Journalof Analytical Toxicology,Vol.

24, January/February 2000

Rapid Analysisof Amphetamine,Methamphetamine, MDA, and MDMA in Urine UsingSolid-Phase Microextraction, Direct On-Fiber Derivatization,and Analysisby GC-MS
C. Jurado*, M.P. Gim~nez, T. Soriano, M. Mendndez,and M. Repetto
Instituto National de Toxicologfa. P.O. Box 863, 41080-Sevilla, Spain

l Abstract I
A rapid, sensitive, and solvent-free procedure for the simultaneous determination of amphetamine, methamphetamine, 3,4-methyleoedioxyamphetamlne (MDA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine was developed using solid-phase microextractlon (SPME)and gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. A headspace vial containing the urine sample, NaOH, NaCI, and amphetamine-d] as the internal standard was heated at 100~ for 20 rain. A polydimethylsiloxane fiber was maintained in the vial headspacefor 10 rain in order to adsorb the amphetaminic compounds, which were subsequently derivatized by exposing the fiber to trifluoroacetic anhydride for 20 rain in the headspace of another vial maintained at 60~ for 20 rain. The trifluoroacetyl derivatives were desorbed in the GC injection port for 5 rain. Several parameters were considered during the method optimization process. These included a comparison of SPMEwith or without headspace, the required derivatization procedure, and the influence of temperature on the headspace extraction and derivatization methods. The optimized method was validated for the four compounds tested. Calibration curves showed linearity in the range 50-1000 ng/mL (r = 0.9946-0.9999). Recoverydata were 71.89-103.24%. The quantitation limits were 10 ng/mL for amphetamine and methamphetamine and 20 ng/mL for MDA and MDMA. All of these data recommend the applicability of the method for use in the analytical routine of a forensic laboratory.

Introduction
Amphetamines are powerful central nervous system stimulants. The most commonly available amphetamines are amphetamine and methamphetamine, which are used to treat mild depression, obesity, and narcolepsy. In the past several years, a new generation of synthetic amphetamines----"designer drugs'--has appeared, which includes, among others, 3,49 Author to whom correspondence should be addressed. Emad quim@sev,inaltox,es,

methylenedioxymethamphetamine (MDMA)and 3,4-methylenedioxyamphetamine (MDA).These substances, also called entactogens (1), have neurotoxic potency (2). The recreational use of these substances, especially MDMA, has dramaticallyincreased in Spain since the late 1980s. During this period of time, numerous cases of abuse, intoxication, and death involving amphetamine compounds have begun to appear. As a result, the analysis of these substances has become more frequent and should now be considered a routine part of forensic toxicologylaboratory procedures. Traditional extraction methods are tedious and time-consuming and, depending on the method, require evaporation of solvents of varying volumes. An additional problem is the risk of losing the analytes of interest during manipulation. Consequently, new methodologies to solve these problems are being studied. Solid-phase microextraction (SPME) is a recently developed extraction technique in which analytes are adsorbed directly from the sample onto a fused-silicafiber that is coated with an appropriate stationary phase. During adsorption the fiber is inserted into the sample matrix or into the headspace above the sample. The extraction is based on the partitioning of analytes between the stationary phase and the sample matrix. Analytes are adsorbed onto the fiber until equilibrium is reached. The fiber is then inserted into the injection port of a gas chromatograph (GC), where it is heated in order for the analytes to be thermally desorbed. In the past few years, considerable research has been carried out on SPME, and this technique has been increasingly used over time. At first SPMEwas performed only on water samples; more recently, it has been demonstrated to be useful when analyzing such biological samples as urine or blood. SPME has been successfully applied in the analysis of environmental contaminants and volatile compounds (3-8); pesticides (9-14); drugs of abuse, especiallyamphetamines (15-20); and prescription drugs, including barbiturates (21), valproic acid (22), antidepressants (23,24), benzodiazepines(25,26), and antihistamines (27).
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Journal of Analytical Toxicology, Vol. 24, January/February 2000

