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Clinical Chemistry 48: 1472-1485, 2002;
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(Clinical Chemistry. 2002;48:1472-1485.)
© 2002 American Association for Clinical Chemistry, Inc.

Drug Testing in Blood: Validated Negative-Ion Chemical Ionization Gas Chromatographic–Mass Spectrometric Assay for Determination of Amphetamine and Methamphetamine Enantiomers and Its Application to Toxicology Cases

Frank T. Peters1, Thomas Kraemer1 and Hans H. Maurera1

1 Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Saarland, D-66421 Homburg (Saar), Germany.

aAuthor for correspondence. Fax 49-6841-16-26051; e-mail hans.maurer{at}uniklinik-saarland.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Enantioselective analysis of amphetamine (AM) or methamphetamine (MA) in urine is already a well-established tool for differentiation of illicit from therapeutic ingestion of AM or MA derivatives. However, because of the increasing importance of plasma or serum in analytical toxicology, a method for enantioselective analysis of AM and MA in these matrices is needed.

Methods: AM and/or MA were extracted from 0.2 mL of blood plasma or serum by mixed-mode solid-phase extraction. After derivatization with S-(-)-heptafluorobutyrylprolyl chloride, the resulting diastereomers were separated by gas chromatography on a HP-5MS column during a 15-min program and detected by mass spectrometry in the negative-ion chemical ionization mode (NICI-GC-MS). The method was fully validated and applied to >50 samples from authentic toxicology cases.

Results: The derivatized AM and MA enantiomers were well separated and sensitively detected. The method was linear from 5 to 250 µg/L per enantiomer with analytical recoveries, accuracy, and within- and between-run precision well within required limits. Extraction yields were 88.9–98.6%. Implications of concentrations and enantiomeric composition of AM and MA in the authentic samples were considered.

Conclusions: This sensitive, reliable, rapid NICI-GC-MS assay is suitable for enantioselective determination of AM and MA in blood plasma or serum samples.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Amphetamine (AM)1 and methamphetamine (MA) are chiral compounds whose S-(+) enantiomers exhibit five times greater pharmacologic potency than the corresponding R-(-) enantiomers. AM and MA are both widely abused as stimulants, but both substances are also metabolites of several (racemic or optically pure) therapeutic drugs or are even used as such themselves, e.g., R-(-)-MA in cold medications and S-(+)-AM within withdrawal treatment programs (1)(2)(3)(4)(5). For the correct interpretation of positive AM and/or MA results, it is important to differentiate AM and/or MA abuse from therapeutic ingestion of AM and/or MA or one of their precursor drugs. Enantioselective analysis of AM and MA not only can provide the information needed for this differentiation, it may also allow discrimination of different synthesis or supply routes. The method is especially useful in identifying the synthesis of illicit MA [e.g., synthesis from ephedrine leads to S-(+)-MA, and synthesis from phenylacetone leads to racemic MA]. Enantioselective analysis of AM and/or MA has, therefore, become an important tool in analytical toxicology. This is also reflected by the great number of publications on this issue, which have been extensively reviewed (3)(4)(5)(6).

Enantioseparation is achieved via three principal ways. The first is separation of enantiomers on chiral stationary phases with or without previous derivatization with achiral reagents, using HPLC or gas chromatography (GC) with different detectors. The second approach is derivatization of enantiomers with chiral reagents to the corresponding diastereomers, which are then separated on common achiral stationary phases. The third alternative is separation of enantiomers by capillary electrophoresis using chiral selectors and ultraviolet or diode array detection (7)(8)(9)(10)(11)(12)(13). However, most authors have described procedures for enantioselective analysis of AM and/or MA in urine; only a few have described chiral AM/MA analysis in other matrices, such as blood plasma or serum (11)(14)(15)(16)(17)(18)(19)(20)(21)(22), hair (10)(12)(23)(24)(25)(26), or saliva (19). However, in recent years, the importance of plasma or serum analysis in clinical and forensic toxicology has grown continuously (27)(28). In many countries, analysis of blood samples is mandatory in the context of driving under the influence of drugs. In the future, it will be important to gather data on enantioselective disposition and/or enantiomeric composition of AM and/or MA in human plasma or serum after illicit as well as after therapeutic ingestion of AM and/or MA or one of their precursor drugs.

An ideal analytical method for this purpose should fulfill the following criteria: high sensitivity, because of the relatively low concentrations of AM and/or MA in blood (16)(29); high selectivity, as is provided by mass spectrometric detection; reliability of quantitative data; small sample volume; and short analysis time. The method of Zhou and Krull (22) lacks sensitivity, with the lowest point of the calibration curve corresponding to 150 µg/L for each AM enantiomer. Hutchaleelaha and coworkers (14)(15) used small volumes of rat serum (0.1 mL) and achieved excellent sensitivity. However, the drawbacks of their methods are the time-consuming sample preparation and the use of fluorescence detection, which is not selective enough for toxicologic applications. The method of Hasegawa et al. (16) also has excellent sensitivity, but it requires 3 mL of plasma, too much considering the limited sample volumes available in forensic toxicology. The method of Matin et al. (19) combines good sensitivity with selective mass spectrometric detection, but a rather high sample volume of 1 mL is needed. Furthermore, this method does not include determination of MA, and its reliability can not be assessed because of the lack of validation data. Other methods lack sensitivity (20) or the detailed description needed for assessment of their applicability for routine analysis (11)(17)(18)(21). Thus, a sensitive, reliable, and rapid assay with selective mass spectrometric detection is needed for the determination of AM/MA enantiomers in small volumes of plasma or serum.

