Methamphetamine Enantiomers Research Articles

Enantiodiscrimination of methamphetamine by circular dichroism using a porphyrin tweezer

Using exciton-coupled circular dichroism (ECCD) spectroscopy, our lab was able to differentiate between the two enantiomers of methamphetamine using a commercially available porphyrin tweezer as an achiral host. The host-guest complex formed with (+)-(S)-methamphetamine produced a negative bisignate-shaped ECCD spectrum, whereas the complex formed with (-)-(R)-methamphetamine produced a positive one. This sensitive technique could serve as an alternative method for the enantiodiscrimination of chiral methamphetamine, a commonly abused drug in the United States.

Negative-Ion Chemical Ionization Gas Chromatography–Mass Spectrometry Assay for Enantioselective Measurement of Amphetamines in Oral Fluid: Application to a Controlled Study with MDMA and Driving Under the Influence Cases

Enantioselective analysis of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) helps interpret toxicological results. Methods have been described for various matrices, but so far not for oral fluid, a matrix of increasing importance in testing for drugs of abuse, especially in the context of driving under the influence of drugs (DUID). After dilution with 200 microL carbonate buffer (pH 9), oral fluid samples (10-50 microL) were derivatized with S-heptafluorobutyrylprolyl chloride. The resulting diastereomers were extracted into 100 microL of cyclohexane, separated by gas chromatography (HP-5MS column), and detected by mass spectrometry in the negative-ion chemical ionization mode (GC-NICI-MS). The method was validated and applied to samples from a controlled study with MDMA and from authentic DUID cases. The derivatized AM, MA, MDA, MDMA, and MDEA enantiomers were well separated from each other. The method was linear from 5-250 microg/L per enantiomer of MDA and from 25-1250 microg/L per enantiomer of AM, MA, MDMA, and MDEA. With the exception of MDEA, analytical recoveries, repeatability, and intermediate precision were within required limits. The analyte concentrations and enantiomer ratios in the application samples correlated only weakly with corresponding published plasma data. This sensitive, reliable, and fast GC-NICI-MS assay enantioselectively measures AM, MA, MDA, and MDMA in oral fluid samples. Prediction of plasma concentrations and enantiomer ratios from respective oral fluid data is not possible.

Human pharmacology of the methamphetamine stereoisomers

To help predict the consequences of precursor regulation, we compared the pharmacokinetics and pharmacodynamics of the methamphetamine (INN, metamfetamine) stereoisomers. In this study 12 methamphetamine abusers received intravenous d-methamphetamine (0.25 and 0.5 mg/kg), l-methamphetamine (0.25 and 0.5 mg/kg), racemic methamphetamine (0.5 mg/kg), or placebo with the use of a 6-session, double-blind, placebo-controlled, balanced crossover design. Pharmacokinetic measures (including area under the plasma concentration-time curve [AUC], elimination half-life, systemic clearance, apparent volume of distribution during the elimination phase, and apparent bioavailability) and pharmacodynamic measures (including heart rate, blood pressure, respiratory rate, and visual analog scale ratings for "intoxication," "good drug effect," and "drug liking") were obtained. Pharmacokinetic parameters for the individual enantiomers given separately were similar, with dose-proportional increases in AUC and maximum plasma concentration. After racemate administration, the AUC for d-methamphetamine was 30% smaller than that for l-methamphetamine (P = .0085). The elimination half-lives were longer for l-methamphetamine (13.3-15.0 hours) than for d-methamphetamine (10.2-10.7 hours) (P < .0001). Compared with placebo, d-methamphetamine (0.25 mg/kg, 0.5 mg/kg, and racemic) increased the heart rate (P < .0001), blood pressure (P < .0001), and respiratory rate (P < .05), and this increase lasted for 6 hours. The peak heart rate changes after racemic methamphetamine and 0.5 mg/kg d- and l-methamphetamine were similar (18.7 +/- 23.4 beats/min, 13.5 +/- 18.5 beats/min, and 10.7 +/- 10.2 beats/min, respectively), but racemic methamphetamine and 0.5 mg/kg d-methamphetamine increased systolic blood pressure more than 0.5 mg/kg l-methamphetamine (33.4 +/- 17.8 beats/min and 34.5 +/- 18.9 beats/min, respectively, versus 19.5 +/- 11.3 beats/min; P < .01). l-Methamphetamine, 0.5 mg/kg, was psychoactive, producing peak intoxication (46.0 +/- 35.3 versus 30.3 +/- 24.9) and drug liking (47.7 +/- 35.1 versus 28.6 +/- 24.8) ratings similar to 0.5 mg/kg d-methamphetamine, but the effects of l-methamphetamine dissipated more quickly (approximately 3 hours versus 6 hours). The effects of 0.25 mg/kg l-methamphetamine were similar to those of placebo. Racemic methamphetamine was similar to d-methamphetamine with regard to most pharmacodynamic measures. The pharmacokinetics of the methamphetamine enantiomers are similar, but there are substantial pharmacodynamic differences between the isomers. At high doses, l-methamphetamine intoxication is similar to that of d-methamphetamine, but the psychodynamic effects are shorter-lived and less desired by abusers. Racemic and d-methamphetamine have similar effects and would be expected to have comparable abuse liabilities.

