Stereospecific Derivatization of Amphetamines, Phenol Alkylamines

The enantiomers of amphetamines, phenol alkylamines, and hydroxyamines are separated by using r-methoxy-r-. (trifluoromethyl)phenylacetyl chloride as ...
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Anal. Chem. 1996, 68, 3015-3020

Stereospecific Derivatization of Amphetamines, Phenol Alkylamines, and Hydroxyamines and Quantification of the Enantiomers by Capillary GC/MS Ho-Sang Shin*,† and Manfred Donike‡

Korea Water Works Institute, 86-3, Yangpyung-dong, Youngdungpo-gu, Seoul, Korea, and Institut fuer Biochemie, Carl-Diem-Weg 6, Koeln 41, Germany

The enantiomers of amphetamines, phenol alkylamines, and hydroxyamines are separated by using r-methoxy-r(trifluoromethyl)phenylacetyl chloride as the chiral derivatizing agent for amino groups. Prior to N-acylation, amine salts are converted into the free bases and hydroxyl groups into O-silyl ethers by reaction with N-methyl-Nsilylamides. N-Methyl-N-(trimethylsilyl)trifluoroacetamide, N-methyl-N-(triethylsilyl)trifluoroacetamide, or Nmethyl-N-(tert-butyldimethylsilyl)trifluoroacetamide was used to protect the hydroxyl groups by TMS, TES, or the tBDMS groups. All these N-methyl-N-silylamides were able to convert amino salts to the free bases. The reaction is selective and rapid, and the diastereomeric derivatives are well separated by capillary gas-liquid chromatography. This procedure is suitable for simultaneous determination by gas chromatography/mass spectrometry with selected-ion monitoring and is also applicable to quantification of the compounds in a biological matrix. Amphetamine, methamphetamine, norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, p-hydroxyamphetamine, pholedrine, p-hydroxynorephedrine, and p-hydroxyephedrine are sympathomimetic agents.1 Each of these compounds exists as a pair of enantiomers (Figure 1), and these enantiomers, like many other drugs possessing chiral centers, have different pharmacological activities. For example, the (S)-(+)-amphetamine has apparently greater phamacological activity as a stimulant2 and hyperthermic agent3 than the (R)-(-)-enantiomer. The central stimulant effect of (1S,2R)-(+)-ephedrine amounts to 80% of the activity of (1R,2S)-(-)-ephedrine.4 Enantiomers of drugs may also differ in the rate of their metabolism. For example, the stereoselective metabolism of amphetamine,5-7 methamphetamine,8,9 and ephedrine10,11 has been described in the literature. Increased †

Korea Water Works Institute. Institut fuer Biochemie. (1) Gilman, A. G.; Goodman, L. S.; Rall, T. W.; Murad, F. The Pharmacological Basis of Therapeutics, 7th ed.; Macmillan: New York, 1985; p 149. (2) Snyder, S. H.; Tayler, K. M. Science 1970, 168, 1487. (3) Hajos, G. T.; Garattin, S. J. Pharm. Pharmacol. 1973, 25, 418. (4) Jenner, P.; Testa, B. Drug Metab. Rev. 1973, 2 (2), 117. (5) Wright, J.; Cho, A. K.; Gal, J. Xenobiotica 1977, 7 (5), 257. (6) Gal, J. J. Pharm. Sci. 1977, 66 (2), 169. (7) Matin, S. B.; Wan, S. H.; Knight, J. B. Biomed. Mass Spectrom. 1977, 4 (2), 118. (8) Morgan, C. D.; Cattaben, F.; Costa, E. J. Pharm. Exp. Ther. 1972, 180, 127. (9) Beckett, A. H. Xenobiotica 1971, 1, 365. (10) Axelrod, J. J. Biol. Chem. 1955, 214, 753. ‡

S0003-2700(96)00365-4 CCC: $12.00

© 1996 American Chemical Society

Figure 1. Chemical structure and absolute configuration of the compounds studied.

