Ultrafast Screening of Synthetic Cannabinoids and Synthetic Cathinones in Urine by RapidFire-Tandem Mass Spectrometry.

Screening for emerging drugs of abuse, specifically synthetic cathinones and synthetic cannabinoids, is difficult for high-throughput laboratories as immunoassay kits are often unavailable. Consequently, most laboratories employ liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening, which can be complex and time consuming as these techniques may require involved sample preparation and lengthy analysis times. The increasing demand for novel psychoactive substance testing necessitates alternative screening methods that are sensitive, fast and versatile. The RapidFire tandem mass spectrometry system (RF-MS-MS) provides a rapid and highly specific screen for these emerging drugs of abuse with minimal sample preparation and an instrumental analysis time of <14 s per sample. Presented here are two RF-MS-MS screening methods used to analyze 28 emerging drugs of abuse, 14 synthetic cannabinoids and 14 synthetic cathinones, in urine with run times of 9 and 12.6 s, respectively. Sample preparation and hydrolysis were performed in a 96-well plate with one multiple reaction monitoring transition used for the identification of each compound. Eighteen thousand urine specimens were screened by liquid-liquid extraction followed by LC-MS-MS analysis, and the results were compared with those obtained using the RF-MS-MS screening method. The analytical data illustrate the advantages of the RF-MS-MS methods.

Detection of 25C-NBOMe in Three Related Cases.

An accidental death associated with the use of the designer drug, 2-(4-chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25C-NBOMe), is reported. A 23-year-old Caucasian male experienced severe respiratory distress and died after being subdued by military law enforcement. At autopsy, remarkable findings upon internal examination included mild to moderate coronary atherosclerosis, biventricular dilation, mild right ventricular hypertrophy and bilateral pulmonary edema and congestion. The decedent's blood contained no drugs, ethanol or other volatile compounds. Pseudoephedrine, nicotine and cotinine were detected in his urine. A LC-QTOF designer drug screen, employing a basic solid-phase extraction, was used to isolate 25C-NBOMe, 25C-NBOH and 2C-C from both blood and urine specimens. Quantitative analysis was performed by LC-MS-MS operating in multiple reaction monitoring mode. 25C-NBOMe and 2C-C were present in the blood (2.07 and 0.12 ng/mL) and in the urine (27.43 ng/mL and 0.38 ng/mL), respectively. 25C-NBOMe concentrations were determined by standard addition in the brain (19.10 ng/g), spleen (27.13 ng/g), lung (25.21 ng/g), liver (15.20 ng/g), kidney (25.06 ng/g) and gastric contents (30.24 µg total in 100 mL submitted). On the basis of decedent case history, autopsy and toxicological findings, the medical examiner ruled the cause of death as 25C-NBOMe toxicity temporally associated with excited delirium and forcible restraint. The manner of death was ruled accidental.

Analysis of Parent Synthetic Cannabinoids in Blood and Urinary Metabolites by Liquid Chromatography Tandem Mass Spectrometry.

Synthetic cannabinoids emerged on the designer drug market in recent years due to their ability to produce cannabis-like effects without the risk of detection by traditional drug testing techniques such as immunoassay and gas chromatography-mass spectrometry. As government agencies work to schedule existing synthetic cannabinoids, new, unregulated and structurally diverse compounds continue to be developed and sold. Synthetic cannabinoids undergo extensive metabolic conversion. Consequently, both blood and urine specimens may play an important role in the forensic analysis of synthetic cannabinoids. It has been observed that structurally similar synthetic cannabinoids follow common metabolic pathways, which often produce metabolites with similar metabolic transformations. Presented are two validated quantitative methods for extracting and identifying 15 parent synthetic cannabinoids in blood, 17 synthetic cannabinoid metabolites in urine and the qualitative identification of 2 additional parent compounds. The linear range for most synthetic cannabinoid compounds monitored was 0.1-10 ng/mL with the limit of detection between 0.01 and 0.5 ng/mL. Selectivity, specificity, accuracy, precision, recovery and matrix effect were also examined and determined to be acceptable for each compound. The validated methods were used to analyze a compilation of synthetic cannabinoid investigative cases where both blood and urine specimens were submitted. The study suggests a strong correlation between the metabolites detected in urine and the parent compounds found in blood.