Veryfew SPME procedures include a derivatization step. Nagasawa et al. (16) made heptafluorobutyramide derivativesfrom amphetamines by injecting heptafluorobutyric anhydride (HFBA) in the injection port just before inserting the fiber. Ugland et al. (18), in their method for amphetamine and methamphetamine, added alkylchloroformate reagent to the urine sample to obtain carbamates, which were then extracted with the fiber. Finally, Gmeiner et al. (28) performed a headspace silylation with N,O-bis[trimethylsilyl]trifluoroacetamide (BSTFA)without any catalyst for on-fiber derivatization of polycyclicaromatic hydrocarbon metabolites. The aim of this study was to developa rapid, reliable, and sensitive method for the simultaneous determination of amphetamine, methamphetamine, MDA, and MDMA in urine samples. This involved headspace-SPME, on-fiber headspace derivatization, and analysis by gas chromatography-mass spectrometry (GC-MS).

above the autotune voltage. The MSD was autotuned daily with perfluorotributylamine. The GC was equipped with an HP-Ultra 1 capillary column (crosslinked methyl-silicone, 25 m x 0.2 ram, 0.33-~um film thickness). The oven temperature was held at 60~ for 1 min, then programmed to 100~ at 40~ then to 200~ at 15~ and finally to 290~ at 40~ and held at this temperature for 5 rain. The injector and GC-MS interface temperatures were 250~ and 280~ respectively. Helium was used as a carrier gas with a flow rate of 1 mL/min. Table I, Selected Ions and Retention Times Compound
Amphetamine Methamphetamine MDA MDMA Amphelamine-d3(I.S.)

Ions* (m/z)
14010o, 11889,9143 1541o o, 11831,9113 1351o0,16238,27518 1541o0,16271,289ts 143100, 12189,9443

R.T: (min)
7.5 8.5 10.1 10.9 7.53

Experimental Reagents and materials Amphetamine, methamphetamine, MDA, MDMA, and amphetamine-d 3 standard solutions (100 p.g/mL in ethanol) were purchased from Radian (Austin, TX) for the preparation of standards and other quality-control materials. Trifluoroacetic anhydride (TFA) was obtained from Sigma (Madrid, Spain). Sodium chloride and sodium hydroxide were obtained from Merck (Barcelona, Spain). An SPME assembly with a replaceable extraction fiber coated with 100 I~m of polydimethylsiloxane and the headspace (5 mL) vials were both purchased from Supelco (Bellefonte, PA). Headspace-SPME method One milliliter of urine and 50 I~Lof deuterated internal standard containing amphetamine-d3 at 10 mg/mL were placed into a 5-mL headspace vial. The sample was alkalinized with 50 I~Lof 2N NaOH, and 0.5 g of NaCI was then added to enhance the vaporization of the amphetaminic compounds. The vials were hermetically sealed and heated at 100~ by using a MultiBlock heater (model 2050-1, Lab-Line Instruments, Inc., Melrose Park, IL). After 10 min (the time required to allow the compounds to reach equilibrium), the SPME device was inserted through the septum of the vial, and the fiber was exposed in the headspace for another 10 min. The needle was then removed and inserted into the headspace of another closed vial that contained TFAand was maintained at 60~ in order to obtain the trifluoroacetyl derivatives of the amphetaminic components present in the sample. After 20 min of exposition in the TFA gas phase, the fiber was thermally desorbed in the GC injection port for 5 min. GC-MS method The GC-MS system consisted of a Hewlett-Packard (Palo Alto, CA) 5890 GC coupled to a 5971A mass selective detector (MSD), which was operated in the selected ion monitoring mode (SIM). The electron multiplier voltage was set at 400 eV 12

" ions used for quantitation are in bold type. R.T. = retention time.

Table II. Estimated RecoveryDAbsolute and Relative to Amphetamine-d3~of Amphetamine, Methamphetamine, MDA, and MDMA from Fortified Urine Samples at Three Different Concentrations Compound
Amphetamine n 6 6 6

Concentration Recovery (%) (ng/mL) absolute relative

50 100 500 93.08 98.79 97.49 103.24 101.64 102.22

Methamphetamine

6 6 6
6 6 6 6 6 6

50 100 500
50 t00 500 50 100 500

85.77 89.16 92.50

87.80 80.78 75.82 76.66 73.15 71.89

75.22 85.26 91.01

90.48 93.17 80.90 90.98 86.09 80.06

MDA

MDMA

Table III. Regression Equations, Correlation Coefficients, and Detection (LOD) and Quantitation (LOQ) Limits Compounds
Amphetamine Methamphetamine MDA MDMA