Sensitive, rapid and validated gas chromatography/negative ion chemical ionization-mass spectrometry assay including derivatisation with a novel chiral agent for the enantioselective quantification of amphetamine-type stimulants in hair☆

A novel chiral derivatisation agent, (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, was used for the indirect resolution of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers using gas chromatography coupled to mass spectrometry operating in the negative-ion chemical ionization mode (GC/MS-NICI). This new chiral derivatisation reagent was readily obtained in optically pure form after a simple two-step synthesis. Optimal derivatisation was accomplished in 15 min at room temperature in a carbonate buffer and the resulting diastereoisomers were base line separated by GC in 12 min only. No racemization was observed during the derivatisation. The method was applied and fully validated for the enantiomeric quantification of amphetamines and methylenedioxylated amphetamines in hair. The analyses of 24 hair specimens from suspected ATS abusers showed that 24 cases were positive for MA and/or AM enantiomers and that in most cases the concentrations of (S)-MA and (S)-AM exceeded those of the corresponding (R)-enantiomers. One hair specimen was tested positive for both enantiomers of MDMA and MDA.

High-Performance Liquid Chromatographic-Mass Spectrometric Determination of Methamphetamine and Amphetamine Enantiomers, Desmethylselegiline and Selegiline, in Hair Samples of Long-Term Methamphetamine Abusers or Selegiline Users

We devised a highly sensitive method for simultaneously determining methamphetamine (MA) and amphetamine (AP) enantiomers, desmethylselegiline (DMSG) and selegiline (SG), in human hair using a derivatization technique and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). MA and AP enantiomers and DMSG were effectively converted to trifluoroacetic acid (TFA) derivatives, and the sensitivity of MA and DMSG increased five times over compared with that of free bases. The TFA derivatives of each compound were stable within one week in a stock solution of methanol or for 24 h in the HPLC mobile phase (mixture of methanol and ammonium formate buffer). Each compound was well separated, and calibration curves were linear in the concentration range 0.04-40 ng/mg for MA enantiomers, SG and DMSG, and 0.2-40 ng/mg for AP enantiomers. The accuracy and precision of the method were evaluated, and relative standard deviations were within 7%. Our method was successfully applied to hair samples obtained from long-term MA abusers and SG users. (+)-MA and (+)-AP were detected from three MA abusers at concentrations of 0.79-20.85 and 0.04-3.30 ng/mg, respectively. On the other hand, (-)-MA, (-)-AP, DMSG, and SG were detected in three SG users at concentrations of 2.48-9.05, 0.72-3.10, 0.12-0.59, and 0-0.04 ng/mg, respectively. Based on our obtained data, discrimination of MA abusers from SG users was considered to be possible by comparing optical isomers of MA and AP, the existence of DMSG and/or SG, and the concentration ratio of AP to MA in hair samples.

