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Analysis of dextrorphan, a metabolite of dextromethorphan, using gas chromatography with electron-capture detection
J Pharmacol Toxicol 41 (1999) 143–146
Analysis of dextrorphan, a metabolite of dextromethorphan, using gas chromatography with electron-capture detection Mahnaz Salsali, Ronald T. Coutts, Glen B. Baker* Neurochemical Research Unit, Department of Psychiatry, University of Alberta, 1E7.44 W Mackenzie Health Sciences Center, Edmonton, AB, T6G 2R7, Canada Received June 1, 1999; revised and accepted July 12, 1999
Abstract Dextromethorphan, a constituent of many over-the-counter cough syrups, is used as a probe drug for phenotyping subjects for their cytochrome P450 2D6 (CYP2D6) enzyme activity and for measuring CYP2D6 activity of preparations such as microsomes. In such studies, formation of the metabolite dextrorphan is used as indicator of the activity of this CYP enzyme. The present report describes an electroncapture gas chromatographic procedure developed for detection and quantification of dextrorphan in human liver microsomal preparations in vitro. After basification of the incubation mixture, dextrorphan was derivatized with pentafluorobenzoyl chloride under aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column, an electron capture detector, and a printer-integrator. Para-hydroxymephenytoin was carried through the procedure as internal standard. The procedure, which involves the derivatization of dextrorphan under aqueous conditions, is rapid and involves the use of the relatively economical procedure of electron-capture gas chromatography. The derivative is stable and possesses excellent chromatographic properties. © 2000 by Elsevier Science Inc. Keywords: Dextromethorphan; Dextrorphan; Electron-capture gas chromatography; Pentafluorobenzoyl chloride
1. Introduction Dextromethorphan (DM) is the (1)-isomer of the codeine analogue levorphanol; however, unlike the (2)-isomer, it has no analgesic or addictive properties and does not act through opioid receptors. The drug acts centrally to elevate the threshold for coughing (Reisine and Pasternak, 1996) and is a constituent of many cough syrups. The metabolism of DM is primarily by O-demethylation to dextrorphan, a reaction that is mediated primarily by the cytochrome P450 enzyme CYP2D6 (Schmid et al., 1985; Jacqz-Aigran et al., 1993; Von Moltke et al., 1998). Dextromethorphan is also metabolized to 3-methoxymorphinan and 3-hydroxymorphinan, but these appear to be minor pathways mediated by CYP2D6 and CYP3A3/4, respectively (Jacqz-Aigran et al., 1993). Because the CYP2D6 enzyme displays polymorphism, DM metabolism to dextrorphan has been used to phenotype subjects (Kupfer et al., 1984; Baumann and Jonzier-Perey, 1988; Guttendorf et al., 1988; Evans et al., 1989; Kiivet et al., 1993; Caslavska et al., 1994). Dextromethorphan has been similarly used in in vitro studies as a probe substrate for CYP2D6 (Kronbach et al., 1987; Kron* Corresponding author. GB Baker. Tel.: (780) 492-6591/7604. Fax: (780) 492-6841. e-mail: [email protected].
bach, 1991; Vielnascher et al., 1996; Rodrigues, 1996). In recent years, there has also been increased concern about metabolic drug-drug interactions involving DM, particularly when it is coadministered with drugs that inhibit CYP2D6 (Butler, 1994; Gaertner, 1994; Skop et al., 1994). DM is a relatively potent inhibitor of reuptake of serotonin in nerve terminals (Henderson and Fuller, 1992), and it is conceivable that its accumulation as a result of inhibition of its metabolism by CYP2D6 by coadministered CYP2D6 inhibitors, such as certain selective serotonin reuptake inhibitor (SSRI) antidepressants, could account for its adverse effects in such situations (Achamallah, 1992; Skop et al., 1994; Nierenberg and Semprebon, 1993). In an effort to study such interactions using human liver microsomes, we have developed a convenient assay for the DM metabolite dextrorphan. Several methods have been reported for the quantification or screening of dextrorphan in urine, plasma, and liver microsomes, including high-performance liquid chromatography (HPLC) (Vielnascher et al., 1996; Park et al., 1984; East and Dye, 1985; Chen et al., 1990; Lam and Rodriguez, 1993; Stavchansky et al., 1995; Mistry et al., 1998), gas chromatography (GC) with selective nitrogen detection (Kintz et al., 1989), capillary electrophoresis (Caslavska et al., 1994), and thin-layer chromatography (Guttendorf et al., 1988). In the present report, a procedure
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that combines rapidity, simplicity, and high sensitivity is described for the determination of dextrorphan in incubates of DM with human liver microsomes. The method permits extractive derivatization of dextrorphan under aqueous conditions and involves the use of the relatively economical procedure of electron-capture GC. 2. Materials and methods 2.1. Materials Dextrorphan, DM, and para-hydroxymephenytoin were obtained from Research Biochemicals International (Natick, MA, USA). Pentafluorobenzoyl chloride (PFBC) was purchased from Sigma-Aldrich Canada Ltd (Oakville, Ontario, Canada). All solvents were HPLC grade (99.9%) and were purchased from Fisher Scientific Ltd. (Nepean, ON, USA). Human liver microsomes were obtained from the International Institute for the Advancement of Medicine (Exton, PA, USA).