In this report, we present a sensitive and selective method for quantification of enantiomers of AM and/or MA that we have developed. In addition, we discuss the implications of AM/MA concentrations and their enantiomeric composition in authentic samples.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
chemicals and reagents
Methanolic solutions (1000 mg/L) of racemic AM-d11 and MA-d5 were obtained from Promochem. Racemic AM hydrochloride, R-(-)-AM hydrochloride, S-(+)-AM hydrochloride, and racemic MA hydrochloride were obtained from Lipomed. R-(-)-MA hydrochloride and S-(+)-MA hydrochloride were supplied by Prof. Pfleger (Homburg, Germany). Isolute Confirm HCX cartridges (130 mg; 3 mL) were obtained from Separtis. Heptafluorobutyric anhydride (HFBA), S-(-)-1-phenylethylamine (S-PEA; S:R >=99.5:0.5), thionyl chloride, and sodium hydrogen carbonate were obtained from Fluka. L-Proline was obtained from Sigma-Aldrich. All other chemicals were obtained from E. Merck. All chemicals were of analytical grade or the highest purity available. The derivatization reagent S-(-)-N-heptafluorobutyrylprolyl chloride (S-HFBPCl) was synthesized in our laboratory according to the procedure described by Lim et al. (30) with modifications (see below).

biosamples
Pooled human blank plasma samples were used for validation of the procedure and were obtained from a local blood bank. Samples were tested for the absence of AM and/or MA before use [gas chromatography–mass spectrometry (GC-MS); limit of detection (LOD), 1 µg/L]. Authentic human blood samples were submitted by various hospitals for toxicologic analysis. Any required Institutional Review Board demands for use of the submitted samples were fulfilled.

synthesis of S-HFBPCL
The synthesis of the derivatization reagent was based on the procedure described by Lim et al. (30) with some modifications. A pear-shaped flask containing 120 mg of L-proline [S-(-)-proline] was placed in an ice bath, and 3 mL of diethyl ether and 0.5 mL of HFBA were subsequently added. The flask was sealed with a Teflon screw cap and left in the ice bath for 10 min. After the flask had sat at room temperature for 2 h, the diethyl ether, excess HFBA, and the byproduct heptafluorobutyric acid were evaporated under reduced pressure at room temperature. Again, the flask was put in an ice bath, and 1.6 mL of thionyl chloride and 3 mL of toluene were added to the residue. The flask was kept at room temperature for 2 h. The toluene and excess thionyl chloride were then evaporated under reduced pressure at room temperature. The residue was dissolved in 1 mL of dichloromethane, which was then evaporated under reduced pressure at room temperature. The last step was repeated, and finally the residue was dissolved in 10 mL of dichloromethane. This solution contained S-HFBPCl at a theoretical concentration of 0.1 mol/L. Aliquots of 1 mL were transferred to autosampler vials that were sealed and stored at -20 °C.

To verify the optical purity of the derivatization reagent, 10 µL of a 10 mg/L solution of optically pure S-PEA was transferred to a reaction vial. After the addition of 0.2 mL of aqueous carbonate buffer (35 g/L sodium bicarbonate–15 g/L sodium carbonate, pH 9) and 10 µL of the derivatization reagent, the reaction vial was sealed and derivatization was carried out as described under the section on sample preparation.

sample preparation for gc-ms analysis
Aliquots (0.2 mL) of plasma or serum were diluted with 2 mL of purified water. After the addition of 0.1 mL of a methanolic solution of the racemic internal standards (IS) AM-d11 and MA-d5 (0.2 mg/L each), the samples were mixed (15 s) on a rotary shaker and loaded on solid-phase extraction (SPE) cartridges previously conditioned with 1 mL of methanol and 1 mL of purified water. After extraction, the cartridges were washed with 1 mL of purified water, 1 mL of 0.01 mol/L hydrochloric acid, and 2 mL of methanol. Reduced pressure was applied until the cartridges were dry, and the analytes were eluted with 1 mL of methanol–aqueous ammonia (98:2 by volume) into 1.5-mL reaction vials. The eluates were evaporated to dryness under a stream of nitrogen at 56 °C. After the addition of 0.2 mL of aqueous carbonate buffer (70 g/L sodium bicarbonate–30 g/L sodium carbonate; 50 g of buffer in 950 mL of water; pH 9) and 6 µL of derivatization reagent (0.1 mol/L S-HFBPCl in dichloromethane), the reaction vials were sealed and left on a rotary shaker at room temperature for 30 min. Thereafter, 0.1 mL of cyclohexane was added, and the reaction vials were sealed again and left on a rotary shaker for 1 min. The phases were separated by centrifugation (10 000g for 1 min), and the cyclohexane phase (upper) was transferred to autosampler vials. Aliquots (3 µL) were injected into the GC-MS system.

enantioselective gc–negative-ion-chemical ionization–ms quantification
Apparatus.
The samples were analyzed using an Agilent Technologies (AT) 6890 Series GC system combined with an AT 5973 network mass selective detector, an AT 7683 series injector, and an AT enhanced Chem Station G1701CA, Ver. C.00.00 21-Dec-1999.

GC conditions.
GC condition were as follows: splitless injection mode; 5% phenyl methyl siloxane column [HP-5MS; 30 m x 0.25 mm (i.d.); 250-nm film thickness]; injection port temperature, 280 °C; carrier gas, helium; flow rate, 1 mL/min; column temperature, 100 °C increased to 180 °C at 30 °C/min, to 230 °C at 5 °C/min, and to 310 °C at 30 °C/min.

