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A carboxyl functionalized organic-inorganic hybrid monolithic column (TMOS-co-CES) was applied as in-tube solid-phase microextraction (SPME) sorbent combining with ultra-performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometer for separation and analyzation of seven typical amphetamine-type stimulants (ATSs), including amphetamine (AM), methamphetamine (MAM), cathinone, methcathinone, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine. The application potential of TMOS-co-CES material to ATSs was preliminarily confirmed by computational simulation by using cathinone as a representative. The influences of various SPME parameters and analytical performance were investigated systematically. As matched with the results of computational simulation, TMOS-co-CES column could capture ATSs under milder near neutral pH condition with high extraction efficiency basing on the adsorption mechanism explained as a mixed mode of electrostatic and hydrophobic interactions. Seven target trace ATSs in spiked sewage, pond water and urine could be rapidly and conveniently separated and enriched by the proposed TMOS-co-CES in-tube SPME method under the optimized conditions with good accuracy, repeatability and resistance to matrix interference. Moreover, AM and MAM had been successfully detected in real urines of suspected drug abusers by TMOS-co-CES in-tube SPME method, which indicated that the proposed method had good application feasibility for drug monitoring. The mild extraction condition and ideal method performance further made the TMOS-co-CES in-tube SPME method more potential in applications for forensic analysis and drug abuse., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Psychological disorders and dramatic social problems are serious concerns regarding the abuse of amphetamine and its stimulant derivatives worldwide. Consumers of such drugs experience great euphoria along with serious health problems. Determination and quantification of amphetamine-type stimulants are indispensable skills for clinical and forensic laboratories. Analysis of low drug doses in bio-matrices necessitates applications of simple and also effective preparation steps. The preparation procedures not only eliminate adverse matrix effects, but also provide reasonable clean-up and pre-concentration benefits. The current review presents different methods used for sample preparation of amphetamines from urine as the most frequently used biological matrix. The advantages and limitations of various sample preparation methods were discussed focusing on the miniaturized methods.

Introduction: Immunoassay urine drug screen (UDS) is frequently used in clinical practice for initial screening process, being generally available, fast, and inexpensive. Exposure to widely prescribed drugs might determine false-positive UDS amphetamines, leading to diagnostic issues, wrong therapeutic choices, impairment of physician-patient relationship, and legal implications., Areas Covered: To summarize and comment on a comprehensive list of compounds responsible for UDS false positives for amphetamines, we conducted a literature review on PubMed along with a comparison with Real-World Data from the Food and Drug Administration Adverse Event Reporting System (FAERS) database analysis between 2010 and 2022. Forty-four articles and 125 Individual Case Safety Reports (ICSR) involving false-positive amphetamine UDS in psychiatric patients were retrieved from FAERS., Expert Opinion: False-positive results were described in literature for antidepressants, atomoxetine, methylphenidate, and antipsychotics, but also for non-psychiatric drugs of common use, such as labetalol, fenofibrate, and metformin. Immunoassay method is usually responsible for false-positive results, and in most cases, mass spectrometry (MS) does not eventually confirm the UDS positivity. Physicians should be aware of immunoassays' limitations and when turning to a confirmatory test. Any new cross-reaction should be reported to pharmacovigilance activities.