Multidrug toxicity involving sumatriptan.

A multidrug fatality involving sumatriptan is reported. Sumatriptan is a tryptamine derivative that acts at 5-HT(1B/1D) receptors and is used for the treatment of migraines. The decedent was a 21-year-old white female found dead in bed by her spouse. No signs of physical trauma were observed and a large number of prescription medications were discovered at the scene. Toxicological analysis of the central blood revealed sumatriptan at a concentration of 1.03 mg/L. Following therapeutic dosing guidelines, sumatriptan concentrations do not exceed 0.095 mg/L. Sumatriptan was isolated by solid-phase extraction and analyzed using liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode. A tissue distribution study was completed with the following concentrations measured: 0.61 mg/L in femoral blood, 0.56 mg/L in iliac blood, 5.01 mg/L in urine, 0.51 mg/kg in liver, 3.66 mg/kg in kidney, 0.09 mg/kg in heart, 0.32 mg/kg in spleen, 0.01 mg/kg in brain, 15.99 mg/kg in lung and 78.54 mg/45 mL in the stomach contents. Carisoprodol, meprobamate, fluoxetine, doxylamine, orphenadrine, dextromethorphan and hydroxyzine were also present in the blood at the following concentrations: 3.35, 2.36, 0.63, 0.19, 0.06, 0.55 and 0.16 mg/L. The medical examiner ruled the cause of death as acute mixed drug toxicity and the manner of death as accident.

Determination of designer drug cross-reactivity on five commercial immunoassay screening kits.

The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), ß-keto amphetamines, substituted amphetamines, piperazines, α-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits.

Evaluation of designer amphetamine interference in GC-MS amine confirmation procedures.

In recent years, a class of new designer drugs commonly referred to as 'bath salts' have made their way to the illicit drug market. The most common drugs encountered are designer amphetamines and cathinones. Many analytical methods for analysis and identification of bath salts have been published, but there has been little reported on their impact on existing gas chromatography-mass spectrometry (GC-MS) amine confirmation methods. Due to structural similarities, the potential exists that designer amphetamines may interfere with methods used for analysis of sympathomimetic amines. Methiopropamine, 4-fluoroamphetamine, 4-fluoromethamphetamine (4-FMA) and 4-methylamphetamine were examined for potential interference with immunoassays and GC-MS confirmation analysis utilizing three derivatization procedures: R(-)-α-methoxy-α-trifluoromethylphenylacetyl chloride (R-MTPAC), heptafluorobutyric anhydride (HFBA) and chlorodifluoroacetic anhydride (ClF(2)AA). Significant cross-reactivity was observed with all the four compounds on the Syva Emit(®) II Plus Amphetamines and Roche KIMS Amphetamines II immunoassays. Laboratories utilizing GC-MS selected-ion-monitoring confirmation methods with R-MTPAC, HFBA or ClF(2)AA derivatives could experience potential chromatographic and mass spectral interferences from 4-fluroamphetamine, 4-FMA and methiopropamine in the form of ion ratio and quantitative failures. Careful ion selection, proper selectivity and specificity studies during method validation and rigid chromatographic and spectral acceptance criteria are required to assure the robustness and accuracy of GC-MS methods.

A fatality involving AH-7921.

A case is presented of a 19-year-old white male who was found dead in bed by a friend. While no anatomic cause of death was observed at autopsy, toxicological analysis of his blood identified AH-7921, a synthetic opioid. AH-7921 was isolated by liquid-liquid extraction into n-butyl chloride from alkalinized samples. Extracts were analyzed and quantified by gas chromatography mass spectrometry in selected ion monitoring mode. The heart blood had an AH-7921 concentration of 3.9 mg/L and the peripheral blood concentration was 9.1 mg/L. In addition to the blood, all submitted postmortem specimens including urine, liver, kidney, spleen, heart, lung, brain, bile and stomach content were quantified. The following concentrations of AH-7921 were reported: 6.0 mg/L in urine, 26 mg/kg in liver, 7.2 mg/kg in kidney, 8.0 mg/kg in spleen, 5.1 mg/kg in heart, 21 mg/kg in lung, 7.7 mg/kg in brain, 17 mg/L in bile and 120 mg/125 mL in the stomach content. The medical examiner reported that the cause of death was opioid intoxication and the manner of death was accident.