interest in stereochemical aspects of pharmacological activity and drug disposition has led to the need for development of new sensitive and specific methods for the detection of enantiomers in biological fluids. A number of procedures have been reported for the determination of enantiomers of these compounds, including highperformance liquid chromatography (HPLC)12-14 and gas chromatography (GC). Separation of the enantiomers by GC has been achieved either by using an optical active stationary phase15-18 or (11) Axelrod, J. J. Pharm. Exp. Ther. 1955, 114, 430. (12) Gal, J. J. Chromatogr. 1984, 307, 220. (13) Nimura, N.; Kasahara, Y.; Kinoshita, T. J. Chromatogr. 1981, 213, 327. (14) Miller, K. J.; Gal, J.; Ames, M. M. J. Chromatogr. 1984, 307, 335. (15) Frank, H.; Nicholson, G. J.; Bayer, E. J. Chromatogr. 1978, 146, 167. (16) Saeed, J.; Sandra, P.; Verzele, M. J. Chromatogr. 1979, 86, 611. (17) Saeed, J.; Sandra, P.; Verzele, M. J. High Resolut. Chromatogr. Chromatogr. Commun. 1980, 3 (1), 35. (18) Koenig, W. A.; Mischnick-Luebecke, P.; Brassat, B.; Lutz, S.; Wenz, G. Carbohydr. Res. 1988, 183, 11.

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by reacting the enantiomers with a suitable asymmetric reagent.6,7,19 The published methods for quantification of enantiomers by GC are not adequate for the determination of trace amounts of enantiomers containing more than one reactive functional group, e.g., hydroxymaines, and especially for the simultaneous quantification of metabolites with similar structures, because of their insufficient resolution and sensitivity or the temperature limitations of certain columns. Donike20-23 has described the selective N-trifluoroacylation-O-trimethylsilylation of phenol alkylamines and hydroxyamines as well as their corresponding hydrochlorides. This paper describes the selective N-acylation of a mixture of amphetamines, phenol alkylamines, and hydroxyamines with chiral reagents. EXPERIMENTAL SECTION Chemicals and Reagents. The (+)- and (-)-enantiomers of amphetamine sulfate, (+)-methamphetamine, and (-)-norpseudoephedrine‚HCl were purchased from Sigma (Deisenhofen, Germany), and (()-norephedrine‚HCl, (()-ephedrine‚HCl, and (+)-pseudoephedrine‚HCl were purchased from Fluka (Neu-Ulm, Germany). Pure standards of (()-p-hydroxyamphetamine, (()pholedrine, (()-p-hydroxynorephedrine, and (()-p-hydroxyephedrine were provided by Tropon (Koeln, Germany). Reagents were purchased from various sources: the chiral derivatizing reagent (()-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride (MTPACl) from JPS Chimie (Bevaiz, Switzerland), and diethyl ether, methanol, acetonitrile, and trifluoroacetic acid from Merck (Darmstadt, Germany). N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N-methyl-N-(triethylsilyl)trifluoroacetamide (MTESTFA), and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBDMSTFA) were synthesized in our laboratory according to the procedure described by Donike.24 Selective Derivatization Procedures. The dry residue was dissolved in 50 µL of a mixture of acetonitrile-trifluoroacetic acid (60:40 v/v) that contained 200 µg/mL of methyl orange. The mixture was titrated with MSTFA, MTESTFA, or MTBDMSTFA until the color of the reaction mixture changed from red to yellow. The sample was heated for 5 min at 60 °C on a heat block. Then, 5 µL of MTPACl was added to the reaction mixture, and the sample was heated for an additional 5 min at 60 °C. Studies on the Function of MSTFA as a HCl Acceptor. To samples of (R,S)-(()-amphetamine sulfate and (S)-(+)-methamphetamine (each 50 µg in 150 µL of acetonitrile) was added 5 µL of MTPACl reagent, and the mixtures were heated at 60 °C. At the appropriate times, 10 µL aliquots were withdrawn, to which 1 mL of 0.06 N hydrochloric acid and 3 mL of diethyl ether were added. The samples were shaken for 5 min prior to centrifugation (5 min, 1200g). The organic layer was dried. To study how the volume of MSTFA influences the yield and the formation rate of diastereomeric derivatives of amphetamine and methamphetamine 10, 20, 30, and 40 µL amounts of MSTFA were added to the reaction solution before heating, and the described procedures were repeated. The residue was dissolved with 50 µL of methanol, and aliquots (2 µL) of this solution were then analyzed by GC and nitrogen-specific detection. (19) Gilbert, M. T.; Brooks, C. J. W. Biomed. Mass Spectrom. 1977, 4 (4), 226. (20) Donike, M. J. Chromatogr. 1975, 103, 91. (21) Donike, M. Chromatographia 1974, 7, 651. (22) Donike, M. J. Chromatogr. 1973, 78, 273. (23) Donike, M. J. Chromatogr. 1975, 115, 591. (24) Donike, M. J. Chromatogr. 1969, 42, 103.