MS conditions.
MS conditions were as follows: transfer line heater, 280 °C; negative-ion-chemical ionization (NICI), methane (2 mL/min); source temperature, 150 °C; solvent delay, 9 min; selected ion monitoring (SIM) mode. The program was as follows: 9–11 min, m/z 399 (target ion), 379, and 439 for AM-d11, and m/z 388 (target ion), 368, and 428 for AM; 11–13 min, electron multiplier voltage increased by 400 V, m/z 407 (target ion), 387, and 447 for MA-d5 and m/z 402 (target ion), 382, and 442 for MA. For reagent purity check, the conditions were as follows: solvent delay, 6 min; SIM mode; m/z 269 (target ion), 394, and 414 for S-PEA.

The enantiomers of AM and MA were quantified by comparison of their peak-area ratios (enantiomer of analyte vs corresponding enantiomer of the IS) with calibration curves in which the peak area ratios of enriched calibrators were plotted vs their concentrations using a weighted least-squares regression model. Validation samples and >50 samples from authentic toxicology cases were analyzed with the described method.

assay validation
The GC-MS assay was validated for quantification of AM and MA in plasma with special emphasis on the aspects of enantioselective analysis. The experimental design used for the validation experiments was similar to the one proposed by Wieling et al. (31) with some modifications.

Preparation of solutions.
Separate aqueous stock solutions (100 mg/L; free bases) of hydrochlorides of racemic AM, R-(-)-AM, S-(+)-AM, racemic MA, R-(-)-MA, and S-(+)-MA were prepared. Methanolic stock solutions of racemic AM-d11 and racemic MA-d5 (100 mg/L each) were prepared from commercially available methanolic solutions (1000 mg/L). A methanolic working solution of the IS (0.2 mg/L each) and aqueous analytical standard solutions containing both racemic AM and racemic MA (0.02, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/L each) were also prepared. Aqueous enrichment solutions containing racemic AM and racemic MA (1.0, 2.0, 20.0, and 40.0 mg/L each) were prepared for racemic quality-control (QC) samples. Aqueous enrichment solutions containing 20.0 mg/L each of R-(-)-AM and R-(-)-MA and 1.0 mg/L each of S-(+)-AM, S-(+)-MA, and an aqueous solution containing 1.0 mg/L each of R-(-)-AM and R-(-)-MA and 20.0 mg/L each of S-(+)-AM and S-(+)-MA were prepared for nonracemic QC samples. Methanolic solutions containing both racemic AM and racemic MA (0.08 and 1.6 mg/L each) were prepared for addition to samples used in extraction efficiency experiments. All solutions were stored at 4 °C.

Preparation of QC samples.
For preparation of QC samples, 0.1-mL aliquots [0.5 mL for the above-calibration range (ACR) sample] of the corresponding enrichment solutions were transferred to volumetric flasks. Blank plasma was added stepwise to reach a final volume of 10.0 mL. Before each addition step and after the final volume had been reached, the samples were thoroughly vortex-mixed to obtain homogeneous samples. Thus, the following QC samples containing both AM and MA were prepared [concentrations (µg/L) of R and S enantiomers, respectively, are given in parentheses]: LQS (5/5), LOW (10/10), MED (100/100), HIGH (200/200), HIGH/LOW (200/10), LOW/HIGH (10/200), ACR (800/800). The QC samples were divided into aliquots (0.45 mL) and stored at -20 °C.

Selectivity.
Blank plasma samples from 10 different sources were prepared as described above to check for peaks that might interfere with the detection of the analytes or the IS. A zero sample (blank sample plus IS) was analyzed to check for the absence of analyte ions in the respective peaks of the IS. Blank plasma samples enriched with other sympathomimetic amines [ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine), norpseudoephedrine, phentermine, gepefrine, and pholedrine (1000 µg/L each)] and authentic plasma samples containing other drugs of abuse likely to be co-ingested with AM and MA (amphetamine-like designer drugs, cannabis, and cocaine) were also checked for interferences.

Linearity.
Aliquots of blank plasma (0.2 mL) were enriched with 0.1 mL of the corresponding analytical standard solutions to obtain calibration samples at concentrations of 5, 50, 100, 150, 200, and 250 µg/L of each enantiomer of AM and MA. Replicates (n = 6) at each concentration were analyzed as described above. The regression line was calculated using a weighted (1/c2) least-squares regression model. Daily calibration curves using the same concentrations (single measurement per concentration) were prepared with each batch of validation and authentic samples. The back-calculated concentrations of all calibration samples were compared with their respective nominal values.

Analytical recovery, accuracy, and precision.
QC samples (LOW, MED, HIGH, HIGH/LOW, LOW/HIGH) were analyzed as described above in duplicate on each of 8 days. The ACR QC samples were analyzed in the same way, but only 0.05 mL of sample was used instead of 0.2 mL. The concentrations of AM and MA enantiomers in the QC samples were calculated based on the daily calibration curves. Analytical recovery was calculated for each enantiomer of AM and MA as the percentage of the mean calculated concentration from the theoretical concentration. Within- and between-run imprecision (as CV) was calculated by one-way ANOVA using day as the grouping variable. For evaluation of accuracy, a certified external reference sample (Medidrug® BTMF 2/99-B S-plus; Medichem) was analyzed, and the calculated concentrations of AM and MA were compared with the certified confidence range.