Evaluation of abalone ß-glucuronidase substitution in current urine hydrolysis procedures.

This study examined the potential of abalone ß-glucuronidase as a viable and cost effective alternative to current hydrolysis procedures using acid, Helix pomatia ß-glucuronidase and Escherichia coli ß-glucuronidase. Abalone ß-glucuronidase successfully hydrolyzed oxazepam-glucuronide and lorazepam-glucuronide within 5% of the spiked control concentration. Benzodiazepines present in authentic urine specimens were within 20% of the concentrations obtained with the current hydrolysis procedure using H. pomatia ß-glucuronidase. JWH 018 N-(5-hydroxypentyl) ß-d-glucuronide was hydrolyzed within 10% of the control concentration. Authentic urine specimens showed improved glucuronide cleavage using abalone ß-glucuronidase with up to an 85% increase of drug concentration, compared with the results obtained using E. coli ß-glucuronidase. The JWH 018 and JWH 073 carboxylic acid metabolites also showed increased drug concentrations of up to 24%. Abalone ß-glucuronidase was able to completely hydrolyze a morphine-3-glucuronide control, but only 82% of total morphine was hydrolyzed in authentic urine specimens compared with acid hydrolysis results. Hydrolysis of codeine and hydromorphone varied between specimens, suggesting that abalone ß-glucuronidase may not be as efficient in hydrolyzing the glucuronide linkages in opioid compounds compared with acid hydrolysis. Abalone ß-glucuronidase demonstrates effectiveness as a low cost option for enzyme hydrolysis of benzodiazepines and synthetic cannabinoids.

Distribution of methylone in four postmortem cases.

Drugs derived from amphetamine, methamphetamine and their methylenedioxy- analogues, although being sold as plant food or bath salts, are being used as legal alternatives to scheduled amphetamine stimulants. These products often contain methylone, mephedrone and methylenedioxypyrovalerone (MDPV)--three amphetamine derivatives shown to have strong pharmacological effects. Four postmortem cases were analyzed for methylone, mephedrone and MDPV, with drug levels quantitated in multiple biological matrices. All four cases had detectable levels of methylone, with heart blood concentrations of 0.740, 0.118, 0.060 and 1.12 mg/L. Analysis of several tissue samples shows that methylone does not sequester in a particular tissue type after death. The average liver-to-blood ratio was 2.68. Two cases also had MDPV present, but insufficient data were collected to formulate a hypothesis on postmortem sequestration or redistribution. Two different extraction methods, as well as analysis of derivatized and underivatized methylone, show that the drug is suitable for analysis in either method. The cases are believed to show one instance of chronic methylone use, with a urine concentration of 38 mg/L.

Dimethylamylamine: a drug causing positive immunoassay results for amphetamines.

The Department of Defense (DoD) operates six forensic urine drug-testing laboratories that screen close to 5 million urine samples for amphetamines yearly. Recently, the DoD laboratories have observed a significant decrease in the confirmation rates for amphetamines because of specimens screening positive by two separate immunoassays and confirming negative by gas chromatography-mass spectrometry (GC-MS). Previous studies conducted by the Division of Forensic Toxicology, Armed Force Institute of Pathology (AFIP) utilizing a GC-MS basic drug screen and a designer drug screen revealed no common compound or compound classes as to the cause of the immunoassay-positive results. Additional information obtained from an immunoassay vendor suggested the anorectic compound dimethylamylamine (DMAA) may be the cause of the false-positive screens. An additional 134 false-positive samples were received and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS-MS) for DMAA. LC-MS-MS analysis revealed the presence of DMAA in 92.3% of the false-positive samples at a concentration of approximately 6.0 mg/L DMAA, causing a positive screen on both immunoassay kits.

A drug toxicity death involving propylhexedrine and mitragynine.