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Selective Derivatization Kinetic Study. A mixture of (()amphetamine, (()-methamphetamine, (()-norephedrine, (()ephedrine, and (-)-pseudoephedrine (each 50 µg) was dried in a test tube. The dry residue was dissolved in 50 µL of acetonitriletrifluoroacetic acid containing methyl orange and titrated with MSTFA until the color of the reaction mixture changed from red to yellow. The sample was heated for 5 min at 60 °C on a heat block. To the reaction mixture was added 5 µL of MTPACl, and the sample was heated to 60 °C. At appropriate times, 2 µL aliquots were withdrawn and injected into the GC-NPD system. Drug Administration and Sample Collection. Doses of 30 mg of famprofazone (two tablets of Gewodin; Ed. Geistlich Soehne AG, Wolhusen, Switzerland) were administered to a male volunteer orally. The subject was a healthy male, age 27. He was free from other medication for the duration of the experiment. Urine samples were collected at various times over 48 h and stored at 4 °C. Quantification of the Enantiomers in Urine. A 3 mL volume of urine was adjusted to pH 9.6 with sodium bicarbonatepotassium carbonate (2:1 w/w), and 50 ng of internal standard (p-chlorphentermine) was added. The mixture was extracted with 8 mL of diethyl ether-tert-butyl alcohol (7:1 v:v). The organic layer was transferred into a 15 mL glass centrifuge tube, 0.4 mL of 0.06 M hydrochloric acid was added, and extraction was performed by mixing for 10 min in a mechanical shaker. The solution was centrifuged for 5 min at 1200g, and the organic layer was aspirated and discarded. The aqueous layer was dried in a desiccator over phosphorus pentoxide-potassium hydroxide. Calibration curves of peak area ratios of standard to internal standard against concentrations of standards were constructed over the concentration range of 1.0-1000 ng/mL of standards in urine. The concentrations of the unknown samples were determined by comparison to peak area ratios from the standard curve. Gas Chromatography. A Hewlett-Packard 5890 GC-NPD was used for the derivatization time course study. Separation was achieved with an HP fused-silica capillary column with cross-linked 5% phenylmethylsilicone (SE-54), ∼17 m length, 0.2 mm i.d., 0.33 µm film thickness. The chromatographic conditions were as follows: detector, NPD at 300 °C; injector temperature, 280 °C; initial oven temperature, 150 °C; ramp, 20 °C/min; final oven temperature, 320 °C for 2 min; carrier gas, helium at a flow of 1.2 mL/min; hydrogen flow, 3 mL/min; air flow, 100 mL/min; split ratio, 1:10. Gas Chromatography/Mass Spectrometry. All mass spectra were obtained with a Hewlett-Packard GC 5890/5971A instrument. The same capillary column as described above was also used for GC/MS. The operating parameters of GC/MS were as follows: detector, mass selective detector in scan or SIM mode; electron impact; ionization at 70 eV; injector temperature, 280 °C; interface temperature, 300 °C; initial oven temperature, 100 °C; ramp, 10 °C/min; final oven temperature, 320 °C for 2 min; carrier gas, helium at a flow of 1.0 mL/min; split ratio, 1:8. RESULTS AND DISCUSSIONS Selective Derivatization. The compounds used in this study (Figure 1) contain amino and hydroxyl groups suitable for derivatization. The hydroxyl groups react readily with each of the three silylating reagents (MSTFA, MTESTFA, and MTBDMSTFA) under mild conditions. The amino groups, on the other hand, were derivatized with diverse chiral reagents to form amides19,25 or urethanes,13,14 compounds that are chemically

