In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

R. Pootrakronchai, M. Kaewklum, P. Wilairat, T. Kusamran and T. Anukarahanonta

Method Validation for d,l-Isomers of Methamphetamine and Amphetamine by GC/MS

National Doping Control Centre, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

Introduction Amphetamine and methamphetamine are potent central nervous system stimulants (1). Since 2006, the differentiation between d- and l-isomers of methamphetamine is required by WADA (2). There are a number of drugs that are metabolized in the body to amphetamine and/or methamphetamine: amphetaminil, benzphetamine, clobenzorex, selegiline, dimethamphetamine, ethylamphetamine, famprofazone, fencamine, fenethylline, fenproporex, furfenorax, mefenorex, mesocarb and prenylamine. Thus, it is important to separate the enantiomers of amphetamine (AP) and methamphetamine (MA) excreted in urine in order to determine the source of the originally administered drugs. The enantiomeric separation of AP and MA can be achieved using the chiral agent, (S)-(-)-N-trifluoroacetyl-prolyl chloride (l-TPC), as derivatising agent (3,4). A GC/MS procedure for separating the d,l- enantiomer of MA and AP was developed and validated. Experimental Blank urine from 5 healthy volunteers were spiked with standard substances to the final concentration of 0.06-1.00 µg/ml. The separation of AP and MA enantiomeric pair was performed by the modified Procedure I (liquid-liquid extraction) (5). 50 µl of 0.1 M (S)-(-)-N- trifluoroacetyl-prolyl chloride (l-TPC) in dichloromethane was added to the extracted organic phase. After 15 min at room temperature, they were washed with 3 ml of 0.01M NaOH for 15 min and centrifuged at 2500 rpm for 15 min. The organic layer was separated and dried under the

N2 stream. The residue was redissolved in 100 µl tert-butyl methyl ether and 2 µl were injected into the GC.

355 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Instrumentation Agilent GC/MSD (6890/5973); column 5% PHME Siloxane (Ultra2), (12.5 m, i.d 0.2 mm, film thickness 0.11 µm); split ratio 10:1; flow at 0.6 ml/min; solvent delay 1.2 min; temperature program, initial 70ºC (0.13 min), 25ºC/min to 300ºC (hold 1.2 min); transfer line temperature 280 ºC Results and Discussion Although m/z 166 is the base ion, it was not selected as the quantifier ion as the ion is derived from l-TPC. The quantifier ions for the derivatised AP and MA are m/z 237 and 251, respectively (4). Figures 1 and 2 show that this method can separate the enantiomers of MA and AP, respectively. An excretion urine for methamphetamine shows only d-AP, and d-MA peaks (Figure 3). An excretion urine for selegiline shows l-AP, and l-MA peaks (Figure 4). Thus separation and identification of the isomer peaks can be used to differentiate the source of the originally administered drugs. Table 1 shows results from the method validation for the enantiomers of AP and MA. Overall recovery was 80%, with %CV less than 15% (a valid range for repeatability) (6) and the LOD was about 10 times less than the minimum required performance limits (MRPL) for the detection of prohibited substances (0.5 µg/ml) (7). Table 1. Summary of results for method validation (n=5) Substance %Recovery %CV LOD (ng/ml) RRT* d-MA 78.63 2.58 60 1.534 l-MA 81.20 1.55 60 1.517 d-AP 72.94 2.33 60 1.398 l-AP 68.34 2.29 60 1.374

*ISTD = Diphenylamine, RT = 4.09 min

356 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Abundance Abundance 6.28 Abundance TIC:B5J1UPC3.D Scan 1046 (6.265 min): B5J1UPC3.D 4.09 TIC:B1J1METH.D 5500000 4.09 166 6.19 6.27 1400000

5000000 7000000 l-MA 1300000 4500000 1200000

6000000 d-MA d-MA 1100000 4000000 ISTD 1000000 5.71 3500000 ISTD 5000000 900000 5.61 58 251 3000000 800000 251 4000000 700000 2500000 5.54 600000 3000000 2000000 5.54 500000 91

1500000 400000 2000000 A 5.03 5.47 B C 4.61 1000000 5.66 B 300000 1000000 4.17 A 200000 500000 119 5.47 6.18 194 5.03 100000 148 225 0 40 273 0 0 295 323343323295 343 371 401 446 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 3.804.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.806.006.20 6.40 6.60 6.80 40 60 80 100120140160180200220240260280300320340360380400420440100120140160180200220240260280300320340360380400420440 Time--> m/z--> Time--> Figure 1. A. TIC of spike sample (1 µg/ml), d-MA-TPC (6.28 min), ISTD (RT= 4.09 min), B. TIC of spike sample (1 µg/ml), l- MA-TPC (6.19 min), d-MA-TPC (6.27 min), ISTD (RT=4.09 min), C. Mass spectrum of d-MA-TPC in spike sample (1μg/ml)