sauga, Ontario, Canada) was employed. The carrier gas was helium at a flow-rate of 2 mL/min, and the make-up gas at the detector was methane-argon (5:95) at a flow rate of 30 mL/min. The injection port and detector temperatures were 2508 and 3258C, respectively. The oven temperature was initially set at 1058C, maintained at that level for 0.5 min, and increased at a rate of 158C/min to a final temperature of 2958C, which was maintained for 10 min. All injections of samples were carried out using the splitless mode of injection with a purge off time of 0.5 min. A standard curve was prepared by subjecting a series of tubes containing a fixed amount of internal standard (1000 ng) and varying amounts of dextrorphan to the same analytical procedure in parallel with each assay run. 2.2.3. Gas chromatography-mass spectrometry Coupled GC-MS (electron-impact mode) was used to confirm the structures of the derivatives of dextrorphan and the
2.2. Methodology 2.2.1. Extraction and derivatization A 100-mL reaction mixture containing 50 mL of a solution of DM (final concentration of DM: 200 mM), 10-mL of a preparation of human liver microsomes (protein content 5 23 mg/mL), 25 mL of a b-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-generating system, and 15 mL of potassium phosphate buffer (100 mM, pH 5 7.4) was incubated at 378C for various periods of time. The NADPHgenerating system contained 1.3 mM NADP1, 3.3 mM glucose 6-phosphate, 0.8 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride, all in 100 mM potassium phosphate buffer (pH 7.4). At the end of incubation period, 1N perchloric acid (50 mL) was added to stop the reaction. After adding para-hydroxymephenytoin (internal standard), the incubation mixture (100 mL each) was basified by adding 2 mL of saturated sodium carbonate solution. A solution (3 mL) of ethyl acetate:acetonitrile:PFBC (9:1:0.02) was then added to each tube. The tubes were shaken vigorously for 10 min and centrifuged (1000 g) for 5 min. The top organic layers were transferred to another set of tubes and taken to dryness under a stream of nitrogen or in a SAVANT evaporator (Savant Instruments, Farmington, NY, USA). Each residue was taken up in 300 mL of toluene. After adding 450 mL of ammonium hydroxide (1N) to each sample, they were vortexed a few seconds and centrifuged briefly. The top layer was transferred to a microfuge tube and 1 mL was used for GC analysis. 2.2.2. Gas chromatography A Hewlett Packard (HP) 5880A gas chromatograph equipped with a 15-mCi 63Ni linear electron-capture detector and a narrow-bore fused silica capillary column (25 m 3 0.32 mm; 1.05 mm film of 5% phenylmethylsilicone as stationary phase) obtained from Hewlett Packard (Missis-
Fig. 1. (A) A GC trace of a derivatized extract of an incubation medium containing no DM. (B) A GC trace of a derivatized extract of an incubation medium containing human liver microsomes, DM, an NADPH-generating system, and 0.1 mM potassium phosphate buffer; the mixture was incubated for 30 min before extraction. (C) A GC trace of PFB derivatives of authentic standards of the internal standard, para-hydroxymephenytoin (a; retention time, 16.78 min), and dextrorphan (b; retention time, 17.82 min).