A death involving abuse of propylhexedrine and mitragynine is reported. Propylhexedrine is a potent α-adrenergic sympathomimetic amine found in nasal decongestant inhalers. The decedent was found dead in his living quarters with no signs of physical trauma. Analysis of his computer showed information on kratom, a plant that contains mitragynine, which produces opiumlike effects at high doses and stimulant effects at low doses, and a procedure to concentrate propylhexedrine from over-the-counter inhalers. Toxicology results revealed the presence of 1.7 mg/L propylhexedrine and 0.39 mg/L mitragynine in his blood. Both drugs, as well as acetaminophen, morphine, and promethazine, were detected in the urine. Quantitative results were achieved by gas chromatography-mass spectrometry monitoring selected ions for the propylhexedrine heptafluorobutyryl derivative. Liquid chromatography-tandem mass spectrometry in multiple reactions monitoring mode was used to obtain quantitative results for mitragynine. The cause of death was ruled propylhexedrine toxicity, and the manner of death was ruled accidental. Mitragynine may have contributed as well, but as there are no published data for drug concentrations, the medical examiner did not include mitragynine toxicity in the cause of death. This is the first known publication of a case report involving propylhexedrine and mitragynine.

Multiple-drug toxicity caused by the coadministration of 4-methylmethcathinone (mephedrone) and heroin.

An accidental death caused by the combined use of a new designer drug, 4-methylmethcathinone (mephedrone), and heroin is reported. A 22-year-old Caucasian male was found unresponsive in his living quarters and was transported to the hospital where he died. During autopsy, needle marks were found along the decedent's lower legs and ankles. Investigators discovered the decedent and his roommate had been using "Black Tar" heroin and mephedrone. Routine toxicological analysis detected morphine in the decedent's blood at 0.06 mg/L. Additionally, 6-acetylmorphine, morphine, codeine, and doxylamine were detected in his urine. A designer drug screen, employing a basic liquid-liquid extraction followed by pentafluropropionic anhydride derivatization, was used to isolate mephedrone from both blood and urine specimens. The derivatized extracts were analyzed by gas chromatography- mass spectrometry (GC-MS) operating in full-scan mode. Quantitative analysis of mephedrone was performed by GC-MS operating in selective ion monitoring mode using methamphetamine-d(14) as an internal standard. Mephedrone was confirmed in the decedent's blood and urine at 0.50 and 198 mg/L, respectively. The physiological and pharmacological effects of mephedrone and any associated toxicity have not been reported. However, because of its structural similarities with methcathinone and the high concentration in the decedent's blood, the overall contribution of mephedrone to the death could not be minimized. Therefore, the medical examiner reported the cause of death as multiple-drug toxicity and the manner of death as accidental.

Detection of 1-benzylpiperazine, 1-(3-trifluoromethylphenyl)-piperazine, and 1-(3-chlorophenyl)-piperazine in 3,4-methylenedioxymethamphetamine-positive urine samples.

Historically, ecstasy tablets contained 3,4-methylenedioxymethamphetamine (MDMA) as the psychoactive component. In recent years, the Drug Enforcement Administration (DEA) and other law enforcement agencies have seized ecstasy tablets that are comprised of psychoactive drugs or drug mixtures other than MDMA. Many jurisdictions have reported the presence of piperazine derivatives including 1-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)-piperazine (TFMPP), and 1-(3-chlorophenyl)-piperazine (mCPP) in ecstasy tablets. These piperazine derivatives produce stimulant and psychoactive effects similar to those produced by MDMA, amphetamine, and methamphetamine. In many countries, their use is not controlled, and therefore they have become a legal alternative to MDMA. For this study, a targeted population of 251 MDMA-positive urine samples were analyzed for designer drugs, including the piperazine derivatives. A basic liquid-liquid extraction followed by pentafluoropropionic anhydride (PFPA) derivatization and a full scan (m/z 42-550) gas chromatography-mass spectrometry analysis was used to screen the urine samples for 33 designer drugs. Overall, in 36% of the specimens analyzed, a stimulant or psychoactive compound other than MDMA and 3,4-methylenedioxyamphetamine (MDA) was detected. BZP, TFMPP, and mCPP were detected in 15%, 7%, and 1% of the samples, respectively.

An overdose death involving the insufflation of extended-release oxymorphone tablets.