Correlation LOD LOQ Regression equation coefficient (r2) (ng/mL) (ng/mL)

y = 2.806x- 0.043 y = 0.979x- 0.036 y= 0.992x- 0.010 y = 1.321x - 0.044 0.9946 0.9967 0.9999 0.9991 3 3
6

10 10 20 20

Journal of Analytical Toxicology,Vol. 24, January/February 2000

Table I shows the ions monitored for the different drugs plus the internal standard (I.S.) (amphetamine-d3), and their respective retention times. The substances were identified by their retention times and relative abundance of the three ions monitored. One ion was used for quantitation, and the others were qualifier ions. Analyteswere quantitated by comparing the ratios of peak areas with that of deuterated amphetamine.

As amphetamine-d3 was used as the only I.S. for all of the analytes, the relative recovery with amphetamine-d3 as the I.S. was also measured. Table II shows the recovery data for the four compounds involved in this study at low (50 ng/mL), medium (100 ng/mL), and high (500 ng/mL) concentrations. The results demonstrated a recovery greater than 70% for all of the analytes considered.

Results and Discussion Recovery Absolute recovery of the analytes was established by comparing the peak areas of control urine (n = 6 for each concentration tested) with the peak areas obtained when pure standard analytes were evaporated to dryness and derivatized with TFA.
L

Table IV. Intra-assayand InterassayPrecision (CV) and Accuracy of the Method at Three Different Concentrations
Intra-assay (n = 6) Interassay(n = 16) Conc. precision accuracy precision accuracy Compounds
Amphetamine (ng/mt)
50 1O0 500 50 100 500 50 100 500 50 100 500

Linearity Linearity was established for all four compounds by standard calibration curves, with a range from 50 ng/mL to 1000 ng/mL. The calibration curves were prepared by adding pure standards to blank urine in order to obtain concentrations of 50, 100, 250, 500, 750, and 1000 ng/mL. The fortified samples were analyzed according to the method already described. The curves were obtained by plotting the peak-area ratios (analyte/amphetamine-d3)against the concentrations. Single linear regression equations and correlation coefficients, shown in Table III, were calculated using a Microsoft Graph software package. Correlation coefficients were between 0.9946 and 0.9999. The assay quantitation limits were 10 ng/mL for amphetamine and methamphetamine and 20 ng/mL for MDAand MDMA.
Precision and accuracy

(%)
1.91 0.36 0.02 4.71 3.81 0.01 2.34 2.32 1.68 3.36 1.24 1,25

(%)
96,76 98.36 97.78 89.6 94.71 95.02 99.83 83.11 79.27 93.26 89.29 86.38

(%)
6.24 4.85 0,75 7.67 5.18 0.56 5.42 4.53 1.46 1.22 6.58 1.65

(%)
95.5 99.23 93.82 92.24 91.35 92.50 92.58 95.16 81.97 81.33 80.41 73.46

Methamphetamine

MDA

MDMA

Intra- and interassay precision of the analytical method were determined by analyzing three different concentrations (50, 100, and 500 ng/mL) of fortified urine samples in batches. Intra-assay precision was established by analyzing the samples (n = 6) daily,whereas analyses for interassay precision were performed monthly over a four-month period (n = 4 per month). Accuracy was evaluated by comparing the assayed analyte concentrations for the intra- and interassay precision samples with their fortified concentrations. The data (coefficientof variation and accuracy) for each assay, all of which can be considered acceptable, are shown in Table IV.

Parameter optimization for the SPMEheadspacemethod Several parameters, such as the effect of salting, equilibrium and adsorption times and temperature were evaluated in order to optimize the method. --t---Amphetamine - E . - Methamphetamlne - k - MDA - a - MDMA ...-IF- 1.8." [ When saturating urine samples with NaCl, I the recovery of all four analytes was consider1 0 0 ~ .............. | .......... ! ably, almost 2-3 times, higher than in unsalted samples. In any headspace method, equilibrium between the gas and liquid phases is essential before extraction. A 5-rain period of equilibrium was not enough to achieve good reproducibility in the results. This problem was solved when the time for equilibration was inO~ I I creased to 10 rain. 7(PC ~ I(~'C 110~ Periods of 5, 10, 15, and 20 rain were Temperature checked to establish adequate adsorption time. Figure 1. Correlation between the recovery percentagesof amphetamine, methamphetamine, After exposing the fiber in the headspace for 10 MDA, MDMA, and the I.S. (amphetamine-d 3) with the different temperaturestested in the rain, the analyte recoveries were approxiheadspace-SPMEmethod. mately 100%.
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Journal of Analytical Toxicology, Vol. 24, January/February 2000