Table 1. Characteristics of the Mass Spectra of Diastereomeric Derivatives derivatives

MW

characteristic ions m/z

amphetamine, N-MTPA methamphetamine, N-MTPA norephedrine, N-MTPA, O-H norephedrine, N-MTPA, O-TMS norephedrine, N-MTPA, O-TES norephedrine, N-MTPA, O-TBDMS ephedrine, N-MTPA, O-H ephedrine, N-MTPA, O-TMS ephedrine, N-MTPA, O-TES ephedrine, N-MTPA, O-TBDMS p-hydroxyamphetamine, N-MTPA, O-TMS p-hydroxyamphetamine, N-MTPA, O-TES p-hydroxyamphetamine, N-MTPA, O-TBDMS pholedrine, N-MTPA, O-TMS pholedrine, N-MTPA, O-TES pholedrine, N-MTPA, O-TBDMS p-hydroxynorephedrine, N-MTPA, bis-O-TMS p-hydroxynorephedrine, N-MTPA, bis-O-TES p-hydroxynorephedrine, N-MTPA, bis-O-TBDMS p-hydroxyephedrine, N-MTPA, bis-O-TMS p-hydroxyephedrine, N-MTPA, bis-O-TES p-hydroxyephedrine, N-MTPA, bis-O-TBDMS

351 365 367 439 481 481 381 453 495 495 439 481 481 453 495 495 527 569 569 541 583 583

189(100), 91(85.5), 119(44.9), 260(39.1), 162(15.7), 234(14.7), 351(1.5) 189(100), 274(66.4), 91(29.2), 119(16.0), 200(7.3), 248(1.5), 365(0.7) 189(100), 186(72.2), 261(43.4), 105(28.7), 229(12.5), 260(12.3), 367(0.1) 179(100), 73(57.7), 333(51.8), 189(23.7), 424(6.0), 260(3.1), 439(0.1) 221(100), 189(42.2), 105(23.2), 375(14.2), 452(12.4), 292(3.1) 221(100), 73(80.4), 189(43.7), 424(22.9), 260(3.2), 375(2.6), 466(0.8) 189(100), 274(34.4), 77(17.5), 105(17.3), 275(11.1), 200(4.6), 342(0.2) 189(100), 274(49.5), 73(23.9), 179(16.2), 347(7.1), 200(6.7), 438(1.8) 189(100), 274(72.3), 221(41.4), 466(6.0), 389(5.8), 332(1.7), 495(0.1) 189(100), 274(49.6), 221(27.0), 73(26.0), 438(13.3), 304(3.6), 480(0.6) 206(100), 189(80.9), 73(43.1), 105(21.3), 260(3.6), 306(2.2), 439(0.3) 248(100), 189(94.8), 191(46.2), 221(17.8), 260(6.7), 292(6.0), 481(0.2) 248(100), 189(64.4), 249(28.5), 221(12.2), 260(5.1), 292(4.6), 481(0.1) 189(100), 206(33.4), 73(15.8), 274(9.9), 105(9.5), 248(1.3), 453(0.1) 189(100), 248(43.3), 274(14.3), 249(12.0), 105(9.2), 342(0.5) 189(100), 248(41.1), 274(16.2), 73(14.8), 200(6.4), 306(0.5), 480(0.1) 267(100), 73(71.8), 189(28.0), 268(27.3), 105(11.8), 333(6.9), 512(0.4) 351(100), 189(22.6), 87(19.0), 352(15.3), 260(2.8), 422(0.9) 351(100), 73(93.0), 352(80.1), 189(23.4), 249(5.6), 554(3.0), 260(1.6) 267(100), 73(64.8), 268(59.2), 189(27.7), 333(15.5), 105(16.0), 512(9.4) 351(100), 352(43.5), 189(42.0), 87(17.9), 353(16.5), 274(3.8), 293(3.3) 351(100), 73(63.3), 352(52.2), 189(39.5), 353(19.4), 293(2.5), 568(0.3)

Figure 3. Function mechanism of MSTFA as a HCl acceptor.