Immunoassays are routinely used to provide rapid urine drug screening results in the clinical setting. These screening tests are prone to false-positive results and ideally require confirmation by mass spectrometry. In this study, we have examined a large number of urine specimens where drugs other than amphetamines may have caused a false-positive amphetamine immunoassay screening result. Urine drug screens (12,250) in a clinical laboratory that used the CEDIA amphetamine/ecstasy method were reviewed for false-positive results over a 6-year period (2015-2020). An additional 3,486 referred samples, for which confirmatory--mass spectrometry was requested, were also reviewed. About 86 in-house samples and 175 referral samples that were CEDIA false-positive screens were further analyzed by an LC-QTOF general unknown screen. Potential cross-reacting drugs were identified, and their molecular similarities to the CEDIA targets were determined. Commercial standards were also analyzed for cross-reactivity in the amphetamine/ecstasy CEDIA screen. Positive amphetamine results in 3.9% of in-house samples and 9.9% of referred tests for confirmatory analysis were false positive for amphetamines. Of these false-positive specimens, on average, 6.8 drugs were detected by the LC-QTOF screen. Several drugs were identified as possible cross-reacting drugs to the CEDIA amphetamine/ecstasy assay. Maximum common substructure scores for 70 potential cross-reacting compounds were calculated. This was not helpful in identifying cross-reacting drugs. False-positive amphetamine screens make up to 3.9-9.9% of positive amphetamine screens in the clinical laboratory. Knowledge of cross-reacting drugs may be helpful when mass spectrometry testing is unavailable., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The rapid sensing of drug compounds has traditionally relied on antibodies, enzymes and electrochemical reactions. These technologies can frequently produce false positives/negatives and require specific conditions to operate. Akin to antibodies, molecularly imprinted polymers (MIPs) are a more robust synthetic alternative with the ability to bind a target molecule with an affinity comparable to that of its natural counterparts. With this in mind, the research presented in this article introduces a facile MIP-based dye displacement assay for the detection of (±) amphetamine in urine. The selective nature of MIPs coupled with a displaceable dye enables the resulting low-cost assay to rapidly produce a clear visual confirmation of a target's presence, offering huge commercial potential. The following manuscript characterizes the proposed assay, drawing attention to various facets of the sensor design and optimization. To this end, synthesis of a MIP tailored towards amphetamine is described, scrutinizing the composition and selectivity (ibuprofen, naproxen, 2-methoxphenidine, quetiapine) of the reported synthetic receptor. Dye selection for the development of the displacement assay follows, proceeded by optimization of the displacement process by investigating the time taken and the amount of MIP powder required for optimum displacement. An optimized dose-response curve is then presented, introducing (±) amphetamine hydrochloride (0.01-1 mg mL -1 ) to the engineered sensor and determining the limit of detection (LoD). The research culminates in the assay being used for the analysis of spiked urine samples (amphetamine, ibuprofen, naproxen, 2-methoxphenidine, quetiapine, bupropion, pheniramine, bromopheniramine) and evaluating its potential as a low-cost, rapid and selective method of analysis.

A simple and rapid pipette-tip solid-phase extraction (PT-SPE) procedure with derivatization prior to gas chromatography triple quadrupole mass spectrometry analysis is developed for the simultaneous determination of amphetamine (AMP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. The PT-SPE procedure using self-made sorbent could extract drugs within 6 min from 100-μL urine samples, requiring low solvent-consumption (<2.0 mL). Besides, the self-made pipette tip could be reused at least five times. Under optimized conditions, the recoveries of four amphetamines at spiked levels (low, medium and high) ranged from 87.7 to 110.4%, with relative standard deviations < 9.5%. The limit of detections and limit of quantifications for AMP, MA, MDA and MDMA were in the range of 2.52-8.25 ng⋅mL-1 and 8.4-27.5 ng⋅mL-1, respectively. Validation results show that the proposed method is suitable for the quantitation of amphetamines and has been successfully applied in the urine samples of suspected drug abusers., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The present paper describes an original method for the online preconcentration and analysis of ring-substituted amphetamines in urine samples, used on the integration of robot-assisted hollow fiber liquid-phase microextraction (HF-LPME), high-performance liquid chromatography (HPLC), and fluorescence detection (FLD). A lab-made autosampler, actuating a 100-μL syringe and equipped with a three-way solenoid microvalve, allowed the acceptor phase to flow through and be withdrawn from the lumen fiber, enabling the automated online transference of the enriched acceptor phase for chromatographic analysis, through a six-port switching valve. The developed online HF-LPME-LC/FLD method demonstrated high analytical throughput and confidence, facilitating the efficient extraction and determination of the target analytes, with minimal solvent consumption and sample manipulation, in a straightforward way. Sample cleanup, analyte uptake, and analysis were carried out in 14.5 min. Under optimal conditions, automated online HF-LPME showed excellent linearity, precision, and trueness, obtaining intraday RSDs between 2.9 and 9.2% (n = 6) and interday RSDs between 5.3 and 9.3% (n = 6). Enrichment factors (EFs) ranged between 14.2 and 15.7, extraction recoveries (ERs) ranged between 17.7 and 19.5%, and the limits of detection (S/N = 3) were 2.0, 3.0, and 3.0 μg L -1 for MDA, MDMA, and MDEA, respectively. The method proved to be an effortless, rapid, reliable, and environment-friendly approach for the determination of drug abuse in urine samples. Graphical abstract.