Processed sample stability.
For estimation of the stability of processed samples under the conditions of GC-MS analysis, LOW and HIGH QC samples (n = 8 for each) were extracted and derivatized as described above. The extracts obtained at each concentration were pooled. Aliquots of these pooled extracts at each concentration were transferred to autosampler vials and injected under the conditions of a regular analytical run at time intervals of 2 h and 25 min. The stability of the derivatives was tested by regression analysis in which the absolute peak areas of each enantiomer of AM and MA at each concentration were plotted vs injection time. Instability of processed samples would be indicated by a negative slope significantly different from zero (P <0.05).

Freeze/thaw stability.
For evaluation of freeze/thaw stability, QC samples (LOW and HIGH) were analyzed before (control samples; n = 6) and after three freeze/thaw cycles (stability samples; n = 6). For each freeze/thaw cycle, the samples were frozen at -20 °C for 21 h, thawed, and kept at ambient temperature for 3 h. The experiments were carried out together with the analytical recovery and precision experiments, and the concentrations of the QC samples were calculated based on the daily calibration curves. Stability was tested against a lower acceptance limit corresponding to 90% of the mean of control samples by a one-sided t-test (P <0.05).

Long-term stability.
The experimental design and procedure for evaluation of long-term stability were similar to those used for freeze/thaw stability. QC samples (LOW and HIGH) were analyzed before (control samples; n = 6) and after storage at -20 °C for 6 months (stability samples; n = 6).

Limits.
The lowest point of the calibration curve was the limit of quantification (LOQ) of the method (5 µg/L for each enantiomer of AM and MA). A LOQ QC sample was analyzed (n = 5) to determine whether the criteria established for LOQ [analytical recovery within 100% ± 20% of the nominal value and a CV <20% (32)(33)] were met at this concentration. The LOD was not systematically evaluated. However, enriched samples (n = 5) containing 1 µg/L each of the enantiomers of AM and MA were analyzed to determine whether this concentration could be detected.

Extraction efficiency.
Extraction samples (n = 5) at low (10 µg/L) and high (200 µg/L) concentrations were prepared by enriching a blank plasma (0.2 mL, previously diluted with 2 mL of purified water) with 0.05 mL of a methanolic solution of racemic AM and MA (0.08 and 1.6 mg/L for the low and high extraction samples, respectively). Samples were loaded on SPE columns and extracted. Before evaporation, 0.1 mL of the IS solution was added to each eluate. For the control samples (n = 5), 0.2 mL of blank plasma was diluted with 2 mL of purified water, loaded on SPE columns, and extracted. Before evaporation, 0.05 mL of a methanolic solution of racemic AM and MA (0.08 and 1.6 mg/L for the low and high control samples, respectively) and 0.1 mL of IS solution were added to the eluates. After evaporation, the residues were derivatized and analyzed as described above. Extraction efficiencies (mean and 95% confidence intervals) were estimated by comparison of the peak-area ratios (analytes vs IS) from extraction samples and control samples for each enantiomer of AM and MA at each concentration.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The aim of this study was to develop and validate an enantioselective assay for sensitive, selective, reliable, and rapid determination of AM and/or MA enantiomers from small volumes of plasma or serum. This assay should be applicable to study the composition and disposition of AM and/or MA enantiomers in clinical and forensic toxicology samples.

derivatization reagent
AM and/or MA are usually derivatized before GC-MS analysis to improve their chromatographic properties (4). It was therefore an obvious choice to use an optically pure chiral reagent for the derivatization step, allowing separation of the resulting diastereomers on a standard achiral column. We chose chiral derivatization with S-HFBPCl because this reagent had been used successfully by other authors for analysis of structurally related compounds. In addition, the derivatives (30)(34)(35)(36)(37)(38) were readily ionized in the NICI mode (35)(39).

Synthesis of the S-HFBPCl.
Synthesis of the S-HFBPCl reagent was based on the procedure of Lim et al. (30), which yielded the reagent in high optical purity. However, some modifications were introduced to this procedure to make it easier and safer. Lim et al. further purified their reagents before use by distillation, which we found unnecessary. Good results were obtained with reagents of commercially available purity. In addition, we used a simple ice bath rather than the dry ice–acetone bath used by Lim et al. (30) before the reaction of HFBA with L-proline. For the second reaction step with thionyl chloride, we substituted benzene with the less toxic toluene. Finally, in contrast to the procedure described by Lim et al., we did not cap the vials containing the final reagent solution under nitrogen.

Lim et al. (30) tested the optical purity of the reagent by derivatizing optically pure enantiomers of PEA ({alpha}-methylbenzylamine) and analyzing the resulting diastereomers. We only used S-PEA of certified high optical purity (S:R >=99.5:0.5), which forms the S,S diastereomer with S-HFBPCl. Thus, the peak corresponding to the reagent impurity (S,R diastereomer) eluted first, allowing better resolution from the slightly tailing main peak (S,S diastereomer) and, therefore, easier detection of even small amounts of impurities. However, it must be mentioned that in this experiment the S,R diastereomer is not separated from the enantiomeric R,S diastereomer, which is formed by derivatization of R-PEA, the optical impurity of S-PEA, with S-HFBPCl. Therefore, it was impossible to differentiate whether the impurity peak corresponded to impurities of the reagent, the S-PEA, or both. However, in a freshly prepared batch of the reagent, the area of the impurity peak corresponded to <0.1% of the total peak area (main peak plus impurity peak). Therefore, the optical purity of the synthesized S-HFBPCl was >=99.9%. In two previously synthesized batches of S-HFBPCl, which had been stored at -20 °C for 4 and 10 months, respectively, the optical purity was >=99.8%. This shows that our modified procedure led to a reagent of high optical purity that was stable for at least 10 months at -20 °C. Furthermore, no or very little racemization of the reagent occurred during synthesis or under the conditions of derivatization.