Two cases are reported involving the abuse of extended-release oxymorphone hydrochloride tablets (Opana® ER) in combination with alprazolam (Xanax®). Two juvenile females were discovered unresponsive and hypoxic by a male acquaintance. The trio had reportedly crushed and snorted Opana ER tablets and consumed Xanax for recreational purposes. Emergency personnel were able to stabilize one female. The second female was pronounced dead at the scene. Blood and urine samples from the surviving female were collected at the trauma center between 48 and 96 h post incident. Toxicology results showed the presence of oxymorphone, doxylamine, dextromethorphan, alprazolam, α-hydroxyalprazolam, oxazepam, and temazepam in her urine. No drugs were detected in her blood. Toxicology on the deceased female revealed the presence of 0.13 mg/L oxymorphone and 0.04 mg/L alprazolam in her blood. Gastric contents contained 0.25 and 0.93 mg/L of oxymorphone and alprazolam, respectively. Oxymorphone, alprazolam, and α-hydroxyalprazolam were present in her urine. Quantitative results were achieved by gas chromatography-mass spectrometry monitoring selected ions for the oxime-oxymorphone-trimethylsilyl derivative, alprazolam, and the α-hydroxyalprazolam tert-butyldimethylsilyl derivative. The established linearity ranges for the opiate and benzodiazepine methods were 0.050-3.000 and 0.025-1.000 mg/L, respectively. The cause of death was reported as multiple drug toxicity, and the manner of death was accidental.

The detection and quantitative analysis of the psychoactive component of Salvia divinorum, salvinorin A, in human biological fluids using liquid chromatography-mass spectrometry.

Salvia divinorum, a member of the mint plant family, has hallucinogenic properties that have become increasingly sought after by recreational drug users. The main psychoactive component, salvinorin A, has potency comparable to lysergic acid diethylamide. Though still legal to possess in most of the United States and much of Europe, little is known regarding the compound's long-term health effects, addiction liability, and pharmacokinetics. Limited data are available in the scientific literature, and few analytical methods are published for the detection in human biological fluids. These factors contribute to the unfamiliarity of the compound and complicate the method development process necessary to accommodate special requested testing for salvinorin A. A sensitive analytical method for the detection and quantitation of salvinorin A in human biological fluids was developed and validated to resolve analytical shortcomings. The method utilizes a solid-phase extraction technique coupled with liquid chromatography-electrospray ionization mass spectrometry operated in selected ion monitoring mode. The assay has a linear range of 5.0-100 ng/mL with a correlation coefficient of 0.997. The limit of detection and limit of quantitation were experimentally determined as 2.5 and 5.0 ng/mL, respectively. The method has been applied to blood and urine samples successfully and can be used to detect the presence of salvinorin A in forensic testing.

Quantitative analysis of the aminosteroidal non-depolarizing neuromuscular blocking agent vecuronium by LC-ESI-MS: A Postmortem Investigation.

The apparent recreational use of an aminosteroidal non-depolarizing neuromuscular blocking agent, vecuronium, is reported in this postmortem investigation. A quantitative method for the analysis of vecuronium and its active metabolite, 3-desacetylvecuronium, in blood and tissue samples was developed using liquid chromatography-electrospray ionization mass spectrometry operated in positive selected ion monitoring mode. Chromatographic separation was performed on a Gemini 5-microm C18 column using a mobile phase of 0.1% formic acid/acetonitrile at 0.700 mL/min. The method was linear from 0.01 to 1.00 mg/L with correlation coefficients of 0.999 and greater for both compounds. The limits of detection and quantitation were determined in blood to be 0.005 and 0.010 mg/L, respectively. The coefficients of variation were less than 10% for both intra- and interday assays. Vecuronium was quantitated in blood at 0.070 mg/L and in the kidney, liver, and spleen at 0.224, 0.045, and 0.080 mg/kg, respectively. The active metabolite 3-desacetylvecuronium was quantitated in blood at 0.100 mg/L, in the urine at 0.040 mg/L and in the kidney, liver, spleen, and lung at 0.271, 0.100, 0.082, and 0.164 mg/kg, respectively.

Detection of 1-benzylpiperazine and 1-(3-trifluoromethylphenyl)-piperazine in urine analysis specimens using GC-MS and LC-ESI-MS.