Background: Substance use during pregnancy is a major medical and public health concern. Determination of the most appropriate screening protocol remains a clinical conundrum. Interviews and/or laboratory drug screens may be costly, inaccurate, and are frequently inadequate to identify patterns of substance use for a given population or geographic area. We compared commercially available urine "dip cup" toxicology screens obtained in the clinic to university hospital drug toxicology results., Methods: 267 observed urine samples were collected from pregnant women with known substance use disorders enrolled in a specialized treatment program that included access to buprenorphine medication-assisted treatment. Each urine sample was tested by commercial dip cup with temperature confirmation and then sent to the university hospital laboratory for analyses. The number of substances detected and cost for each screening method were compared., Results: Uniformly, the dip cup had comparable detection of amphetamines, barbiturates, cocaine, methadone, opiates, and tetrahydrocannabinol to the university hospital laboratory with the exception of benzodiazepines. In addition, the dip cup detected use of buprenorphine (a commonly misused opiate receptor ligand not included in the hospital screen) and was significantly less expensive., Conclusions: Commercially available urine dip cups are cost-effective, equally comparable to hospital based screening, and provide 'real time' results germane to clinical care and treatment planning., (Copyright © 2019. Published by Elsevier B.V.)

There has been only a few reports regarding aripiprazole causing false positive urine amphetamine drug screens, exclusively on children accidently ingesting aripiprazole. Herein, we present the first reported case of a 40 year old woman affected by Bipolar I Disorder, treated with aripirazole at therapeutic oral dose ranging from 15 mg/day to 30 mg/day, in the context of a depressive episode with mixed and psychotic features, showing a false positive urine amphetamine drug screen. We document the relationship between aripiprazole-dose, plasma concentration and amphetamines values in toxicologic urine examinations over time. Awareness of potential false positive urine amphetamine drug screens during aripiprazole treatment can condition therapeutic choices and prevent legal implications., (Copyright © 2019. Published by Elsevier Inc.)

Despite the efforts to prevent the spread of new psychoactive substances (NPS) such as synthetic amphetamine derivatives, it is apparent that newer types of NPS are still emerging on the market in recent years. Due to high potential for their abuse, reliable analytical methods are required to determine these substances in biological samples. The objective of this study was to develop and validate the gas chromatography-mass spectrometric (GC-MS) method for the simultaneous determination of 13 amphetamine-related NPS (amphetamine; AP, 4-fluoroamphetamine; 4FA, methamphamine; MA, 4-fluoromethamphetamine; 4FMA, 4-chloroamphetamine; 4CA, para-methoxyamphetamine; PMA, 4-chloromethamphetamine; 4CMA, 6-(2-aminopropyl)benzofuran; 6APB, 4-methylenedioxyamphetamine; MDA, para-methoxymethamphetamine; PMMA, 6-(2-methylaminopropyl)benzofuran; 6MAPB, 3,4-methylenedioxymethamphetamine; MDMA, 5,6-methylenedioxy-2-aminoindane; MDAI) in urine. The analytes were extracted at pH 7.4 by liquid-liquid extraction prior to their trifluoroacetyl derivatives and then analyzed by GC-MS. The validation parameters included selectivity, linearity, lower limits of quantification (LLOQ), intra and interday precision and accuracy, recovery and stability. The linear ranges were 2-100 ng/mL for AP, 4FA, 4FMA, 4CA, PMA, 6APB, MDA, and MDAI, 2-250 ng/mL for 4CMA, PMMA, and 6MAPB and 25-1,000 ng/mL for MA and MDMA, with acceptable coefficients of determination (r2 > 0.9963). The intra and interday precision were within 11.9 and 12.5%, while the intra and interday accuracies ranged from -10.6% to 13.0% and -11.0% to 6.8% for the nominal concentration at all studied levels, respectively. The LLOQs for each analyte were 2.0-25 ng/mL. The recoveries ranged from 69.3% to 96.4%. The short- and long-term variations of the analytes in urine were lower than 8.5 and 12.7%, indicating that the analytes are stable at least for 16 h at room temperature and for 7 days at 4°C, respectively. The applicability of the method was examined by analyzing urine samples from drug abusers and was determined to be effective for detecting multiple drug use.