sample preparation
Extraction procedure.
A mixed-mode (reversed-phase C8 and strong cation-exchange) SPE procedure was developed, which led to very clean extracts, even from hemolyzed blood samples. The use of water rather than buffer for dilution of samples and the second conditioning step led to somewhat higher extraction efficiencies for AM and/or MA, which can be explained by the competition of buffer cations (Na+ or K+) and protonated analyte ions at the cation-exchange sites on the sorbent. Furthermore, use of buffer was not necessary because plasma or serum is physiologically buffered. During the washing steps, nonbasic compounds were effectively separated from the basic analytes. These were eluted from the cation-exchange sorbent with methanol–aqueous ammonia (98:2 by volume), and the eluates were carefully evaporated to dryness under mild conditions to prevent evaporative loss of AM and/or MA.

Derivatization procedure.
Derivatization of AM and/or MA enantiomers to the corresponding R,S and S,S diastereomers, whose structures and NICI mass spectra are shown in Fig. 1 , was carried out under aqueous alkaline (Schotten–Baumann) conditions. Derivatization under these conditions had already been described by Lim et al. (30) to have advantages over derivatization in organic solvents: little or no racemization occurred and excess reagent was destroyed, eliminating GC-MS interference. The latter fact also improved column life. No loss of column performance was observed in our study after hundreds of analyses.



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  		Figure 1. Structures and NICI mass spectra of S-HFBP derivatives of R-(-)-AM-d11 (panel 1), R-(-)-AM (panel 2), S-(+)-AM-d11 (panel 3), S-(+)-AM (panel 4), R-(-)-MA-d5 (panel 5), R-(-)-MA (panel 6), S-(+)-MA-d5 (panel 7), and S-(+)-MA (panel 8).

No obvious differences in derivatization yields of the enantiomers of AM and/or MA were observed. Small differences in the peak areas of diastereomers corresponding to the AM and MA enantiomers were attributable to slightly different fragmentation patterns in the full-scan NICI mass spectra shown in Fig. 1Up . The derivatives were extracted from the aqueous phase with 0.1 mL of cyclohexane. This provided a further cleanup of the final extract, as only rather lipophilic compounds were extracted from the aqueous phase by this solvent.

enantioselective nici-gc-ms quantification
GC conditions.
The HP-5MS GC column is a standard achiral GC column with especially low bleed characteristics, which is advantageous for sensitive MS detection. Initial temperature and the oven ramp temperature program were optimized to achieve baseline separation of both AM and MA enantiomers within 15 min and 20 s. This run time was short compared with previously described methods (11)(15)(16)(17)(18)(19)(20)(21)(22).

MS conditions.
Derivatization with S-HFBPCl led to AM and/or MA derivatives that were readily ionized in the NICI ionization mode because of the electronegativity of the heptafluorobutyryl moiety (39). The high sensitivity of this ionization technique was further increased by operating the MS in the SIM mode. However, the ionization properties of the MA derivatives were not quite as good as those of the AM derivatives. Therefore, the electron multiplier voltage was increased by 400 V in the time window of MA derivatives to reach similar peak heights for both AM and MA derivatives. For the AM and AM-d11 enantiomers, the two most abundant fragment ions and the respective molecular ions were chosen from the full-scan NICI mass spectra for monitoring in the SIM mode. The corresponding (homologous) ions were chosen for MA and MA-d5 enantiomers. The most abundant ions for AM (m/z 388), MA (m/z 402), AM-d11 (m/z 399), and MA-d5 (m/z 407) were chosen as the target ions for quantification (see Fig. 1Up ). Because of the relatively high m/z values of the ions, the observed noise was low. This low noise, combined with the excellent ionization properties in the NICI mode, allowed sensitive detection of AM and/or MA enantiomers, even in a small sample volume (0.2 mL). Shown in Fig. 2 are merged mass fragmentograms of a blank plasma (Fig. 2A ) and an enriched sample (Fig. 2B ) containing 5 µg/L each of the enantiomers of AM and MA after SPE and derivatization with HFBPCl. Even at this low concentration, the peak abundances of both enantiomers of AM and MA were much higher than the background noise (signal-to-noise ratio >>10).



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  		Figure 2. Merged mass fragmentograms of plasma samples after SPE and derivatization with S-HFBPCl.

(A), blank plasma sample. The peaks indicate matrix peaks in the mass fragmentograms of ions m/z 368 (left) and 387 (right). (B), blank plasma sample enriched with 5 µg/L each of R-(-)-AM (peak 2), S-(+)-AM (peak 4), R-(-)-MA (peak 6), and S-(+)-MA (peak 8). Peaks 1, 3, 5, and 7 correspond to 50 µg/L each of R-(-)-AM-d11, S-(+)-AM-d11, R-(-)-MA-d5, and S-(+)-MA-d5, respectively. Peak numbers correspond to the panels in Fig. 1Up . Inset, enlargement of area containing peaks 6 and 8.

assay validation
The GC-MS assay was validated for quantification of AM and MA in plasma with special consideration to aspects of enantioselective analysis. The experimental design used for the validation experiments was similar to the one proposed by Wieling et al. (31). Some modifications were introduced where it seemed reasonable.