Pharmacokinetics of intravenous d- and l-methamphetamine in healthy volunteers

Background/Aims Methamphetamine has a chiral structure. The aim of the present study was to assess the pharmacokinetics of methamphetamine enantiomers in humans. Methods In a randomized, double-blind, six-period crossover study, methamphetamine (0.25 mg/kg d- or l-, 0.5 mg/kg d-, l- or racemic (1:1) methamphetamine and placebo) was given intravenously to 12 healthy methamphetamine users. Results d-Methamphetamine showed 30% smaller AUC compared to l-methamphetamine after administration of the racemic form. However, AUCs were similar between d- and l-methamphetamine when given on separate occasions at both doses (0.25 and 0.5 mg/kg). AUC ratios for d-methamphetamine/l-methamphetamine were 0.910 (90% CI; 0.837–0.984) for the 0.25 mg/kg dose, 0.894 (0.821–0.967) for the 0.5 mg/kg dose, and 0.679 (0.623–0.736) for the racemic dose. AUC and Cmax at 0.5 mg/kg dose were twofold greater than those at 0.25 mg/kg dose for both enantiomers. The elimination half-lives were slightly longer for l-methamphetamine. Vd was similar for both formulations. AUC ratios for amphetamine/methamphetamine were significantly higher for the d-enantiomer (16–17%) than the l-enantiomer (3–4%). Conclusions d- and l-Methamphetamine are apparently bioequivalent in terms of AUC when given separately, with linear pharmacokinetics between 0.25 and 0.5 mg/kg doses. However, administration of d-methamphetamine results in a higher metabolite concentration compared to the l-form. NIDA DA12521 and DA12393. Clinical Pharmacology & Therapeutics (2005) 77, P37–P37; doi: 10.1016/j.clpt.2004.12.034

Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography–mass spectrometry

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography–mass spectrometry (GC–MS) is described. Hair specimens were digested with 1 M sodium hydroxide at 100 °C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with ( S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC–MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1–45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios ( R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of ( R)-MA and ( R)-AM than those of the corresponding ( S)-enantiomers.

Simultaneous Chiral Analysis of Methamphetamine and its Metabolites by Capillary Electrophoresis/Mass Spectrometry with Direct Injection of Urine

We describe a rapid and simple method for the analysis of the d- and l-isomers of seven methamphetamine-related compounds and the d-isomer of pseudoephedrine (pseudoEP) by capillary electrophoresis/mass spectrometry (CE/MS) with direct injection of urine. The compounds were methamphetamine (MA), amphetamine (AP), dimethylamphetamine, ephedrine, norephedrine, methylephedrine, p-hydroxymethamphetamine (pOHMA). The electrolyte was 1 M formic acid/1 M ammonium formate (10/0.2, v/v) (pH 2.0) containing 1.5 mM heptakis-(2,6-diacetyl-6-sulfato)-β-cyclodextrin. The 14 enantiomers and d-pseudoEP were completely separated within 30 min. A urine sample was mixed with an equal volume of internal standard solution, filtered with a 0.45 μm filter and then injected into the CE/MS system. In an analysis of urine sample from a healthy person spiked with racemic MA and AP, the reproducibilities (n = 6) of the migration times and peak areas after correction by an internal standard were under 0.08% and under 3.6%, respectively. The detection limits using selected ion monitoring were 0.01 μg/ml (which corresponds to 0.02 μg/ml urine) for the enantiomers of MA, AP and pOHMA. The detection yields of the enantiomers of MA, AP and pOHMA from urine were in the range of 97.7-108.8%. The proposed method was successfully used for the chiral analysis of urine samples from MA and dimethylamphetamine (DMA) addicts and patients under selegiline pharmacotherapy.