Among the emerging new psychoactive substances (NPS), compounds carrying an N-ortho-methoxybenzyl substituent, the so-called NBOMes, represented a highly potent group of new hallucinogens. Recently, 3,4-dimethoxyamphetamine (3,4-DMA)-NBOMe and 4-methylmethamphetamine (4-MMA)-NBOMe occurred, but no data on their pharmacokinetics were available. According to other NBOMes, they are expected to be extensively metabolized. For detection and identification of their phase I and II metabolites, nano liquid chromatography coupled to high resolution tandem mass spectrometry (nanoLC-HRMS/MS) was used. Rat urine was prepared by simple dilution and incubation mixtures with pooled human liver S9 fraction by precipitation. Furthermore, the results concerning detectability using the new nanoLC approach were compared to those obtained by conventional ultra-high performance LC (UHPLC). In addition, the detectability of the compounds by standard urine screening approaches (SUSAs) routinely used by the authors with UHPLC-HRMS/MS, LC-MS n , and GC-MS was tested. Both NBOMes were extensively metabolized mainly by O-demethylation and conjugation with glucuronic acid (3,4-DMA-NBOMe) or oxidation of the tolyl group to the corresponding carboxylic acid (4-MMA-NBOMe). The developed nanoLC-HRMS/MS approach was successfully applied for identification of 38 3,4-DMA-NBOMe metabolites and 33 4-MMA-NBOMe metabolites confirming its detection power. Furthermore, the solvent saving nanoLC system showed comparable results to the UHPLC-HRMS/MS approach. In addition, an intake of an estimated low common user's dose of the compounds was detectable by all SUSAs only via their metabolites. Suggested targets for urine screening procedures were O-demethyl- and O,O-bis-demethyl-3,4-DMA-NBOMe and their glucuronides and carboxy-4-MMA-NBOMe and its glucuronide and N-demethyl-carboxy-4-MMA-NBOMe., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Carbon nanotubes (CNTs) have attracted a lot of attention as effective sorbents due to their strong sorption properties and several potential applications in many fields. In this work, the acid oxidized multiwalled carbon nanotubes (MWCNTs-COOH) was coated onto a stainless steel wire by a simple physical adhesion approach to develop solid-phase microextraction (SPME) fibers. By combination of the MWCNTs-COOH coated fiber-based headspace SPME and gas chromatography-mass spectrometry (GC-MS), the developed method demonstrates a good enhancement factor (288-651), low limits of detection (LODs, 0.2-1.3μg/L) for determination of amphetamine-type stimulant drugs (ATSs) in urine samples. The recoveries of the spiked ATSs (5, 50 and 500μg/L) were in the range of 88-107%, the calibration curve was linear for concentrations of analytes in the range from 0.5 to 1000μg/L (R=0.963-0.999). Furthermore, single fiber repeatability and fiber-to-fiber reproducibility were in the range of 2.3%-6.2% (n=6) and 5.7%-9.8% (n=3), respectively. The MWCNTs-COOH coated fiber is highly thermally stable and can be used over 150 times. The method was successfully applied to the forensic determination of amphetamine (AMP) and methamphetamine (MAMP) in human urine samples and satisfactory results were achieved., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Monitoring of amphetamine-type stimulant (ATS) confronts clinical labs with a high number of samples involving a variety of biological matrices. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), routinely used for confirmation of ATS abuse, requires of laborious and matrix-dependent sample treatment methods, this increasing analysis time and cost. In this work, a universal and single-step sample treatment, based on supramolecular solvents (SUPRAS), was proposed for simplifying ATS confirmation in seven biological matrices. The SUPRAS was synthesized in situ in the sample (900 µL of basified oral fluid, urine, serum, sweat or breast milk or 50 mg of digested hair or fingernails) by the addition of hexanol (200 µL) and tetrahydrofuran (900 µL). The mixture was vortex-shaken and centrifuged and the SUPRAS extract was subsequently analyzed by positive ion mode electrospray LC-MS/MS. The method was fully validated for amphetamine (AMP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), N-ethyl-3,4-methylenedioxyamphetamine (MDEA) and N-methyl-3,4-methylenedioxyamphetamine (MDMA). Maximum ion suppression or enhancement was 9% and 7%, respectively, and extraction recoveries (87-111%) and within- (0.1-6.7%) and between-day (0.3-9.7%) CVs were all within required values. The lower limits of quantification (LLOQ) for biological fluids (5 ng/mL), and hair and fingernails (100 ng/g) were all well below the cut-offs established by worldwide organizations. Confirmation of MDA was carried out in five urine samples that tested positive for ATS by immunoassay. The SUPRAS-LC-MS/MS methodology succeeded in developing a hitherto unexplored and universal tool for quantifying ATS in a comprehensive pool of biological matrices of interest in forensic and clinical samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The number of reports of serious adverse effects and intoxication after the use of the new drug 4-fluoroamphetamine (4-FA) increases. At the Emergency Department of the OLVG-Oost Hospital in Amsterdam, an