Preparation of QC samples.
QC samples for analytical recovery and precision experiments were prepared at three concentrations (LOW, MED, and HIGH) covering the calibration range, as has become the international standard (31)(33)(40)(41). To account for the high concentrations expected in toxicology cases, an ACR sample containing the analytes at concentrations above the highest points of the calibration curves was also prepared. Only 0.05 mL, rather than the usual sample volume (0.2 mL), was analyzed so that the amounts of analytes were within the ranges of the calibration curves. Additionally, QC samples containing high concentrations of one enantiomer each of AM and MA and low concentrations of the other enantiomer (HIGH/LOW and LOW/HIGH) were prepared to evaluate the analytical recovery and precision for the quantification of low concentrations of one enantiomer in the presence of high concentrations of the other.

Selectivity.
Ten blank plasma samples from different sources were prepared as described above to check for peaks that might interfere with the detection of the analytes or the IS. Typical mass fragmentograms of a blank sample after SPE and derivatization are shown in Fig. 2AUp . Only two small peaks are present in these fragmentograms, which might have derived from endogenous compounds. The peak on the left (in the mass fragmentogram of ion m/z 368) was well separated from the AM enantiomers (peaks 2 and 4 in Fig. 2BUp ) and did not, therefore, interfere with the analysis. The peak on the right (in the mass fragmentogram of ion m/z 387) had a retention time similar to the S-(+)-MA-d5 derivative. However, this did not cause interference with the analysis because this ion is only a qualifier ion of S-(+)-MA-d5. Furthermore, the peak abundance of the matrix peak could be neglected in comparison with the peak abundance of the S-(+)-MA-d5 derivative in samples containing the IS. The absence of analyte ions in the peaks of the IS in a zero sample (blank sample plus IS) demonstrated that the IS did not contain relevant amounts of nondeuterated substance and that the mass spectra contained no fragment ions identical to the monitored analyte ions. No interference was detected in any of the enriched samples containing other sympathomimetic amines or in any of the authentic samples tested that contained other drugs of abuse (see Materials and Methods).

Calibration model.
Calibration samples were prepared at six evenly distributed concentrations (5–250 µg/L) for each enantiomer of AM and MA. In our experience, this range covers concentrations that are to be expected for most authentic samples. The inverse of the squared concentration (1/c2) was an appropriate weighting factor to account for unequal variances (heteroscedasticity) over the calibration range. Evaluation of weighted linear and second-order regression models indicated a slight curvature in the data and a better fit of the second-order model. However, Hartmann et al. (40) have proposed that simpler linear models be accepted if the data for precision and analytical recovery are within acceptable limits. Because this was the case for our data, we decided to accept the weighted linear regression model. The slopes and y-intercepts (including 95% confidence intervals of both parameters) and coefficients of determination for each enantiomer of AM and MA are listed in Table 1 . The narrow confidence intervals of the regression parameters justified confinement to single measurements for daily calibration curves, which were prepared with each batch of samples. The back-calculated concentrations of all calibration samples were within 100% ± 15% of the nominal concentrations, thus fulfilling the criteria established by Shah et al. (33).


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  		Table 1. y-Intercepts, slopes, and coefficients of determination of weighted (1/c2) linear calibration models for AM and MA enantiomers.1

Analytical recovery, accuracy, and precision.
QC samples were analyzed in duplicate on each of 8 days as has been proposed by Hartmann et al. (40). The results of the analytical recovery experiments are given in Table 2 . All except the low concentrations of AM enantiomers in the LOW/HIGH and the HIGH/LOW samples were well within 100% ± 15% of the nominal concentrations, which has been proposed as an acceptance limit for this parameter (32)(33). The high values for analytical recoveries of AM enantiomers in the LOW/HIGH and HIGH/LOW samples were attributable to optical impurities in the R-(-)-AM hydrochloride and S-(+)-AM hydrochloride used for the preparation of enrichment solutions of these samples; they contained ~2.0% and ~1.6% of their corresponding optical antipodes, respectively. Thus, these samples actually contained ~14 µg/L R-(-)-AM and ~13.2 µg/L S-(+)-AM, respectively, instead of the nominal 10 µg/L. In Table 2 , the numbers in parentheses indicate the correspondingly corrected nominal values and the resulting analytical recoveries. These data show that the analytical recoveries were acceptable after this correction. Accuracy was evaluated by analysis of a certified reference sample. The concentration of total AM was calculated to be 78.3 µg/L. This value lay within the certified confidence range of 59.7–96.5 µg/L and was virtually identical to the target value for total AM (78.1 µg/L). The target value for MA was not certified, and no confidence range was given. However, the calculated value (106.9 µg/L) for MA lay very close to the target value for MA (105.6 µg/L). Additionally, this sample was found to contain racemic AM, whereas MA was present in the form of pure S-(+)-MA.


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  		Table 2. Analytical recovery data for the determination of AM and MA enantiomers in human plasma.

Within-run precision (repeatability) and between-run precision were calculated by one-way ANOVA using day as the grouping variable. According to Wieling et al. (31), within-run precision was calculated as the CV within groups. Between-run precision was calculated, accordingly, as the CV between groups. All precision data lay within the established acceptance limits of 15% (32)(33), and most were <5% (Table 3 ). These data show that possible variations during sample preparation were well compensated by the use of deuterated analogs as IS. The results obtained for analytical recovery, accuracy, and precision for both internal and external QC samples confirmed the reliability of the described method.


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  		Table 3. Within- and between-run precision data for the determination of AM and MA enantiomers in human plasma.