Simultaneous Chiral Analysis of Methamphetamine and Related Compounds by Capillary Electrophoresis/Mass Spectrometry Using Anionic Cyclodextrin

A capillary electrophoresis/mass spectrometry method for the simultaneous chiral analysis of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA), ephedrine (EP), norephedrine (NE) and methylephedrine (ME) in urine has been developed. The background electrolyte was 1 M formic acid (pH 1.7). Using 0.85 mM heptakis(2,6-diacethyl-6-sulfato)-beta-cyclodextrin as the chiral selector, the 12 enantiomers were completely separated within 25 min. The detection limits were 0.01 microg mL(-1) for the enantiomers of MA, AP, DMA, EP and ME, and 0.02 microg mL(-1) for the enantiomers of NE using selected ion monitoring. The reproducibilities of within-run (n = 4) for the migration times and peak areas of the standard mixture were under 0.58% and 7.83%, respectively. The calibration curves of the peak areas of the 12 enantiomers were linear in the range of 0.05 - 10 microg mL(-1). This method was applicable to the analysis of urine samples.

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

A simple, rapid, reliable, and economic analytical scheme starting with in situ liquid–liquid extraction and asymmetric (or diastereomeric) chemical derivatization (ChD) followed by gas chromatography (GC)-isotope dilution mass spectrometry (MS) is described for the simultaneous determination of d- and l-amphetamine (AP) and methamphetamine (MA) in urine which could have resulted from the administration of various forms of questioned amphetamines or amphetamines-generating drugs. By using l- N-trifluoroacetyl-1-prolyl chloride ( l-TPC) as chiral derivatizing agent, resolutions of 2.2 and 2.0 were achieved for the separation of AP and MA enantiomeric pairs, respectively, on an ordinary HP-5MS capillary column. The GC–MS quantitation was carried out in the selected ion monitoring (SIM) mode using m/ z 237 and 251 as the quantifier ions for the respective diastereomeric pairs of AP- l-TPC and MA- l-TPC. The calibration curves plotted for the two pairs of analytes stretch with good linearity down to 45 ng/mL, and the limits of detection and quantitation determined were as low as 40 and 45 ng/mL, respectively. Also, a comparative study using 10 real-case urine specimens previously screened as positive for MA administration showed mostly tolerable biases between the two sums (of concentration) of d- and l-MA obtained via an asymmetric l-TPC-ChD approach and via an ordinary pentafluoropropionylation (PFPA-ChD) approach, respectively, as well as between the two sums of d- and l-AP obtained thereupon, thus validating the proposed analytical scheme as a promising forensic protocol for the detailed analysis of enantiomeric amphetamines in urine.

Effect of amine mobile phase additives on chiral subcritical fluid chromatography using polysaccharide stationary phases

Increased retention and selectivity in the subcritical fluid chromatography (SFC) of various amine compounds on polysaccharide chiral stationary phases (CSP) was observed upon incorporation of cyclic amines into the modifier. The retention increases are most pronounced with 2-propanol and are almost absent when methanol is used as modifier. This suggests that the effect may arise from a restriction to the modifier access to the binding site required to effect elution. The effect of the amine additives in SFC does not remain after their removal from the mobile phase. Findings were applied to the development of a 5 min separation of amphetamine and methamphetamine enantiomers.

Liquid chromatographic method for the determination of enantiomeric composition of amphetamine and methamphetamine in hair samples

Interest in hair analysis as an alternative or complementary approach to urinalysis for drug abuse detection has grown in recent years. Hair analysis can be particularly advantageous for drugs such as amphetamine and methamphetamine that are rapidly excreted. Confirmation of abuse of these stimulants is complicated by the fact that some forms are found in legitimate medications. Examination of the enantiomeric composition of amphetamine and methamphetamine in hair samples can provide valuable assistance in interpreting drug testing results. In this work, we developed a liquid chromatographic method for the separation of amphetamine and methamphetamine enantiomers isolated from human hair samples. The drug enantiomers were separated on a chiral stationary phase after derivatization with an achiral fluorescent agent. The methodology was evaluated with a Standard Reference Material that contained several drugs of abuse including amphetamine and methamphetamine.

Simultaneous chiral determination of methamphetamine and its metabolites in urine by capillary electrophoresis-mass spectrometry.