Figure 2. Kinetics of the reaction of (+)-methamphetamine with MTPA(-)-Cl.

relatively stable and not easy to cleave. MTPACl has been shown recently to be a useful chiral reagent for GC26,27 and HPLC14 separation of the amines. In the present work, the mixture compounds of amines (e.g., amphetamine and methamphetamine), phenol alkylamines, and amino alcohols were examined after conversion to the diastereomeric amides, prepared respectively by selective O-silylation with the three silylation reagents, followed by selective N-acylation with MTPACl. By titrating with silylating agents until the color change of the reaction mixture, use of excess silylating agents was avoided. N-MTPA-O-silylated diastereomers are well suitable for identification and quantification by GC/MS. This technique of selective derivatization can be applied to salts of these amines without prior liberation of the bases. Study on the Function of MSTFA as a HCl Acceptor. It was observed that the content of MSTFA in the reaction mixture influenced the yield and the formation rate of the diastereomeric derivatives of (()-amphetamine and (+)-methamphetamine. This study was performed by varying the MSTFA amount in the reaction mixture between 10 and 40 µL and observing the (25) Caccia, S.; Chiabrando, C.; De Ponte, P.; Fanelli, R. J. Chromatogr. Sci. 1978, 16, 543. (26) Ames, M. M.; Frank, S. K. Biochem. Phamacol. 1982, 31, 5. (27) Dellaria, J. F.; Santarsiero, B. D. J. Org. Chem. 1989, 54, 3916.

formation rates of the three derivatives. To exclude the reaction in the injection port, the excess reagents were removed by reextraction into 0.06 N HCl. In attempts to derivatize secondary amines, e.g., methamphetamine, Gal6 reported that the reaction of these amines with MTPACl was considerably slower than the reaction of primary amines and that they remained essentially underivatized, even though pyridine was used as a base. This fact was in accord with our results when pyridine was used as a base for the reaction. In contrast, the maximum obtainable yield of the derivatives as determined by GC was obtained as soon as 30 µL of MSTFA was added to the reaction solution. The result for methamphetamine is demonstrated in Figure 2. The y-axis gives the peak height relative to the mean value of the peaks within 5% of the maximum peak height. This fact shows that MSTFA can possibly be used as a strong HCl acceptor for the acylation with acid chloride by the mechanism presented in Figure 3. MSTFA can also be used to convert amino salts to the free bases. Selective Derivatization Time Course Study. The relationship between the yield of the N-MTPA, O-silylated derivatives of the test compounds and the reaction time were evaluated as described in the Experimental Section. Maximum yield of the derivatives was determined by GC immediately after addition of the acyl reagents, and no trace amount of underivatized substance was detected in the reaction mixture after addition of reagents. Because the reaction in the injection port of the GC could not be excluded, the kinetics of the real reaction during derivatization was not studied. But, it can be deduced that maximum yield of Analytical Chemistry, Vol. 68, No. 17, September 1, 1996

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Figure 5. Migration of the TMS or TES group to the carbonyl oxygen. R1 ) H or CH3; R2 ) TMS or TES.