Designer piperazines, such as 1-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)-piperazine (TFMPP), are widely available and have become popular party drugs throughout the world. Used in many countries as legal alternatives to methamphetamine and ecstasy, these designer piperazines exhibit several of the same stimulant and psychoactive properties of their illicit counterparts. Presented is a case study of seven urine analysis specimens analyzed for designer piperazines. A full scan gas chromatography-mass spectrometry screen detected the presence of BZP and TFMPP in all seven specimens. Confirmation using liquid chromatography-electrospray ionization-mass spectrometry operating in selected ion monitoring mode (SIM) yielded urinary concentrations ranging from 13.0 to 429.1 mg/L and 0.79 to 25.4 mg/L for BZP and TFMPP, respectively.

The detection of hydromorphone in urine specimens with high morphine concentrations.

A previous study suggested that small amounts of morphine are metabolically converted to hydromorphone. In the present study, morphine positive urine specimens obtained from a postmortem laboratory and a random urinalysis program were tested for morphine, codeine, hydromorphone, hydrocodone, oxymorphone, and oxycodone to assess the possibility that small amounts of hydromorphone are produced from the metabolism of morphine. The opioids were analyzed by gas chromatography-mass spectrometry as their respective trimethylsilyl derivatives following solid phase extraction. The limit of detection for hydromorphone was 5 ng/mL. A total of 73 morphine positive urine specimens were analyzed, with morphine concentrations ranging from 131 to 297,000 ng/mL. Hydromorphone was present at a concentration > or =5 ng/mL in 36 of these specimens at concentrations ranging from 0.02% to 12% of the morphine concentration. Hydrocodone was not detected in these specimens at the assay detection limit of 25 ng/mL. These results support earlier work suggesting that the detection of hydromorphone in urine specimens does not necessarily mean that exogenous hydromorphone or hydrocodone was used.

Modern instrumental methods in forensic toxicology.

This article reviews modern analytical instrumentation in forensic toxicology for identification and quantification of drugs and toxins in biological fluids and tissues. A brief description of the theory and inherent strengths and limitations of each methodology is included. The focus is on new technologies that address current analytical limitations. A goal of this review is to encourage innovations to improve our technological capabilities and to encourage use of these analytical techniques in forensic toxicology practice.

Quantitative and isomeric determination of amphetamine and methamphetamine from urine using a nonprotic elution solvent and R(-)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid chloride derivatization.

Forensic Urine Drug Testing Laboratories often requires two confirmatory methods for a methamphetamine positive screen. First, methamphetamine is identified and quantitated using gas chromatography-mass spectrometry. If the total methamphetamine concentration is above the administrative cutoff level, the isomeric composition must be determined. This eliminates a possible contribution by over-the-counter cold medications that contain l-methamphetamine (Vick's inhalers). Products that contain only the l-isomer of methamphetamine must be distinguishable from prescription or illicitly manufactured methamphetamine, which consists mainly of the d-isomer. Optically impure derivatizing reagents will produce an impure mixture from a pure isomeric compound. Therefore, methods utilizing impure reagents can prove problematic when interpreting results. Use of an optically pure chiral derivatizing reagent, such as R(-)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid chloride, allows for the creation and measurement of chromatographically separable isomeric compounds. The novel method described here utilizes a polymer-based solid-phase column adapted to a positive pressure manifold extraction system and a one-step derivatization process that occurs directly in the elution solvent. This methodology eliminates an elution solvent dry-down step that may adversely affect recovery of volatile amphetamine compounds. Although the method was designed for the quantitative analysis of the isomers of amphetamine and methamphetamine, it can be adapted for use with a wide range of phenethylamines including methylenedioxyamphetamine, N-methylenedioxymethamphetamine, and possibly N-methylenedioxyethylamphetamine. The linear range for quantitation was 25-10,000 ng/mL for d,l-methamphetamine and d,l-amphetamine, and correlation coefficients were 0.997 or better. The coefficient of variation for all four analytes did not exceed 2.8%. Concentrations analyzed ranged from 500 to 4000 ng/mL (n=40). The method allows for a simple and accurate quantitation and isomeric determination of amphetamine and methamphetamine using a process that eliminates extraction and derivatization complications common in current methods.