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internal standard. A HP MSD mass selective detector was linked to a HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA, USA). Operating conditions were as follows: ion source temperature, 2008C; interface temperature, 2958C; column pressure, 34.5 kPa; accelerating voltage, 2200 eV; ionization voltage, 70 eV; scan speed, 200 amu/sec; and dwell time, 200 msec. 3. Results and discussion The procedure is rapid and the derivative is stable and has excellent chromatographic properties (Fig. 1). The retention times of the PFB derivatives of dextrorphan and para-hydroxymephenytoin were 17.82 and 16.78 min, respectively. The standard curve of the PFB derivative of dextrorphan was linear from 10 to 2000 ng (r2 . 0.99 obtained routinely). The limit of detection was ,5 ng per 1 mL sample (, 17 pg “on column”). The mean recovery, determined using 100 ng of dextrorphan, was virtually quantitative. Intra-assay and inter-assay coefficients of variation, determined at 100 ng of dextrorphan, were 5.5% and 7.3%, respectively (N 5 6). Derivatized samples were shown to be stable for at least 48 hours, when stored at 2808C. The proposed electron-impact mass fragmentation of the PFB derivative of dextrorphan was consistent with the structure shown in Figure 2. The method involves extractive derivatization of dextrorphan with PFBC, followed by quantification by GC-ECD. Such extractive derivatization with PFBC under aqueous conditions has been shown in the past to be particularly use-
Fig. 3. Time-dependent production of dextrorphan (DXR) from dextromethorphan (200 mM), in an incubation medium containing human liver microsomes and an NADPH-generating system. Results are expressed as means 6 SEM (n 5 3).
ful for analysis of drugs and neurochemicals containing phenolic groups and/or primary or secondary amines (Nazarali et al., 1987a; Cristofoli et al., 1982; Nazarali et al., 1984, 1986; Nazarali et al., 1987b; Rao et al., 1987). In summary, a rapid, inexpensive procedure has been developed for extraction and quantification of dextrorphan formed from incubation of DM with human liver microsomes and an NADPH-generating system in drug metabolism studies. Figure 3 demonstrates the application of this method to the quantification of dextrorphan formed from DM in a time-course study of metabolite production using human liver microsomes. Although the method has been applied to quantify dextrorphan in microsomal experiments, it should be readily adaptable to other studies such as investigation of levels of this metabolite in tissues or body fluids. Acknowledgments Funding was provided by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research (grant and studentship) and a University of Alberta Ph.D. Recruitment Scholarship. The authors are grateful to Dr. D. F. LeGatt for conducting the mass spectral analysis and Ms. Gail Rauw for expert technical advice. References
Fig. 2. Proposed electron-impact mass fragmentation of the PFB derivative of dextrorphan. Values in parentheses are relative abundances. *Expulsion of hydrocarbon radicals from the molecular ion with structural rearrangements gives these fragments for which it is not possible to provide exact molecular structures.
Achamallah, N.S. (1992). Visual hallucination after combining fluoxetine and dextromethorphan. Am J Psychiatry 194, 1406. Baumann, P., & Jonzier-Perey, M. (1988). GC and GC-MS procedures for simultaneous phenotyping with dextromethorphan and mephenytoin. Clin Chim Acta 171, 211–222. Butler, K. (1994). Opinion: letters. Can Pharm J 127, 271. Caslavska, J., Hufschmid, E., Theurillat, R., Desiderio, C., Wolfisberg, H., & Thormann, W. (1994). Screening for hydroxylation and acetylation polymorphisms in man via simultaneous analysis of urinary metabo-