Abundance Abundance Abundance TIC:B2J12AMP.D 5.63 6500000 4.09 TIC:B1J1AMP.D Scan 931 (5.718 min): B2J12AMP.D 7500000 166 6000000 1400000 7000000 4.09 d-AP 1300000 5500000 6500000 ISTD 5.72 1200000 6000000 5000000 l-AP l-AP 5.62 1100000 5500000 4500000 1000000 5000000 ISTD 4000000 900000 4500000 3500000 800000 4000000 5.56 237 237 3000000 700000 3500000 600000 3000000 5.56 2500000 194 500000 2500000 2000000 A B 400000 C 2000000 1500000 91 118 300000 1500000 A 1000000 B C 200000 1000000 5.70 4.16 5.03 4.164.18 5.03 5.48 69 139 5.47 500000 4.61 100000 44 500000 4.61 212 259 313281 333355 391 446 0 0 0 3.804.004.204.404.604.805.005.205.405.605.80 40 60 80 100120140160180200220240260280300320340360380400420440 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 Time--> m/z--> Time--> Figure 2. A. TIC of spike sample (1 µg/ml), l-AP-TPC (5.63min), ISTD (RT= 4.09 min), B. TIC of spike sample (1 µg/ml), l- AP-TPC (5.62 min), d-AP-TPC (5.72 min), ISTD (RT= 4.09 min), C. Mass spectrum of d-AP-TPC in spike sample (1μg/ml)

TIC: B1A1UPC1.D Abundance Scan 1035 (6.211 min): B1A1UPC1.D Scan 920 (5.664 min): B1A1UPC1.D Abundance 6.21 Abundance 166 4.05 4.59 4.85 5.00 5.51 150000 17000 166 140000 ISTD 16000 550000 d-MA 130000 15000 500000 120000 14000 110000 13000 450000 100000 12000 400000 11000 d-AP 90000 73 58 10000 350000 80000 9000 300000 A 70000 251 B 8000 C 250000 60000 7000 5.66 50000 91 6000 41 237 200000 5000 91 194 40000 150000 4000 118 30000 3000 100000 20000 2000 119 139 50000 194 1000 10000 256 40 148 225 213 277 298 322 341 368 415 446 0 284 304 323341368 390 412 446 0 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 m/z--> m/z--> Time--> Figure 3. A. TIC of positive methamphetamine sample, B. Mass spectrum of d-MA-TPC (RRT= 1.533), C. Mass spectrum of d-AP- TPC (RRT= 1.397)

357 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Abundance Abundance Abundance

Scan 1016 (6.119 min): SELEGIDERI.D 650000 166 Scan 894 (5.539 min): SELEGIDERI.D 4.02 TIC: SELEGIDERI.D 140000 166 600000 130000

3500000 550000 120000

500000 110000 58 3000000 ISTD 6.12 450000 100000

400000 90000 2500000 80000 350000 2000000 70000 l-MA 300000 251 60000 237 251 250000 91 237 1500000 5.77 50000 l-AP 194 200000 40000 1000000 A B 91 C 5.60 150000 4.81 30000 5.42 118 5.54 100000 5.86 500000 4.22 20000 69 119 41 50000 194 139 10000 148 225 0 40 281 304323341 369 403 429447 212 257 281299 327 355373 399 429429399 447 0 0 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 40 60 80 100120140160180200220240260280300320340360380400420440100120140160180200220240260280300320340360380400420440 40 60 80 100120140160180200220240260280300320340360380400420440100120140160180200220240260280300320340360380400420440 m/z--> Time--> m/z--> Figure 4. A. TIC of an excretion urine of selegiline, B. Mass spectrum of l-MA (RRT= 1.522), C. Mass spectrum of l-AP (RRT= 1.378)

References 1. Kramer, T., Maurer, HH. (1998) Determination of amphetamine, methamphetamine and amphetamine-derived designer drugs or medicaments in blood and urine. J Chromatogr B 713, 163-187. 2. World Anti-Doping Agency. The 2006 Prohibited List. International Standard, Montreal (2006) http//www.wada-ama.org/rtecontent/document/2006_LIST.pdf 3. John, TC. (1996) Enantiomeric composition of amphetamine and methamphetamine derived from the precursor compound famprofazone. Forensic Sci Int. 80, 189-199. 4. Sheng, MW., Ting, CW., Yun, SG.(2005) Simultaneous determination of amphetamine and methamphetamine enantiomers in urine by simultaneous liquid-liquid extraction and diastereomeric derivatization followed by gas chromatographic-isotope dilution mass spectrometry. J Chromatogr B 816, 131-143. 5. Hemmersbach, P., de la Torre, R. (1996) Stimulant, narcotics and ß-blockers: 25 years of development in analytical techniques for doping control. J Chromatogr B 687, 221-238. 6. Jimenez, C., Ventura, R., Segura, J. (2002) Validation of qualitative chromatographic methods: strategy in antidoping control laboratories. J Chromatogr B 767, 341-351. 7. Minimum required performance limits for detection of prohibited substances. (2004) WADA technical document -TD2004MRPL version 1.0

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