A capillary electrophoresis-mass spectrometry method for the simultaneous chiral determination of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA) and p-hydroxymethamphetamine (pOHMA), in urine has been developed. The internal standards used were 2-phenylethylamine and 1-amino4-phenylbutane. The electrolyte was 1 M formic acid (pH 2.2). The chiral selector, which was added to the electrolyte, was a mixture of 3 mM beta-cyclodextrin and 10 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin. The detection limits were 0.03 microg ml(-1) for the enantiomers of MA and AP and 0.05 microg ml(-1) for the enantiomers of pOHMA using selected ion monitoring. In the analysis of healthy adult urine samples spiked with MA, AP and pOHMA, the precision of within-run assays (n = 4) for the migration time after correction with two internal standards were under 0.04%, and the detection yields utilizing solid phase extraction were 95-105%. This method was applicable to the analysis of urine samples of MA addicts and DMA addicts.

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

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. 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. The derivatized AM and MA enantiomers were well separated and sensitively detected. The method was linear from 5 to 250 micro g/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. This sensitive, reliable, rapid NICI-GC-MS assay is suitable for enantioselective determination of AM and MA in blood plasma or serum samples.

Quantification of methamphetamine, amphetamine and enantiomers by semi-micro column HPLC with fluorescence detection; applications on abusers' single hair analyses.

Achiral and chiral semi-micro column high-performance liquid chromatographic methods with fluorescence detection to determine methamphetamine and amphetamine in human hair are described. These compounds were extracted into 5% trifluoroacetic acid (TFA) in methanol, derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride and separated either on a 250 x 1.5 mm i.d. octadecyl-silane (ODS) or a 150 x 2 mm i.d. OD-RH column. Linear calibration curves extending over a wide range of concentration that covers the practical samples were obtained for amphetamine, methamphetamine and their enantiomers (r = 0.999). Resolution values for amphetamine and methamphetamine enantiomers were 3.4 and 1.1, respectively. Intra- and inter-day variations of both the methods were not larger than 8.9% expressed as relative standard deviations (n >/= 5). The limits of detection at a signal-to-noise ratio of 3 obtained by both the methods were in the range of 1.0-4.7 fmol/5 microL injection with the achiral method being more sensitive. Abusers' hair samples were analyzed by the two methods and only the S(+)-enantiomers were found in eight Japanese abusers' hair samples. The achiral method was used to study the concentrations of these compounds in single black and white hair strands of abusers.

Enantiomer-specific high-performance liquid chromatography with fluorescence detection of methamphetamines in abusers' hair and urine

Enantiomer-specific high-performance liquid chromatography with fluorescence detection using 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride as a fluorescence labeling reagent was applied to determine methamphetamine and its metabolites in abusers' hair and urine. Hair samples were segmentally analyzed based on 1 cm long segments. In four hair samples, only the S(+)-enantiomers of methamphetamine and its N-demethylated metabolite, S(+)-amphetamine were detected. Satisfactory correlation (r = 0.901) between the results of high-performance liquid chromatography-fluorescence and those of gas chromatography-nitrogen phosphorous detection was obtained (n = 19). In an abuser's urine sample, the S(+)- and R(-)-enantiomers of methamphetamine, amphetamine and para-hydroxymethamphetamine were detected. The degree of N-demethylation of S(+)-methamphetamine into the corresponding metabolite of amphetamine was significantly higher than that of the R(-)-enantiomer.

Stereoselective screening for and confirmation of urinary enantiomers of amphetamine, methamphetamine, designer drugs, methadone and selected metabolites by capillary electrophoresis