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lites of mephenytoin, dextromethorphan and caffeine by capillary electrophoretic procedures. J Chromatiogr B Biomed Appl 656, 219–231. Chen, Z.R., Somogyi, A.A., & Bochner, F. (1990). Simultaneous determination of dextromethorphan and three metabolites in plasma and urine using high-performance liquid chromatography with application to their disposition in man. Ther Drug Monit 12, 97–104. Cristofoli, W.A., Baker, G.B., Coutts, R.T., & Benderly, A. (1982). Analysis of a monofluorinated analogue of amphetamine in brain tissue using gas chromatography with electron-capture detection. Prog Neuropsychopharmacol Biol Psychiatry 6, 373. East, T., & Dye, D. (1985). Determination of dextromethorphan and metabolites in human plasma and urine by high-performance liquid chromatography with fluorescence detection. J Chromatogr 338, 99–112. Evans, W.E., Relling, M.V., Petros, W.P., Meyer, W.H., Mirro, J. Jr., & Crom, W.R. (1989). Dextromethorphan and caffeine as probes for simultaneous determination of debrisoquin-oxidation and N-acetylation phenotypes in children. Clin Pharmacol Ther 45, 568–573. Gaertner, R. (1994). Opinion: letters. Can Pharmaceut J 127, 271. Guttendorf, R.J., Wedlund, P.J., Blake, J., & Chang, S.L. (1988). Simplified phenotyping with dextromethorphan by thin-layer chromatography: application to clinical laboratory screening for deficiencies in oxidative drug metabolism. Ther Drug Monit 10, 490–498. Henderson, M.G., & Fuller, R.W. (1992). Dextromethorphan antagonizes the acute depletion of brain serotonin by p-chloroamphetamine and H75/12 in rats. Brain Res 594, 323-326. Jacqz-Aigran, E., Funck-Bretano, C., & Cresteil, T. (1993). CYP2D6 and CYP3A-dependent metabolism of dextromethorphan in humans. Pharmacogenetics 3, 197–204. Kiivet, R.A., Svensson, J.O., Bertilsson, L., & Sjoqvist, F. (1993). Polymorphism of debrisoquine and mephenytoin hydroxylation among Estonians. Pharmacol Toxicol 72, 113–115. Kintz, P., Mangin, P., Lugnier, A., & Chaumont, AJ. (1989). Determination of dextromethorphan and its major metabolite dextrorphan by gas chromatography. Ann Biol Clin (Paris) 47, 193–195. Kronbach, T. (1991). Bufuralol, dextromethorphan, and debrisoquine as prototype substrates for human P450 2D6. Methods Enzymol 206, 509– 517. Kronbach, T., Mathys, D., Gut, J., Catin, T., & Meyer, UA. (1987). Highperformance liquid chromatographic assays for bufuralol 11-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver. Anal Biochem 162, 24–32. Kupfer, A., Schmid, B., Preisig, R., & Pfaff, G. (1984). Dextromethorphan as a safe probe for debrisoquine hydroxylation polymorphism. Lancet 2, 517–518. Lam, Y.W., & Rodriguez, S.Y. (1993). High-performance liquid chromatography determination of dextromethorphan and dextrorphan for oxidation phenotyping by fluorescence and ultraviolet detection. Ther Drug Monit 15, 300–304. Mistry, B., Leslie, J., & Eddington, N.E. (1998). A sensitive assay of metoprolol and its major metabolite alpha-hydroxymetoprolol in human plasma and determination of dextromethorphan and its metabolite dextrorphan in urine with high performance liquid chromatography and fluorometric detection. J Pharm Biomed Anal 16, 1041–1049.
Nazarali, A.J., Baker, G.B., & Boisvert, D.P. (1986). Metabolism and disposition of N-(2-cyanoethyl)amphetamine (fenproporex) and amphetamine: study in the rat brain. J Chromatogr B Biomed Appl 380, 393–400. Nazarali, A.J., Baker, G.B., Coutts, R.T., & Greenshaw, A.J. (1987a). Para-hydroxytranylcypromine: presence in rat brain and heart following administration of tranylcypromine and an N-cyanoethyl analogue. Eur J Drug Metab Pharmacokinet 12, 207–214. Nazarali, A.J., Baker, G.B., Coutts, R.T., Pasutto, F.M., & Cristofoli, W.A. (1984). Simultaneous analysis of N-(2-cyanoethyl)amphetamine (fenproporex), amphetamine and para-hydroxyamphetamine in rat brain. Res Commun Subst Abuse 5, 317– 320. Nazarali, A.J., Baker, G.B., Coutts, R.T., Yeung, J.M., & Rao, T.S. (1987b). Rapid analysis of 2-phenylethylamine in tissues and body fluids utilizing pentafluorobenzoylation followed by electron-capture gas chromatography. Prog Neuropsychopharmacol Biol Psychiatry 11, 251–258. Nierenberg, D.W., & Semprebon, M. (1993). The central nervous system serotonin syndrome. Clin Pharmacol Ther 53, 84–88. Park, Y.H., Kullberg, M.P., & Hinsvark, O.N. (1984). Quantitative determination of dextromethorphan and three metabolites in urine by reverse-phase high-performance liquid chromatography. J Pharm Sci 73, 24–29. Rao, T.S., Baker, G.B., Coutts, R.T., Yeung, J.M., McIntosh, G.J.A., & Torok-Both, G.A. (1987). Analysis of the antidepressant phenelzine in brain tissue and urine using electron-capture gas chromatography. J Pharmacol Methods 17, 297–304. Reisine, T., & Pasternak, G. (1996). Opioid analgesics and antagonists. In J. Hardman, L. Limbird, P.B. Molinoff, R.W. Ruddon, & A. Goodman