on-site drug test, the Triage TOX Drug Screen, is available to assist in a rapid diagnosis. In less urgent cases, an EMIT II Plus immunoassay is used to determine semiquantitatively the presence of drugs of abuse (DOA). The antibodies in these immunoassays are designed to detect classic DOA and its urinary metabolites. Amphetamine-like drugs that may cause serious toxicity or impairment may not cross-react with the available immunoassay kits, and therefore may stay undetected. The question arises as to whether 4-FA and paramethoxymethamphetamine (PMMA) are detectable in the toxicological screening procedures commonly used for testing in urine., Methods: Synthetic urine was spiked with the drug under investigation to create spiking standards of 50, 100, 250, 500, 2500, and 5000 ng/mL. Urine drug screens were performed on the automated analyzer Biosite Triage MeterPro using the Triage TOX Drug Screen test and on a Siemens Drug Testing System Viva-E using the EMIT II Plus Ecstasy Assay and the EMIT II Plus Amphetamines Assay., Results: In this concentration range, the EMIT II Plus did not screen positive for PMMA, but there was some cross-reactivity for PMMA on the EMIT II Plus Ecstasy assay. The Triage TOX Drug Screen did test positive for PMMA at a concentration of 2500 ng/mL. The EMIT II Plus Amphetamines did test positive for 4-FA at a concentration of 5000 ng/mL, whereas the Ecstasy [3,4-methylenedioxymethamphetamine (MDMA)] assay only showed low cross-reactivity for 4-FA. The Triage TOX Drug Screen did not test positive for 4-FA., Conclusions: The available immunoassays lack sensitivity to detect 4-FA and PMMA in lower urine concentrations. Awareness of the fact that novel DOA may lead to false-negative urinary drug tests is of great importance.

Study Objectives: Multiple sleep latency testing (MSLT) and the maintenance of wakefulness test (MWT) are gold-standard objective tests of daytime sleepiness and alertness; however, there is marked variability in their interpretation and practice. This study aimed to determine the incidence of positive drug screens and their influence on MSLT, MWT, and polysomnographic variables., Methods: All patients attending Eastern Health Sleep Laboratory for MSLT or MWT over a 21-mo period were included in the study. Urinary drug screening for amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, methadone, and opiates was performed following overnight polysomnography (PSG). Demographics and PSG variables were compared., Results: Of 69 studies, MSLT (43) and MWT (26), 16% of patients had positive urinary drug screening (7 MSLT; 4 MWT). Drugs detected included amphetamines, cannabinoids, opiates, and benzodiazepines. No patient self-reported use of these medications prior to testing. No demographic, MSLT or MWT PSG data or overnight PSG data showed any statistical differences between positive and negative drug screen groups. Of seven MSLT patients testing positive for drug use, one met criteria for the diagnosis of narcolepsy and five for idiopathic hypersomnia. On MWT, three of the four drug-positive patients had a history of a motor vehicle accident and two patients were occupational drivers., Conclusions: These findings indicate drug use is present in patients attending for daytime testing of objective sleepiness and wakefulness. These data support routine urinary drug screening in all patients undergoing MSLT or MWT studies to ensure accurate interpretation in the context of illicit and prescription drug use., (© 2016 American Academy of Sleep Medicine)

Urine drug testing (UDT) has become an essential component in the management of patients prescribed opioid analgesics for the treatment of chronic non-malignant pain. Several laboratory methods are available to monitor adherence with the pharmacological regimen and abstinence from illicit or unauthorized medications. Immunochemical screening methods are rapid and economical, but they have limitations, including lack of specificity, and confirmatory methods are often necessary to verify presumptive positive results. We analyzed the results of confirmatory assays in an outpatient setting to determine the predictive value of presumptive positive urine drug screen results using an automated immunoassay for eight common drugs or drug classes. Positive predictive values (PPVs), in descending order, were as follows: cannabinoids (100%), cocaine (100%), opiates (86.8%), benzodiazepines (74.6%), oxycodone (67.6%), methadone (44.1%) and amphetamines (9.3%). The number of positive barbiturate results was too small to be included in the statistical analysis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A sensitive and rapid CZE-UV method was developed to determine drugs and their metabolites' presence in human urine. Ten drugs of abuse were analyzed including four amphetamines, cocaine, cocaethylene, heroin, morphine, 6-monoacetylmorphine, and 4-methylmethcathinone. An MSS (micelle to solvent stacking) approach was evaluated to enhance method sensitivity. This method considers composition of the micellar sample solution matrix and the injection time. Several analytical conditions influencing the resolution of the drugs mixture as pH and buffer concentration, organic solvent content, were also investigated. The base-line separation of