During the initial development of this assay we tried to apply our published analytical procedure for the extraction of amphetamine and methamphetamine by the headspace method (29). When heating the vial containing the urine sample at 70~ the recovery data for the amphetamine and methamphetamine were quite good (87% and 76%, respectively).However, the data for MDA and MDMAwere very low. For this reason we compared the relative efficiencyof several temperatures for the extraction of amphetamine-related compounds. The recovery data for the four compounds, plus the I.S., at the different temperatures tested (70~ 80~ 100~ and 110~ are illustrated in Figure 1. A temperature of 100~ was chosen because it permitted an acceptable recovery from the urine of all four compounds.
Derivatization The novelty of this method lies in the application of headspace methodology to perform the extraction and derivatization of all the compounds involved in the study. The need for derivatization was demonstrated by comparing the chromatograms of underivatized (batch A) and derivatized (batch B) extracts of urine fortifiedwith the four compounds. It should be noticed that the chromatographic peaks from the trifluoroacetamide derivatives (Figure 2B) had better peak shape, greater resolution, and higher abundance than the underivatized compounds (Figure 2A). As previously mentioned, only two other studies used derivatization after amphetamine extraction by SPME. We tried to avoid Nagasawa et al.'s (16) derivatization with HFBAbecause use of this reagent may result in damage to the stationary

phase of the column and thus result in a shorter column halflife. The derivatization procedure proposed by Ugland et al. (18) involved adding alkylchloroformate to the urine prior to fiber immersion. As we planned our method to be performed by headspace, we decided not to consider this method. Gmeiner et al. (28) performed a headspace silylation for the analysis of hydrocarbon metabolites using BSTFA. After considering the volatility of the fluorinated derivatizing agents, we tried to perform the derivatization and extraction by headspace. We chose TFA because it is the most reactive and volatile of all the fluorinated anhydrides. Moreover, it is the derivatizing agent routinely applied in our laboratory for amphetamine analyses and thus easier for us to use. To accomplish full derivatization, several temperatures were tested. At room temperature derivatization was incomplete, as demonstrated by the simultaneous appearance of the derivative peaks as well as the peaks corresponding to the parent compounds themselves in the same chromatogram. For this reason, we compared derivatization efficacy at 50~ 60~ 70~ and 80~ A temperature of 60~ was chosen because it was the lowest temperature that allowed the complete derivatization of all compounds. In our opinion, derivatization begins when the SPME fiber is exposed to the TFA headspace at 60~ and ends in the injection port of the GC system. An exposure time of 5 min in the injection port of the GC system permitted complete derivatization and, at the same time, released all of the derivatized compounds adsorbed by the fiber. When shorter times were employed, the fiber retained traces of the different compounds, which were detectable when a blank was subsequently injected.

600000
8 48ooooi

A
3000000' [ 2400000' 1800000' 1200000 600000' 0
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A
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~360000
~240000

120000i
4.00

7,00 8.00 9.00 lO.O0 ll.O0 12.00 13.00 14.00 15,00

6.00

8.00 Time (rain)

I 0.00

12.00

Time (rain)

300O000
2400000

2
4

3500000 I 2800000
1

B
4

2100000 1400000 700000 0

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1800000
1200000 600000

7 O0 8.00 9.00 lO00 11.00 12.00 13.00 14.00 15.00

. . . . . . . . . . . . . . . . .

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LO0 6.00 7.00 8.00 9.00 I0.00 11.00 12.00

Time (mln) Figure 3. Influence of the urine matrix on the extraction. Selected ion chromatograms of fortified urine samples analyzed by headspace-SPME (A) or by SPME (B). Peak identification: I, amphetamine; 2, methamphetamine; 3, MDA; and 4, MDMA.