Figure 4. EI spectra of (A) norephedrine, N-MTPA, OTMS derivative, (B) norephedrine, N-MTPA, OTES derivative, and (C) ephedrine, N-MTPA, OTMS derivative.

the derivatives may also be attributed to the function of MSTFA as a HCl acceptor, as described before. The derivatization is easily completed at room temperature; therefore, this method is also effective in distinguishing the enantiomers of the thermally racemizable compounds by GC. The reactions of the enantiomers proceeded at the same rate. Mass Spectrometry. The principal ions of the derivatives in mass spectrometric detection with electron impact ionization at 70 eV are given in Table 1. No significant differences were found between the spectra of the diastereoisomeric amides. Molecular ions were generally of low abundance under the given experimental conditions. N-MTPA derivatives afforded the characteristic ion at m/z 189, due to cleavage adjacent to the carbonyl group from the derivative reagents and the charge was preferentially retained on the fragment containing the silyl ether group in the spectra of the N-MTPA bis-silylate derivatives. An abundant ion at m/z 333 appeared in the spectra of the N-MTPA, O-TMS norephedrine (or norpseudoephedrine) derivatives. Gilbert and Brooks19 had proposed that the TMS group could possibly be transferred either to the nitrogen or to the carbonyl oxygen. This ion from our study changed to m/z 375 in the spectra of the N-MTPA, O-TES norephedrine derivatives and to m/z 347 in the spectra of the N-MTPA, O-TMS ephedrine derivatives, representative of a secondary amine (Figure 4). These changes are due to the difference of the silyl group and the presence of a methyl group on a nitrogen atom. This characteristic peak also appeared in the spectra of norephedrine and ephedrine derivatized by other acylating agents, such as N-methylbistrifluoroacetamide and phenylbutyric anhydride, but no peak from the migration appeared in the spectra of the O-TMS norephedrine, ephedrine, and methylephedrine derivatives, which have no carbonyl oxygen. The 3018 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996

Figure 6. Gas chromatographic separation of N-MTPA(+), OTMS derivatives of amphetamine phenol alkylamines and hydroxyamines on capillary column. Key to peak numbering: 1, (+)-amphetamine; 2, (-)-amphetamine; 3, (+)-methamphetamine; 4, (-)-methamphetamine; 5, (+)-norpseudoephedrine; 6, (-)-norpseudoephedrine; 7, (-)-norephedrine; 8, (+)-norephedrine; 9, (-)-ephedrine; 10, (+)ephedrine; 11, (+)-pseudoephedrine; 12, (-)-pseudoephedrine; 13, (+)-p-hydroxyamphetamine; 14, (-)-p-hydroxyamphetamine; 15, (+)p-hydroxymethamphetamine; 16, (-)-p-hydroxymethamphetamine; 17, (-)-p-hydroxynorephedrine; 18, (+)-p-hydroxynorephedrine; 19, (-)-p-hydroxyephedrine; 20, (+)-p-hydroxyephedrine.

Figure 7. Gas chromatographic separation of N-MTPA(+), OTES derivatives of amphetamine phenol alkylamines and hydroxyamines on capillary column. See Figure 6 caption for key to peak numbering.

basicity of nitrogen in the amide will be not favorable for the migration of silyl groups rather than the amines. From the facts, the ions may result from a migration of the TMS or TES group to the carbonyl oxygen, as represented in Figure 5. Also, an ion at m/z 206 appeared in the spectra of the N-MTPA, O-TMS norephedrine, ephedrine, p-hydroxyamphetamine, and pholedrine derivatives. N-MTPA, O-TMS derivatization of phydroxynorephedrine and p-hydroxyephedrine caused a shift of the peak to m/z 294 due to the presence of the p-hydroxyl group in the phenyl group. The ions at m/z 206 and 294 are due to

Figure 8. Gas chromatographic separation of N-MTPA(+), OTBDMS derivatives of amphetamine phenol alkylamines and hydroxyamines on capillary column. See Figure 6 caption for key to peak numbering. Table 2. Resolution (R)a for Diastereoisomers N-MTPA, N-MTPA, N-MTPA, N-MTPA, O-H OTMS OTES OTBDMS

compound amphetamine methamphetamine norephedrine norpseudoephedrine ephedrine pseudoephedrine p-hydroxyamphetamine pholedrine p-hydroxynorephedrine p-hydroxyephedrine

3.39 1.53 3.42 3.39

3.88 3.84 2.73 3.97 4.60 2.85 3.48 2.77

4.05 4.07 3.03 4.69 4.40 2.79 3.11 2.35

3.94 3.92 2.81 4.71 4.60 2.89 3.25 2.43

a Resolution factor (R) is defined as 2d/(w + w ), where d is the 1 2 separation between the peak maxima and w1 and w2 are the widths of the peaks at baseline.