Gilman (Eds.), Goodman & Gilman’s The Pharmacological Basis of Therapeutics. (p. 551) New York: McGraw-Hill. Rodrigues, A.D. (1996). Measurement of human liver microsomal cytochrome P450 2D6 activity using [O-methyl-14C] dextromethorphan as substrate. Methods Enzymol 272, 186–195. Schmid, B., Bircher, J., Preisig, R., & Kupfer, A. (1985). Polymorphic dextromethorphan metabolism: co-segregation of oxidative O-demethylation with debrisoquin hydroxylation. Clin Pharmacol Ther 38, 618– 624. Skop, B.P., Finkelstein, J.A., Mareth, T.R., Magoon, M.R., & Brown, T.M. (1994). The serotonin syndrome associated with paroxetine, an overthe-counter cold remedy, and vascular disease. Am J Emerg Med 12, 642–644. Stavchansky, S., Demirbas, S., Reyderman, L., & Chai, CK. (1995). Simultaneous determination of dextrorphan and guaifenesin in human plasma by liquid chromatography with fluorescence detection. J Pharm Biomed Anal 13, 919–925. Vielnascher, E., Spatzenegger, M., Mayrhofer, A., Klinger, P., & Jager, W. (1996). Metabolism of dextromethorphan in human liver microsomes: a rapid HPLC assay to monitor cytochrome P450 2D6 activity. Pharmazie 51, 586–588. Von Moltke, L., Greenblatt, D.J., Grassi, J.M., Granda, B.W., Venkatakrishnan, K., Schmider, J., Harmatz, J.S. (1998). Multiple human cytochromes contribute to biotransformation of dextromethorphan invitro: role of CYP2C9, CYP2C19, CYP2D6, and CYP3A. J Pharm Pharmacol 50, 997–1004.
Analysis of dextrorphan, a metabolite of dextromethorphan, using gas chromatography with electron-capture detection Mahnaz Salsali, Ronald T. Coutts, Glen B. Baker* Neurochemical Research Unit, Department of Psychiatry, University of Alberta, 1E7.44 W Mackenzie Health Sciences Center, Edmonton, AB, T6G 2R7, Canada Received June 1, 1999; revised and accepted July 12, 1999
Abstract Dextromethorphan, a constituent of many over-the-counter cough syrups, is used as a probe drug for phenotyping subjects for their cytochrome P450 2D6 (CYP2D6) enzyme activity and for measuring CYP2D6 activity of preparations such as microsomes. In such studies, formation of the metabolite dextrorphan is used as indicator of the activity of this CYP enzyme. The present report describes an electroncapture gas chromatographic procedure developed for detection and quantification of dextrorphan in human liver microsomal preparations in vitro. After basification of the incubation mixture, dextrorphan was derivatized with pentafluorobenzoyl chloride under aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column, an electron capture detector, and a printer-integrator. Para-hydroxymephenytoin was carried through the procedure as internal standard. The procedure, which involves the derivatization of dextrorphan under aqueous conditions, is rapid and involves the use of the relatively economical procedure of electron-capture gas chromatography. The derivative is stable and possesses excellent chromatographic properties. © 2000 by Elsevier Science Inc. Keywords: Dextromethorphan; Dextrorphan; Electron-capture gas chromatography; Pentafluorobenzoyl chloride
1. Introduction Dextromethorphan (DM) is the (1)-isomer of the codeine analogue levorphanol; however, unlike the (2)-isomer, it has no analgesic or addictive properties and does not act through opioid receptors. The drug acts centrally to elevate the threshold for coughing (Reisine and Pasternak, 1996) and is a constituent of many cough syrups. The metabolism of DM is primarily by O-demethylation to dextrorphan, a reaction that is mediated primarily by the cytochrome P450 enzyme CYP2D6 (Schmid et al., 1985; Jacqz-Aigran et al., 1993; Von Moltke et al., 1998). Dextromethorphan is also metabolized to 3-methoxymorphinan and 3-hydroxymorphinan, but these appear to be minor pathways mediated by CYP2D6 and CYP3A3/4, respectively (Jacqz-Aigran et al., 1993). Because the CYP2D6 enzyme displays polymorphism, DM metabolism to dextrorphan has been used to phenotype subjects (Kupfer et al., 1984; Baumann and Jonzier-Perey, 1988; Guttendorf et al., 1988; Evans et al., 1989; Kiivet et al., 1993; Caslavska et al., 1994). Dextromethorphan has been similarly used in in vitro studies as a probe substrate for CYP2D6 (Kronbach et al., 1987; Kron* Corresponding author. GB Baker. Tel.: (780) 492-6591/7604. Fax: (780) 492-6841. e-mail: [email protected].