Time (mln)

Figure 2. Influence of derivatizalion. Selected ion chromatograms of fortified urine samples without derivatization (A) and after derivatization with TFA (B). Peak identification: 1, amphetamine; 2, methamphetamine; 3, MDA; and 4, MDMA.

14

Journalof AnalyticalToxicology,Vol. 24, January/February 2000

Comparison between SPME and SPME-headspacemethods

An additional experiment was performed in order to compare the SPME method with and without headspace. Urine blank was fortifiedwith the four amphetamines (200 ng/mL). The sample was homogenized and divided into two batches, each of which was submitted to the followingextraction procedures: for batch A, we used the previouslyapplied headspace extraction method. For batch B, 2 mL of urine plus the I.S. were placed into a vial and alkalinized with 50 I~L of 2N NaOH. The SPME fiber was then inserted into the urine, which was stirred with a magnetic stirrer. After 10 rain, the fiber was removed from the urine and the adsorbed compounds were derivatizedwith TFAby using the same headspace derivatization procedure as in batch A. The recovery percentages of both methods were similar. Figure 3 shows the chromatograms after injecting both batches. We preferred the headspace method because the chromatograms were cleaner. In addition, the half-life of the polydimethylsiloxanefiber was longer because it was never in touch with the biologicalmatrix. This may be of particular importance when applying this methodology to blood samples.

Conclusions
A method for the simultaneous analysis of amphetamine, methamphetamine, MDA, and MDMAin urine samples has been developed and validated using headspace-SPMEfollowed by headspace on-fiber derivatization and analysis by GC-MS. It has been demonstrated to be simple, only two steps are involved; rapid, a quantitative result is possible in less than 55 rain; sensitive, LOQs are 10 and 20 ng/mL; and specific, the analysis is performed by GC-MS after derivatization. This method has proved to be suitable for application in the analytical routine of a forensic laboratory.

Acknowledgment
The authors thank Glenn Figueroa for his assistance.

References
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5. N. Perchiazzi and R. Ferrari.Application of solid-phasemicro-extraction (SPME)to the determination of volatile organic impurities in pharmaceutical substances.Boil. Chim. Rarm. 135:434-441 (I 996). 6. M. Moder, S. Schraeder,U. Franck,and P. Popp. Determination of phenolic compounds in waste-water by solid-phase microextraction (SPME). Frenesius J. Anal. Chem. 357:326-332 (1997). 7. M i . Chen, I.F. Mao, and C.C. Hsu. Determination of trihalomethanes in drinking water by solid-phase microextraction and gas chromatography with electron-captured detection. Toxicol. Environ. Chem. 60:39--45 (1997). 8. W.E. Brewer, R.C. Galipo, S.L. Morgan, and H.K. Habben. The confirmation of volatiles by solid-phase microextraction and GCMS in the investigation of two traffic fatalities. J. Anal. Toxicol. 21: 286-290 (1997). 9. K. Jinno, T. Muramatsu, Y. Saito, S. Magdic, and J. Pawliszyn. Analysis of pesticides in environmental water samples by solidphase micro-extraction-high-performanceliquid chromatography. J. Chromatogr. A 754:137-144 (1996). 10. K.A. Bucholski, G. Winneke, and L. Dunemann. Determination of byphenils and chlorinated pesticidesin human body fluids and tissues. J. Chromatogr. A 754:479-485 (I 996). 11. T.K. Choudhury, K.O. Gerhardt, and T.R Mawhinney. Solid-phase microextraction of nitrogen- and phosphorous-containing pesticides from water and gas chromatographic analysis. Environ. Sci. Technol. 30:3259-3265 (1996). 12. H. Seno, T. Kumazawa, A. Ishii, M. Nishikaza, K. Watanabe, H. Hattori, and O. Suzuki. Determination of some carbamate pesticides in human body fluids by headspacesolid phase microextraction and gas chromatography. Jpn. J. Forensic Toxicol. 14: 199-203 (1996). 13. M.T. Sng, F.K.Lee, and H.A. Lakso. Solid-phasemicroextraction of organophosphorous pesticides from water. J. Chromatogr. 759: 225-230 (1997). 14. A. Namera, M. Yashiki, N. Nagasawa, Y. Iwasaki, and T. Kojima. Rapid analysisof malathion in blood using head space-solidphase microextraction and selected ion monitoring. Forensic 5cL Int. 88: 125-131 (1997). I 5. M. Yashiki, T. Kojima, T. Miyazaki, N. Nagasawa, Y. lwasaki, and K. Hara. Detection of amphetamines in urine using head spacesolid phase microextraction and chemical ionization selected ion monitoring. Forensic Sci. Int. 76:169-I 77 (I 995). 16. N. Nagasawa, M. Yashiki, Y. lwasaki, K. Hara, and T. Kojima. Rapid analysis of amphetamines in blood using head space-solid phase microextraction and selected ion monitoring. Forensic Sci. Int. 78:95-102 (1996). 17. F. Centini, A. Masti, and I. Barni-Comparini. Quantitative and qualitative analysis of MDMA, MDEA, MA and amphetamine in urine by headspace/solid phase micro-extraction (SPME) and GC/MS. Forensic 5ci. Int. 83:161-166 (1996). 18. H.G. Ugland, M. Krogh, and K.E. Rasmunssen.Aqueous alkylchloroformate derivatization and solid-phase microextraction: determination of amphetamines in urine by capillary gas chromatography. J. Chromatogr. B Biomed. Appl. 701:29-38 (1997). 19. H i . Lord and J. Pawliszyn. Method optimization for the analysis of amphetamines in urine by solid-phase microextraction. Anal. Chem. 69:3899-3906 (1997). 20. C. Battu, P. Marquet, Ai. Fauconnet,E. Lacassie,and G. Lach~ttre. Screening procedure for 21 amphetamine-related compounds in urine using solid-phase microextraction and gas chromatography-mass spectrometry.J. Chromatogr. Sci. 36:I-7 (1998). 21. B.J.Hall and J.S. Brodbelt. Determination of barbituratesby solidphase microextraction (SPME) and ion trap gas chromatography-mass spectrometry.J. Chromatogr. A 777:275-282 (I 997). 22. M. Krogh, K. Johansen, F. Tonnesen,and K.E. Rasmussen.Solidphase microextraction for the determination of the free concentration of valproic acid in human plasma by capillary gas chromatography. J. Chromatogr. B Biomed Appl. 673:299-305 (1995).