Table 3. Linear Relationship and Precision compound

regression line (r)a

peak area ratio to IS, mean ( SD (RSD, %)b

(-)-amphetamine (+)-amphetamine (-)-norephedrine (+)-norephedrine (-)-ephedrine (+)-ephedrine

y ) 0.632x - 0.011 (1.000) y ) 0.644x - 0.012 (1.000) y ) 2.350x - 0.011 (1.000) y ) 2.341x + 0.024 (1.000) y ) 2.303x + 0.014 (1.000) y ) 2.312x - 0.011 (1.000)

0.31 ( 0.10 (3.0) 0.33 ( 0.01 (1.4) 1.62 ( 0.02 (1.3) 1.64 ( 0.03 (2.0) 1.52 ( 0.03 (2.2) 1.58 ( 0.04 (2.3)

a

Correlation coefficient. b Relative standard deviation.

C-N cleavage with hydrogen transfer. But the C-N cleavage in the spectra of the N-MTPA amphetamine and methamphetamine derivatives gave an abundant ion at m/z 119 without hydrogen transfer. The ions selected in this study by quantitative selected-ion monitoring were m/z 206, 260, 267, 274, and 333 for N-MTPA, O-TMS derivatives and m/z 221, 248, 260, 274, and 351 for N-MTPA, O-TES or N-MTPA, O-TBDMS derivatives. Chromatogram and Resolution. Figures 6-8 show respectively the chromatograms of the N-MTPA, O-silylated derivatives of amphetamines, phenol amines, and hydroxyamines when MSTFA, MTESTFA, and MTBDMTFA were used for the silylation. The elution order was found to be dependent only on the stereochemistry at the amine chiral center; i.e., enantiomeric pairs

Figure 9. GC/MS(SIM) chromatograms of the metabolites in urine 2-4 h after administration of famprofazone. Key to peak numbering: 1, (+)-amphetamine-N-MTPA(+); 2, (-)-amphetamine-N-MTPA(+); 3, (+)-methamphetamine-N-MTPA(+); 4, (-)-methamphetamineN-MTPA(+); 5, (-)-ephedrine-N-MTPA(+)-OTMS; 6, (+)-ephedrineN-MTPA(+)-OTMS; 7, (+)-pseudoephedrine-N-MTPA(+)-OTMS; 8, (-)-pseudoephedrine-N-MTPA(+)-OTMS; 9, (+)-norpseudoephedrine-N-MTPA(+)-OTES; 10, (-)-norephedrine-N-MTPA(+)-OTES; 11, (-)-norpseudoephedrine-N-MTPA(+)-OTMS; 12, (+)-norephedrine-N-MTPA(+)-OTES; 13, (+)-p-hydroxyamphetamine-N-MTPA(+)-OTES; 14, (-)-p-hydroxyamphetamine-N-MTPA(+)-OTES; 15, (+)-pholedrine-N-MTPA(+)-OTES; 16, (-)-pholedrine-N-MTPA(+)OTES; 17, (-)-p-hydroxynorephedrine-N-MTPA(+)-bis-OTES; 18, (+)-p-hydroxynorephedrine-N-MTPA(+)-bis-OTES; I, internal standard (p-chlorphentermine-N-MTPA(+)).

were eluted in the sequence 2R- before 2S-form when MTPA(-)-Cl was used, but in the opposite sequence when MTPA(+)Cl was used. Under the same chromatographic conditions, the reagent peaks were well separated from those of the diastereomers and did not interfere with the detection, in spite of the repeated injection of samples. Therefore, it is not necessary to evaporate the reaction medium. These derivatives also do not show any adsorption effects in the GC system. All diastereomers were well separated through conversion to the N-MTPA, O-silylated derivatives. To compare separation Analytical Chemistry, Vol. 68, No. 17, September 1, 1996