Data presented in this paper demonstrate that a competitive binding, electrokinetic capillary-based immunoassay previously used for screening of urinary amphetamine and analogs cannot be employed to distinguish between the enantiomers of amphetamine and methamphetamine. However, capillary zone electrophoresis with a pH 2.5 buffer containing (2-hydroxypropyl)-beta-cyclodextrin as chiral selector is shown to permit the enantioselective analysis of urinary extracts containing methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (Ecstasy) and other designer drugs, and methadone together with its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. In that approach, enantiomer identification is based upon comparison of extracted polychrome UV absorption data and electropherograms obtained by rerunning of spiked extracts with spectra and electropherograms monitored after extraction of fortified blank urine. The suitability of the described chiral electrokinetic capillary method for drug screening and confirmation is demonstrated via analysis of unhydrolyzed quality control urines containing a variety of drugs of abuse. Furthermore, in a urine of a patient under selegiline pharmacotherapy, the presence of the R-(-)-enantiomers of methamphetamine and amphetamine could be unambiguously identified. Direct intake of an R-enantiomer or ingestion of drugs that metabolize to the R-enantiomers can be distinguished from the intake of S-(+)-enantiomers (drug abuse) or prescribed drugs that metabolize to the S-enantiomers of methamphetamine and amphetamine. The described approach is simple, reproducible, inexpensive and reliable (free of interferences of other major basic drugs that are frequently found in toxicological urines) and could thus be used for screening for and confirmation of urinary enantiomers in a routine laboratory.

Direct determination of methamphetamine enantiomers in urine by liquid chromatography with a strong cation-exchange precolumn and phenyl-β-cyclodextrin-bonded semi-microcolumn

Methamphetamine enantiomers in drug abusers’ urine were clearly separated by semi-microcolumn liquid chromatography using a column-switching system. The system consists of two separation processes: firstly, a strong cation-exchange precolumn removes neutral and anionic substances in urine, and then methamphetamine enantiomers trapped in the column are transferred to and separated in a phenyl-β-cyclodextrin-bonded semi-microcolumn (Chiral Drug™, 150 mm×1.5 mm I.D.). ( S)-(+)-Methamphetamine was baseline separated from ( R)-(−)-methamphetamine within 25 min, directly from urine samples. The detection limit for both enantiomers was 0.1 μg/ml.

Direct Analysis of Methamphetamine and Amphatamine Enantiomers in Human Urine by Semi-microcolumn HPLC/Electrospray Ionization Mass Spectrometry.

Semi-microcolumn high performance liquid chromatography (HPLC) with a columnswitching system was connected to electrospray ionization mass spectrometry (ESI-MS) for the analysis of methamphetamine (MA) and amphetamine (AP), a main metabolite of MA. Urine samples were directly introduced to the system. After removal of other components of the sample matrix by the column-switching process, both MA and AP were successfully analyzed with enantiomeric resolution by the use of β-cyclodextrin phenylcarbamate-bonded (phCD) semi-microcolumn, accompanied by spectral information obtained with ESI-MS. Linear calibration curves were obtained for all the analytes throughout the concentration range from 1 ng/ml to 5 μg/ml. The relative standard deviation for peak area ranged from 1.41 to 2.07% at 0.1 μg/ml for the four enantiomer peaks. Detection limits observed for MA and AP enantiomers were 20 ng/ml and 50 ng/ml in the spectrum scanning mode, and 0.5 ng/ml and 1 ng/ml in the single ion monitoring (SIM) mode, respectively. The HPLC/ESI-MS system studied here appeared highly sensitive and useful towards its forensic applications.

Enantioselective high-performance liquid chromatography with fluorescence detection of methamphetamine and its metabolites in human urine.

A simple, rapid and highly sensitive high-performance liquid chromatographic method with fluorescence detection for determining the enantiomers of methamphetamine and its major metabolites, amphetamine and p-hydroxymethamphetamine, in urine samples was developed. Using a newly developed reagent for amines, namely, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride, six enantiomers were derivatized under mild conditions (i.e., 10 min at room temperature, pH 9.0) and separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column following a pre-separation on an ODS column within 42 min, and the effluent was monitored at 440 nm (lambda ex 330 nm). Calibration curves for these derivatives using spiked human urine were linear in the range 0.05-100 mumol dm-3 with correlation coefficients > or = 0.999. The detection limits at a signal-to-noise ratio of 3 were 2.8-8.8 fmol per 5 microliters injection. The relative standard deviations of within- (n = 6) and between-day (n = 5) variations were < or = 7.4%. The method was successfully applied to discriminate between (S)-(+)-methamphetamine and its corresponding metabolites found in abusers' urine and their antipodes in a sample taken from a Parkinsonian patient on selegiline (Deprenyl) therapy.