bach, 1991; Vielnascher et al., 1996; Rodrigues, 1996). In recent years, there has also been increased concern about metabolic drug-drug interactions involving DM, particularly when it is coadministered with drugs that inhibit CYP2D6 (Butler, 1994; Gaertner, 1994; Skop et al., 1994). DM is a relatively potent inhibitor of reuptake of serotonin in nerve terminals (Henderson and Fuller, 1992), and it is conceivable that its accumulation as a result of inhibition of its metabolism by CYP2D6 by coadministered CYP2D6 inhibitors, such as certain selective serotonin reuptake inhibitor (SSRI) antidepressants, could account for its adverse effects in such situations (Achamallah, 1992; Skop et al., 1994; Nierenberg and Semprebon, 1993). In an effort to study such interactions using human liver microsomes, we have developed a convenient assay for the DM metabolite dextrorphan. Several methods have been reported for the quantification or screening of dextrorphan in urine, plasma, and liver microsomes, including high-performance liquid chromatography (HPLC) (Vielnascher et al., 1996; Park et al., 1984; East and Dye, 1985; Chen et al., 1990; Lam and Rodriguez, 1993; Stavchansky et al., 1995; Mistry et al., 1998), gas chromatography (GC) with selective nitrogen detection (Kintz et al., 1989), capillary electrophoresis (Caslavska et al., 1994), and thin-layer chromatography (Guttendorf et al., 1988). In the present report, a procedure
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that combines rapidity, simplicity, and high sensitivity is described for the determination of dextrorphan in incubates of DM with human liver microsomes. The method permits extractive derivatization of dextrorphan under aqueous conditions and involves the use of the relatively economical procedure of electron-capture GC. 2. Materials and methods 2.1. Materials Dextrorphan, DM, and para-hydroxymephenytoin were obtained from Research Biochemicals International (Natick, MA, USA). Pentafluorobenzoyl chloride (PFBC) was purchased from Sigma-Aldrich Canada Ltd (Oakville, Ontario, Canada). All solvents were HPLC grade (99.9%) and were purchased from Fisher Scientific Ltd. (Nepean, ON, USA). Human liver microsomes were obtained from the International Institute for the Advancement of Medicine (Exton, PA, USA).
sauga, Ontario, Canada) was employed. The carrier gas was helium at a flow-rate of 2 mL/min, and the make-up gas at the detector was methane-argon (5:95) at a flow rate of 30 mL/min. The injection port and detector temperatures were 2508 and 3258C, respectively. The oven temperature was initially set at 1058C, maintained at that level for 0.5 min, and increased at a rate of 158C/min to a final temperature of 2958C, which was maintained for 10 min. All injections of samples were carried out using the splitless mode of injection with a purge off time of 0.5 min. A standard curve was prepared by subjecting a series of tubes containing a fixed amount of internal standard (1000 ng) and varying amounts of dextrorphan to the same analytical procedure in parallel with each assay run. 2.2.3. Gas chromatography-mass spectrometry Coupled GC-MS (electron-impact mode) was used to confirm the structures of the derivatives of dextrorphan and the
2.2. Methodology 2.2.1. Extraction and derivatization A 100-mL reaction mixture containing 50 mL of a solution of DM (final concentration of DM: 200 mM), 10-mL of a preparation of human liver microsomes (protein content 5 23 mg/mL), 25 mL of a b-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-generating system, and 15 mL of potassium phosphate buffer (100 mM, pH 5 7.4) was incubated at 378C for various periods of time. The NADPHgenerating system contained 1.3 mM NADP1, 3.3 mM glucose 6-phosphate, 0.8 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM magnesium chloride, all in 100 mM potassium phosphate buffer (pH 7.4). At the end of incubation period, 1N perchloric acid (50 mL) was added to stop the reaction. After adding para-hydroxymephenytoin (internal standard), the incubation mixture (100 mL each) was basified by adding 2 mL of saturated sodium carbonate solution. A solution (3 mL) of ethyl acetate:acetonitrile:PFBC (9:1:0.02) was then added to each tube. The tubes were shaken vigorously for 10 min and centrifuged (1000 g) for 5 min. The top organic layers were transferred to another set of tubes and taken to dryness under a stream of nitrogen or in a SAVANT evaporator (Savant Instruments, Farmington, NY, USA). Each residue was taken up in 300 mL of toluene. After adding 450 mL of ammonium hydroxide (1N) to each sample, they were vortexed a few seconds and centrifuged briefly. The top layer was transferred to a microfuge tube and 1 mL was used for GC analysis. 2.2.2. Gas chromatography A Hewlett Packard (HP) 5880A gas chromatograph equipped with a 15-mCi 63Ni linear electron-capture detector and a narrow-bore fused silica capillary column (25 m 3 0.32 mm; 1.05 mm film of 5% phenylmethylsilicone as stationary phase) obtained from Hewlett Packard (Missis-