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factors between silyl ethers, the resolutions (R) of the derivatives are listed in Table 2. The largest R value occurs for N-MTPA, O-TES derivatives of ephedrine and norephedrine and the smallest for N-MTPA, O-H derivatives, which carry no silyl group. Smaller R values occur for N-MTPA, bis-O-TES derivatives of p-hydroxy compounds than for N-MTPA, bis-O-TMS derivatives of those compounds. These facts may be attributed to the sterically crowded branching of the asymmetric center, which may increase the conformational rigidity, and to the size differential of groups attached to the alcoholic asymmetric carbon atom, as described by Feibush.28 For exact quantitative correlations between structural properties of enantiomers, larger sets of molecules and adequate parametrization are required. Application. The possibility of applying this method for quantitative analysis was studied. The extraction procedure developed by Javaid et al. was used.29 This extraction method has been used routinely as the screening method in horse doping. The sample was extracted with 8 mL of n-pentane-2-propanol (97: 3) and reextracted with 0.4 mL of 0.06 M hydrochloric acid. The aqueous layer was dried, and the residue was selectively derivatized. After conversion to N-MTPA(+), O-TMS derivatives, (-)norpseudoephedrine and (-)-norephedrine, and (+)-pholedrine and (-)-p-hydroxynorephedrine, were overlapped. But, after conversion to N-MTPA(+), O-TES derivatives, these pairs were well separated. Various amounts of (()-amphetamine, (()-norephedrine, and (()-ephedrine (5 ng-5 µg) were added to 3 mL urine and extracted as described in the Experimental Section. The dried extracts were reacted to N-MTPA, O-TMS or N-MTPA, O-TES derivatives and injected into the GC/MS(SIM). The calibration curves were obtained by plotting the ratio of the peak area of the diastereoisomers to that of the internal standard (p-chlorphentermine) against known amounts of the compounds. There was a good linear relationship between the injection amounts and the (28) Feibush, B. Anal. Chem. 1971, 43, 1098. (29) Javaid, J. I.; Dekirmenjian, H.; Liskevych, U.; Lin, R.-L.; Davis, J. M. J. Chromatogr. Sci. 1981, 19, 439. (30) Shin, H.-S. J. Chromatogr., submitted.

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detector response (Table 3). The precision was evaluated by analyzing five samples (at 0.5 µg/mL) according to the experimental procedure described before. The coefficients of variation are reported in Table 3. In our previous investigation on the metabolism of famprofazone30, nine metabolites, e.g. (()-amphetamine, (()-methamphetamine, (()-norpseudoephedrine, (()-norephedrine, (()-pseudoephedrine, (()-ephedrine, (()-p-hydroxyamphetamine, (()pholedrine, and (()-p-hydroxynorephedrine, were identified in human urine. In this study, the urinary excretion of the enantiomers after oral administration of racemic famprofazone was studied. As shown in Figure 9, all enantiomers of the metabolites are well separated, and no interfering peaks from endogeneous substances are present near the peaks of the metabolites and internal standard. The lower limits of detection in urine for 18 metabolites were ∼0.1 ng/mL (signal-noise ratio ) 3). These facts show that the method described could be successfully applied to quantitative analysis of the enantiomers of amphetamine derivatives in urine. CONCLUSIONS The proposed method is well suited for the rapid and simultaneous determination the enantiomers of amphetamine, phenol alkylamines, and hydroxyamines like the ephedrines. This method is applicable to the resolution of optical isomers of the substances containing more than one reactive functional group, e.g., β-blockers, catecholamines, and amino acids. The derivatives are useful for the simultaneous quantification of these enantiomers in a biological matrix by selected-ion monitoring. Additional application of the method to quantitative measurement of these compounds in biological samples is currently under study in our laboratory. Received for review April 16, 1996. 1996.X

Accepted May 9,

AC960365V X

Abstract published in Advance ACS Abstracts, June 15, 1996.