Method Validation for d,l-Isomers of Methamphetamine and Amphetamine by ...

[Pages:4]In: W Sch?nzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strau? - K?ln 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 ?g/ml. The separation of AP and MA enantiomeric pair was performed by the modified Procedure I (liquid-liquid extraction) (5). 50 ?l of 0.1 M (S)-(-)-Ntrifluoroacetyl-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 ?l tert-butyl methyl ether and 2 ?l were injected into the GC.

355

In: W Sch?nzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strau? - K?ln 2007

Instrumentation

Agilent GC/MSD (6890/5973); column 5% PHME Siloxane (Ultra2), (12.5 m, i.d 0.2 mm, film thickness 0.11 ?m); split ratio 10:1; flow at 0.6 ml/min; solvent delay 1.2 min; temperature program, initial 70?C (0.13 min), 25?C/min to 300?C (hold 1.2 min); transfer line temperature 280 ?C

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 ?g/ml) (7). Table 1. Summary of results for method validation (n=5)

Substance d-MA l-MA d-AP l-AP

%Recovery 78.63 81.20 72.94 68.34

%CV 2.58 1.55 2.33 2.29

LOD (ng/ml) 60 60 60 60

RRT* 1.534 1.517 1.398 1.374

*ISTD = Diphenylamine, RT = 4.09 min

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In: W Sch?nzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strau? - K?ln 2007

Abundance

4.09

7000000

6000000

5000000

TIC: B1J1METH.D

ISTD

6.28 d-MA

4000000

3000000

5.54

2000000

1000000

4.17

5.03

5.47

A A

6.18

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 6.80 Time-->

Abundance

5500000 5000000 4500000 4000000 3500000 3000000

4.09

TIC: B5J1UPC3.D

6.19 6.27

l-MA

d-MA

ISTD

5.71

5.61

2500000

2000000 1500000 1000000

5.03 4.61

5.54

5.47 5.66

B B

500000

Time-->

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 6.80

Abundance

1400000

Scan 1046 (6.265 min): B5J1UPC3.D 166

1300000

1200000

1100000

1000000 900000 800000 700000

58

251

251

600000

500000 400000 300000

91

C

200000 100000

0 m/z-->

119

194

40

148

225

273 295 323343 371 401

446

40 60 80 100120140160180200220240260280300320340360380400420440

Figure 1. A. TIC of spike sample (1 ?g/ml), d-MA-TPC (6.28 min), ISTD (RT= 4.09 min),

B. TIC of spike sample (1 ?g/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 (1g/ml)

Abundance

7500000 7000000 6500000 6000000 5500000 5000000 4500000 4000000 3500000 3000000 2500000 2000000 1500000 1000000

500000 0 3.80

Time-->

4.09

TIC: B1J1AMP.D

ISTD

44.1.168

5.03

4.61

4.00 4.20 4.40 4.60 4.80 5.00 5.20

5.63 l-AP

5.56

5.70 5.47

A A

5.40 5.60 5.80

Abundance 6500000 6000000 5500000 5000000 4500000 4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 3.80

Time-->

4.09

TIC: B2J12AMP.D

ISTD

d-AP l-AP 5.625.72

5.56

4.16

5.03

5.48

4.61

4.00

4.20

4.40

4.60

4.80

5.00

5.20

5.40

5.60

5.80

B B

Abundance

1400000

Scan 931 (5.718 min): B2J12AMP.D 166

1300000

1200000

1100000

1000000

900000 800000 700000

237 237

600000

500000

400000 300000

91 118

200000 69

100000 44

139

194

C C

m/z-->

0

212

259 281 313333355 391

446

40 60 80 100120140160180200220240260280300320340360380400420440

Figure 2. A. TIC of spike sample (1 ?g/ml), l-AP-TPC (5.63min), ISTD (RT= 4.09 min), B.

TIC of spike sample (1 ?g/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 (1g/ml)

Abundance 550000

4.05 4.59 ISTD

4.85 5.00

TIC: B1A1UPC1.D 5.51

6.21 d-MA

500000

450000

400000

d-AP

350000

300000

A

250000

5.66

200000

150000

100000

50000

Time-->

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 6.80

Figure 3. A. TIC of positive

Abundance

150000 140000 130000 120000 110000 100000

90000 80000 70000 60000 50000 40000 30000 20000 10000

0 m/z-->

Scan 1035(6.211min): B1A1UPC1.D

Abundance 166

17000

16000

15000

14000

13000

12000

11000

58

10000

251 B

9000 8000

7000

91

6000

5000

4000

3000

119

194

2000 1000

40

148

225

284 304 323 341 368 390 412 446

0

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

m/z-->

Scan 920(5.664min): B1A1UPC1.D 166

73

C

41 91

237 237

194

118

139

213

256 277 298 322 341 368

415 446

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

methamphetamine sample, B. Mass spectrum of d-MA-TPC

(RRT= 1.533), C. Mass spectrum of d-AP- TPC (RRT= 1.397)

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In: W Sch?nzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strau? - K?ln 2007

Abundance

3500000 3000000

4.02

TIC: SELEGIDERI.D

ISTD

6.12

Abundance

650000 600000 550000 500000 450000

58

Scan 1016 (6.119 min): SELEGIDERI.D 166

Abundance

140000 130000 120000 110000 100000

Scan 894 (5.539 min): SELEGIDERI.D 166

2500000

400000

90000

80000

350000

2000000

l-MA 300000

251

70000

237

1500000 1000000

l-AP 5.77

250000

91

251

60000 50000

237

A

200000

B

40000

91

194

C

4.81

5.545.60

5.42

150000

30000

118

500000

4.22

5.86

100000

50000

119

194

20000 41

69

10000

139

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 0

148

225

281 304323341 369 403 429447

40 60 80 100120140160180200220240260280300320340360380400420440

0

212

257 281299 327 355373 399 429447

40 60 80 100120140160180200220240260280300320340360380400420440

Time-->

m/z-->

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//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 ?-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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