Processed sample stability.
The design of the experiments and evaluation procedures were according to Wieling et al. (31). Eight extracts of each LOW and HIGH QC sample were pooled separately and aliquoted to obtain identical processed samples at both concentrations. These samples were injected under the conditions of a routine analytical run at fixed time intervals. The stability of the derivatives was tested by regression analysis in which the absolute peak areas of each diastereomer of AM and MA at each concentration were plotted vs injection time. Instability of processed samples would be indicated by a negative slope significantly different from zero (P <0.05). No instability was observed during a time interval of 17 h, which was ~2–3 h longer than the maximum run time needed for analysis of one batch of samples in our study.

Freeze/thaw stability.
Freezing of QC samples for only 21 h instead of 24 h (40) and leaving them at ambient temperature for 3 h instead of 1 h (40) allowed simultaneous evaluation of benchtop stability, i.e., stability of the analytes in the matrix at ambient temperature over the expected maximum period of time needed for preparation of a batch of samples. Stability was tested by comparing the mean value of the stability samples to an acceptance limit corresponding to 90% of the mean value of control samples by a one-sided t-test (P <0.05). These criteria were fulfilled by both enantiomers of AM and MA at low and high concentrations.

Long-term stability.
The experimental design for evaluation of long-term stability was similar to the one used for freeze/thaw stability. The same acceptance limit was applied. Again, both enantiomers of AM and MA fulfilled these criteria at both low and high concentrations.

Limits.
Several definitions for the LOQ can be found in the literature. One is to define LOQ as the lowest concentration of analyte in a sample that can be quantified with acceptable analytical recoveries and precision. Analytical recoveries within 100% ± 20% and a CV <=20% have been proposed as acceptance limits (32)(33). Replicates (n = 5) of an LQS sample were analyzed to determine whether these criteria were met. The calculated analytical recoveries were 102% for both AM enantiomers, 112% for R-(-)-MA, and 106% for S-(+)-MA. CVs were 5% for both AM enantiomers, 10% for R-(-)-MA, and 6.2% for S-(+)-MA. These values indicate that the criteria for LOQ were fulfilled at the lowest point of the calibration curve. However, because no calibration points <5 µg/L were measured, this concentration was the practical LOQ. Typical mass fragmentograms of a LQS sample after SPE and derivatization are shown in Fig. 2BUp . It can be seen that the peak abundances are far above the noise. The peaks corresponding to the MA enantiomers are somewhat obscured by the IS in the main chromatogram and therefore are enlarged in the inset.

A systematic evaluation of the LOD of the described method does not seem reasonable because even small optical impurities of the derivatization reagent would make the LOD of one enantiomer dependent on the concentration of its optical antipode. In other words, if one enantiomer is present in the sample at a rather high concentration, small impurities of the derivatization reagent would cause a signal at the retention time of the optical antipode even if it was not present in the sample (see the discussion of reagent purity check with S-PEA). Nevertheless, a sample enriched with racemic AM and MA containing 1 µg/L each of the enantiomers of AM and MA was analyzed to determine whether this concentration would still be detectable. In this sample, the signal-to-noise ratio was still >3.

Extraction efficiency.
For estimation of extraction efficiency, the AM and MA enantiomers were added to blank plasma samples and to the eluates of blank plasma samples for preparation of extraction and control samples, respectively. Methanolic solutions (0.05 mL) containing high and low concentrations of racemic AM and MA were used for this purpose because aqueous solutions might have caused losses of AM and/or MA during evaporation of the enriched blank plasma eluate. The IS was added to the eluates of both extraction and control samples. This procedure allowed estimation of extraction efficiency by comparison of the peak-area ratios (analyte vs IS) of extraction samples with those of the control samples. The results, including 95% confidence intervals, are listed in Table 4 . As expected, the results were similar for corresponding enantiomers of AM and MA. The fact that all values lay between 88.9% and 98.6% and the narrow 95% confidence intervals emphasize the suitability of the described SPE procedure for the extraction of AM and MA from plasma samples.


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  		Table 4. Extraction yields of the SPE procedure for extraction of AM and MA enantiomers from human plasma at high and low concentrations.

application
NICI-GC-MS results.
The described GC-MS method was applied to samples from 51 authentic clinical toxicology cases. In approximately one-half of these cases (n = 23), AM and/or MA were found during routine drug screening analysis (screening samples), but the patients showed no symptoms of AM and/or MA ingestion or intoxication. The other half (n = 26) were samples from cases in which the patients showed typical symptoms of AM and/or MA intoxication, e.g., tachycardia, hypertension, agitation, and hallucinations (intoxication samples). In the remaining two cases, AM and/or MA were found after therapeutic ingestion of selegiline. Shown in Fig. 3 are merged mass fragmentograms of plasma samples from three authentic cases after SPE and derivatization with S-HFBPCl [cases 48 (Fig. 3A ), 34 (Fig. 3B ), and 50 (Fig. 3C ) in Tables 5 and 6 ]. Complete lists of the results for AM and MA are given in Tables 5 and 6 , respectively. As expected, the two samples after ingestion of selegiline (cases 50 and 51) contained only R-(-)-AM and R-(-)-MA. All other samples contained mixtures of enantiomers of AM only (n = 33) or of AM and MA (n = 16). The ratios of AM enantiomers (R vs S) ranged from 0.97 to 1.66 (see Table 5 ), with a mean value of 1.15 (n = 36). No significant difference (P >0.05) was detected between the AM enantiomer ratios of screening (n = 12) and intoxication (n = 24) samples. The findings were similar for the MA enantiomers. Their ratios (R vs S) ranged from 1.02 to 1.63 (see Table 6 ), with a mean value of 1.33 (n = 5). These findings are in accordance with the literature and can be attributed to the faster disposition kinetics of the S-(+) enantiomers compared with the R-(-) enantiomers of AM and especially MA (5)(19). One can expect enantiomer ratios to increase with time after ingestion of racemic AM and/or MA. However, important information, e.g., dosage, time of ingestion, and/or sampling time, was unreliable or not available for most of the samples, as is often the case in clinical toxicology. Therefore, a correlation between enantiomer ratios and time after ingestion could not be established.