Fig. 1. (A) A GC trace of a derivatized extract of an incubation medium containing no DM. (B) A GC trace of a derivatized extract of an incubation medium containing human liver microsomes, DM, an NADPH-generating system, and 0.1 mM potassium phosphate buffer; the mixture was incubated for 30 min before extraction. (C) A GC trace of PFB derivatives of authentic standards of the internal standard, para-hydroxymephenytoin (a; retention time, 16.78 min), and dextrorphan (b; retention time, 17.82 min).
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internal standard. A HP MSD mass selective detector was linked to a HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA, USA). Operating conditions were as follows: ion source temperature, 2008C; interface temperature, 2958C; column pressure, 34.5 kPa; accelerating voltage, 2200 eV; ionization voltage, 70 eV; scan speed, 200 amu/sec; and dwell time, 200 msec. 3. Results and discussion The procedure is rapid and the derivative is stable and has excellent chromatographic properties (Fig. 1). The retention times of the PFB derivatives of dextrorphan and para-hydroxymephenytoin were 17.82 and 16.78 min, respectively. The standard curve of the PFB derivative of dextrorphan was linear from 10 to 2000 ng (r2 . 0.99 obtained routinely). The limit of detection was ,5 ng per 1 mL sample (, 17 pg “on column”). The mean recovery, determined using 100 ng of dextrorphan, was virtually quantitative. Intra-assay and inter-assay coefficients of variation, determined at 100 ng of dextrorphan, were 5.5% and 7.3%, respectively (N 5 6). Derivatized samples were shown to be stable for at least 48 hours, when stored at 2808C. The proposed electron-impact mass fragmentation of the PFB derivative of dextrorphan was consistent with the structure shown in Figure 2. The method involves extractive derivatization of dextrorphan with PFBC, followed by quantification by GC-ECD. Such extractive derivatization with PFBC under aqueous conditions has been shown in the past to be particularly use-
Fig. 3. Time-dependent production of dextrorphan (DXR) from dextromethorphan (200 mM), in an incubation medium containing human liver microsomes and an NADPH-generating system. Results are expressed as means 6 SEM (n 5 3).
ful for analysis of drugs and neurochemicals containing phenolic groups and/or primary or secondary amines (Nazarali et al., 1987a; Cristofoli et al., 1982; Nazarali et al., 1984, 1986; Nazarali et al., 1987b; Rao et al., 1987). In summary, a rapid, inexpensive procedure has been developed for extraction and quantification of dextrorphan formed from incubation of DM with human liver microsomes and an NADPH-generating system in drug metabolism studies. Figure 3 demonstrates the application of this method to the quantification of dextrorphan formed from DM in a time-course study of metabolite production using human liver microsomes. Although the method has been applied to quantify dextrorphan in microsomal experiments, it should be readily adaptable to other studies such as investigation of levels of this metabolite in tissues or body fluids. Acknowledgments Funding was provided by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research (grant and studentship) and a University of Alberta Ph.D. Recruitment Scholarship. The authors are grateful to Dr. D. F. LeGatt for conducting the mass spectral analysis and Ms. Gail Rauw for expert technical advice. References
Fig. 2. Proposed electron-impact mass fragmentation of the PFB derivative of dextrorphan. Values in parentheses are relative abundances. *Expulsion of hydrocarbon radicals from the molecular ion with structural rearrangements gives these fragments for which it is not possible to provide exact molecular structures.
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