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journal of Analytical Toxicology,Vol. 24, January/February 2000 23. X.P. Lee, T. Kumazawa, K. Sato, and O. Suzuki. Detection of antidepressants in whole blood by headspace solid-phase microextraction and capillary gas chromatography. J. Chromatogr. Sci. 35:302-308 (1997). 24. S. Ulrich and J. Martens. Solid-phase microextraction with capillary gas-liquid chromatography and nitrogen-phosphorousselective detection for the assay of antidepressant drugs in human plasma. J. Chromatogr. B Biomed. Appl. 696:217-234 (1997). 25. H. Seno, T. Kumazawa, A. Ishii, K. Watanabe, H. Hattori, and O. Suzuki. Detection of benzodiazepines in urine by direct immersion solid-phase microextraction and gas chromatography. jpn. J. Forensic Toxicot. 15:16-20 (1997). 26. M. Krogh, H. Grefslie, and K.E. Rasmussen. Solvent-modified solid-phase microextraction for the determination of diazepam in human plasma samples by capillary gas chromatography. J. Chromatogr. B Biomed. AppL 689:357-364 (1997). 27. M. Nishikawa, H. Seno, O. Suzuki, T. Kumazawa, K. Watanabe, and H. Hattori. Simple analysisof diphenilmethane antihistaminics and their analogues in bodily fluids by headspace solid-phase microextraction-capillary gas chromatography.J. Chromatogr. Sci. 35:275-279 (1997). 28. G. Gmeiner, C. Krassnig,E. Schmid, and H. Tausch. Fastscreening method for the profile analysis of polycyclic aromatic hydrocarbon metabolites in urine using derivatization--solid-phase microextraction. J, Chromatogr. B Biomed. AppL 705:132-138 (1998). 29. D. Martinez and M,P. Gimenez. Determination of amphetamine and methamphetamine by gas-liquid chromatography (head space). Hum. Toxicol. 2:391-393 (1989). Manuscript received November 1(3, 1998; revision received March 9, 1999.

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