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  		Figure 3. Merged mass fragmentograms of authentic plasma samples after SPE and derivatization with S-HFBPCl.

(A), case 48 in Table 5Up (intoxication with racemic AM). Peaks 2 and 4 correspond to 193.7 µg/L R-(-)-AM and 186.9 µg/L S-(+)-AM, respectively. (B), case 34 in Tables 5Up and 6Up (intoxication with racemic AM and MA). Peaks 2, 4, 6, and 8 correspond to 20.6 µg/L R-(-)-AM, 17.8 µg/L S-(+)-AM, 20.8 µg/L R-(-)-MA, and 14.1 µg/L S-(+)-MA, respectively. (C), case 50 in Tables 5Up and 6Up (therapeutic ingestion of selegiline). Peaks 2 and 6 correspond to 13.7 µg/L R-(-)-AM and 45.6 µg/L R-(-)-MA, respectively. In all panels, peaks 1, 3, 5, and 7 correspond to 50 µg/L each of R-(-)-AM-d11, S-(+)-AM-d11, R-(-)-MA-d5, and S-(+)-MA-d5, respectively.


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  		Table 5. Plasma AM concentrations (single enantiomers and total) and enantiomer ratios from authentic toxicology cases.


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  		Table 6. Plasma MA concentrations (single enantiomers and total) and enantiomer ratios from authentic toxicology cases.

Plasma concentrations of S-(+)-AM/MA and total AM/MA from screening and intoxication samples were compared. Only intoxication samples from patients whose symptoms were likely to be attributable to AM and/or MA were included in this evaluation. The corresponding data in Table 5Up are indicated. Other samples from intoxicated patients, in whom the symptoms might also have been attributable to other drugs detected in a previous screening analysis, were excluded. Concentrations of S-(+)-AM (total AM) ranged from below the LOQ to 107.4 (218.4) µg/L in screening samples (n = 23) and from 6.4 (13.0) to 186.9 (398.7) µg/L in intoxication samples (n = 17). Approximately 80% of the samples with concentrations >=50 (100) µg/L but only ~20% of those with concentrations <=10 (20) µg/L were intoxication samples. This is in good accordance with the findings of Pizarro et al. (29), who found a maximum plasma concentration for total AM of 69.1 µg/L after a 40-mg dose of racemic AM. This was the highest dose tested in this study, and it caused clear psychomotor activity and subjective effects. However, the overlap of concentrations from our screening and intoxication samples indicates that interpretation of AM plasma concentration is rather difficult in the concentration range between 10 (20) and 50 (100) µg/L.

In only one of the screening samples (n = 6) did the concentration of S-(+)-MA (total MA) exceed the LOQ, with a value of 6.1 (16.1) µg/L. In intoxication samples (n = 7), the concentrations ranged from below the LOQ to 18.4 (37.2) µg/L. Concentrations greater than the LOQ were measured in only three samples: 5.4 (11.1), 14.1 (34.9), and 18.4 (37.2) µg/L. As expected, plasma concentrations from intoxication samples tended to be higher than those from screening samples. However, because AM was present in all of these samples and because we did not know whether it had been ingested or was present only as a metabolite of MA, we could not draw any reliable conclusions on the correlation of MA concentrations and symptoms of intoxication.

Implications of enantioselective AM/MA analysis.
Concerning interpretation of AM and/or MA results from plasma, enantioselective analysis provides useful information. Taking into account the different pharmacologic potencies of the AM and/or MA enantiomers, observed symptoms should be attributable mainly to the S-(+) enantiomers. However, the actual concentrations of these enantiomers can not be estimated from the results of achiral analysis. This is especially important because both racemic and optically pure enantiomers are available as medicaments or illicit drugs, especially in the case of MA.

Furthermore, ingestion of selegiline might lead to positive immunoassay screening results. Achiral confirmation analysis of such samples, as is common in forensic toxicology, would lead to the unjustified conclusion of illicit MA ingestion.

In conclusion, the presented highly sensitive method was fully validated with special consideration to aspects of enantioselectivity. Applicability was demonstrated by analysis of >50 authentic samples. The information on the enantiomeric composition of AM and MA may be helpful for the interpretation of results. The method will also be useful for the evaluation of enantioselective metabolism and/or pharmacokinetic studies of AM and/or MA and their precursor drugs.


   Acknowledgments
 
We thank Peter Wollenberg and Armin A. Weber for their assistance.


   Footnotes
 
1 Nonstandard abbreviations: AM, amphetamine; MA, methamphetamine; GC, gas chromatography; HFBA, heptafluorobutyric anhydride; S-PEA, S-(-)-1-phenylethylamine; S-HFBPCl, S-(-)-N-heptafluorobutyrylprolyl chloride; MS, mass spectrometry; LOD, limit of detection; IS, internal standard; SPE, solid-phase extraction; AT, Agilent Technologies; NICI, negative-ion chemical ionization; SIM, selected ion monitoring; QC, quality control; ACR, above- calibration range; and LOQ, limit of quantification.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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