+)-4bwas complete within 20 min at ambient temperature. This procedure was also suitable for the determination of CP initially present in plasma. Determination of CP Enantiomers in Plasma. The methodology established for the determination of CP enantiomers in aqueous solution was found to be applicable to these determinations in spiked plasma samples, as interference-free chromatograms exhibiting good resolution between the two diastereomers resulted (Figure 3). A calibration curve, based on chromatographic peak height values versus enantiomer concentration, was established over the concentration range of 0.99-49.94 pg/mL by using the average values of duplicate
determinations at four different concentrations, with the duplicate determinations varying by