all studied analytes in the same run was achieved within 18 min in an uncoated fused silica capillary (50 μm id × 60 cm) using a background solution containing 50 mM phosphate buffer pH 2.5 and 30% ACN v/v. Other experimental parameters such as applied voltage and capillary temperature were set up at 20 kV and 20°C, respectively. LOD values ranging between 15 and 75 ng/mL for all studied compounds were obtained. From a comparison with conventional CZE, the proposed method provides an increase of sensitivity (39- to 55-fold enhancement factor). Under optimal MSS-CZE conditions, good linearity was achieved (R 2 ≤ 0.9998). The method was finally applied to the analysis of urine samples spiked with a standard mixture after a sample pretreatment, reaching satisfactory recovery values., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A gas chromatography-mass spectrometry method for the determination of amphetamines in urine samples by means of liquid-phase microextraction was validated, including calculation of measurement uncertainty. After extraction in the three-phase mode, acceptor phase was withdrawn from the fiber and the residue was derivatized with trifluoroacetic anhydride. The method showed to be very simple, rapid and it required a significantly low amount of organic solvent for extraction. The limits of detection were 10 and 20μg/L for amphetamine and methamphetamine, respectively. The calibration curves were linear over the specified range (20μg/L to 1400μg/L; r(2)>0.99). The method showed to be both precise and accurate and a relative combined uncertainty of 2% was calculated. In order of importance, the factors which were more determinant for the calculation of method uncertainty were: analyte concentration, sample volume, trueness and method precision., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The counter current salting-out homogenous liquid-liquid extraction (CCSHLLE) joined with the dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) has been developed as a high preconcentration technique for the determination of different drugs in urine samples. Amphetamines were employed as model compounds to assess the extraction procedure and were determined by high performance liquid chromatography-ultraviolet detection (HPLC-UV). In this method, initially, NaCl as a separation reagent is filled into a small column and a mixture of urine and acetonitrile is passed through the column. By passing the mixture, NaCl is dissolved and the fine droplets of acetonitrile are formed due to salting-out effect. The produced droplets go up through the remained mixture and collect as a separated layer. Then, the collected acetonitrile is removed with a syringe and mixed with 30.0μL 1-undecanol (extraction solvent). In the second step, the 5.00mLK2CO3 solution (2% w/v) is rapidly injected into the above mixture placed in a test tube for further DLLME-SFO. Under the optimum conditions, calibration curves are linear in the range of 1-3000μgL(-1) and limit of detections (LODs) are in the range of 0.5-2μgL(-1). The extraction recoveries and enrichment factors ranged from 78 to 84% and 157 to 168, respectively. Repeatability (intra-day) and reproducibility (inter-day) of method based on seven replicate measurements of 100μgL(-1) of amphetamines were in the range of 3.5-4.5% and 4-5%, respectively. The method was successfully applied for the determination of amphetamines in the actual urine samples. The relative recoveries of urine samples spiked with amphetamine and methamphetamine are 90-108%., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In recent years, the abuse of synthetic cathinones has increased considerably. This study proposes a method, based on gas chromatography/mass spectrometry (GC-MS), to analyze and quantify six synthetic cathinones in urine samples: mephedrone (4-MMC), methylone (bk-MDMA), butylone, ethylone, pentylone and methylenedioxypyrovalerone (MDPV). In our procedure, the urine samples undergo solid-phase extraction (SPE) and derivatization prior to injection into the GC-MS device. Separation is performed using a HP-5MS capillary column. The use of selective ion monitoring (SIM mode) makes it is good sensitivity in this method, and the entire analysis process is within 18 min. In addition, the proposed method maintains linearity in the calibration curve from 50 to 2,000 ng/mL (r(2) > 0.995). The limit of detection of this method is 5 ng/mL, with the exception of MDPV (20 ng/mL); the limit of quantification is 20 ng/mL, with the exception of MDPV (50 ng/mL). In testing, the extraction performance of SPE was between 82.34 and 104.46%. Precision and accuracy results were satisfactory <15%. The proposed method was applied to six real urine samples, one of which was found to contain 4-MMC and bk-MDMA. Our results demonstrate the efficacy of the proposed method in the identification of synthetic cathinones in urine, with regard to the limits of detection and quantification. This method is highly repeatable and accurate., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The 22 amphetamine-derived synthetic drugs (ADSDs), mostly cathinones, were examined by gas chromatography with mass spectrometry using two different derivatization methods with (i) heptafluorobutyric anhydride (HFBA) and (ii) pentafluorobenzoyl chloride (PFBCl). Both developed derivatization approaches were evaluated and compared for urine and serum samples. Extraction procedures proved to give satisfactory results with regard to recoveries and extract purity, even though both derivatization methods reached acceptable sensitivity for the intended use. The derivatization with PFBCl showed better results with respect to retention and response stability, thus the PFBCl method was selected for validation. Calibration curves were linear over the tested concentration range of 20-1,000 ng/mL with the R(2) values ranging from 0.994 to 0.998. Intra- and interday precisions and accuracies were within 20% for all concentrations in the linear range. The limit of detection was determined to be lower than 2 ng/mL for all 22 analytes. The method proved to be a useful analytical tool in the course of systematic toxicological analysis., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Urine drug screens (UDSs) are used to identify the presence of certain medications. One limitation of UDSs is the potential for false-positive results caused by cross-reactivity with other substances. Amphetamines have an extensive list of cross-reacting medications. The literature contains reports of false-positive amphetamine UDSs with multiple antidepressants and antipsychotics. We present 2 cases of presumed false-positive UDSs for amphetamines after ingestion of aripiprazole. Case 1 was a 16-month-old girl who accidently ingested 15 to 45 mg of aripiprazole. She was lethargic and ataxic at home with 1 episode of vomiting containing no identifiable tablets. She remained sluggish with periods of irritability and was admitted for observation. UDS on 2 consecutive days came back positive for amphetamines. Case 2 was of a 20-month-old girl who was brought into the hospital after accidental ingestion of an unknown quantity of her father's medications which included aripiprazole. UDS on the first day of admission came back positive only for amphetamines. Confirmatory testing with gas chromatography-mass spectrometry (GC-MS) on the blood and urine samples were also performed for both patients on presentation to detect amphetamines and were subsequently negative. Both patients returned to baseline and were discharged from the hospital. To our knowledge, these cases represent the first reports of false-positive amphetamine urine drug tests with aripiprazole. In both cases, aripiprazole was the drug with the highest likelihood of causing the positive amphetamine screen. The implications of these false-positives include the possibility of unnecessary treatment and monitoring of patients., (Copyright © 2015 by the American Academy of Pediatrics.)

In this work, an ordered mesoporous carbon (OMC)/Nafion coated fiber for solid-phase microextraction (SPME) was prepared and used as the working electrode for electro-enhanced SPME (EE-SPME) of amphetamines. The EE-SPME strategy is primarily based on the electro-migration and complementary charge interaction between fiber coating and ionic compounds. Compared with traditional SPME, EE-SPME exhibited excellent extraction efficiency for amphetamine (AP) and methamphetamine (MA) with an enhancement factor of 7.8 and 12.1, respectively. The present strategy exhibited good linearity for the determination of AP and MA in urine samples in the range of 10-1000ngmL(-1) and 20-1000ngmL(-1), respectively. The detection limits were found to be 1.2ngmL(-1) for AP and 4.8ngmL(-1) for MA. The relative standard deviations were calculated to be 6.2% and 8.5% for AP and MA, respectively. Moreover, the practical application of the proposed method was demonstrated by analyzing the amphetamines in urine and serum samples with satisfactory results., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The emergence of novel psychoactive substances is an ongoing challenge for analytical toxicologists. Different analogs are continuously introduced in the market to circumvent legislation and to enhance their pharmacological activity. Although detection of drugs in blood indicates recent exposure and link intoxication to the causative agent, urine is still the most preferred testing matrix in clinical and forensic settings. We developed a method for the simultaneous quantification of 8 piperazines, 4 designer amphetamines and 28 synthetic cathinones and 4 metabolites, in urine by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Data were acquired in full scan and data dependent MS(2) mode. Compounds were quantified by precursor ion exact mass, and confirmed by product ion spectra library matching, taking into account product ions' exact mass and intensities. One-hundred μL urine was subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with gradient mobile phase of 0.1% formic acid in water and in acetonitrile in 20 min. The assay was linear from 2.5 or 5 to 500 μg/L. Imprecision (n=15) was <15.4%, and accuracy (n=15) 84.2-118.5%. Extraction efficiency was 51.2-111.2%, process efficiency 57.7-104.9% and matrix effect ranged from -41.9% to 238.5% (CV<23.3%, except MDBZP CV<34%). Authentic urine specimens (n=62) were analyzed with the method that provides a comprehensive confirmation for 40 new stimulant drugs with specificity and sensitivity., (Published by Elsevier B.V.)

4-methyl-N-ethcathinone (4-MEC), the N-ethyl homologue of mephedrone, is a novel psychoactive substance of the beta-keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4-MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N-deethylation, hydroxylation of the 4-methyl group followed by further oxidation to the corresponding 4-carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N-methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N-deethyl-dihydro isomers and the 4-carboxy-dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4-MEC should be detectable in urine by the GC-MS and LC-MS(n) standard urine screening approaches at least after overdose., (Copyright © 2014 John Wiley & Sons, Ltd.)

Two microextraction techniques based on hollow fiber liquid-phase microextraction (HF-LPME) and ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) had been applied for the determination of drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, methcathinone, ketamine, meperidine, and methadone) in urine and blood samples by gas chromatography-mass spectrometry. Parameters affecting extraction efficiency have been investigated and optimized for both methods. Under the optimum conditions, linearities were observed for all analytes in the range 0.0030-10 μg/ml with the correlation coefficient (R) ranging from 0.9985 to 0.9995 for HF-LPME and in the range 0.0030-10 μg/ml with the R ranging from 0.9985 to 0.9994 for DLLME. The recovery of 79.3-98.6% with RSDs of 1.2-4.5% was obtained for HF-LPME, and the recovery of 79.3-103.4% with RSDs of 2.4-5.7% was obtained for DLLME. The LODs (S/N=3) were estimated to be in the range from 0.5 to 5 ng/ml and 0.5 to 4 ng/ml, respectively. Compared with HF-LPME, the UA-LDS-DLLME technique had the advantages of less extraction time, suitability for batches of sample pretreatment simultaneously, and higher extraction efficiency, while HF-LPME has excellent sample clean-up effect, and is a robust and suitable technique for various sample matrices with better repeatability. Both methods were successfully applied to the analysis of drugs of abuse in real human blood sample., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Immunoassay is currently the most common approach for urine drug screening. However, the continuous emergence of new psychoactive substances (NPS) and their low urinary concentrations have challenged the scope and sensitivity of immunoassays. Consequently, specialized toxicology laboratories rely more and more on mass spectrometry (MS) based techniques. Ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOF-MS) is an especially attractive technique for comprehensive drug screening. The objective was to compare the performances of immunoassay and UHPLC-HR-TOF-MS in terms of scope, flexibility, sensitivity, and reliability of substance identification. A total of 279 post-mortem urine samples were analyzed using a method representative of each technique. The immunoassay method was an Emit II Plus enzyme immunoassay for the following drug groups: amphetamines, benzodiazepines, buprenorphine, cannabis, and opiates. The UHPLC-HR-TOF-MS method was a recently published method covering hundreds of drugs: conventional drugs of abuse, abused prescription drugs, and NPS of various classes. UHPLC-HR-TOF-MS produced a lower number of false positive (FP) results for the drug groups covered by immunoassay. Many of the false negative (FN, n = 40) and FP (n = 22) immunoassay results were obviously due to the higher cut-off concentrations and interfering matrix, respectively. Moreover, the wider scope of UHPLC-HR-TOF-MS allowed detection of NPS and prescription drugs. UHPLC-HR-TOF-MS gave FP results related to a few particular substances. The future option of adjusting all compound-specific reporting parameters individually would allow the method's sensitivity and specificity to be fully exploited., (Copyright © 2014 John Wiley & Sons, Ltd.)

Aim: Exhaled breath has recently been identified as a possible matrix for drug testing. This study explored the potential of this new method for compliance monitoring of patients being treated for dependence disorders., Methods: Outpatients in treatment programs were recruited for this study. Urine was collected as part of clinical routine and a breath sample was collected in parallel together with a questionnaire about their views of the testing procedure. Urine was analyzed for amphetamines, benzodiazepines, cannabis, cocaine, buprenorphine, methadone and opiates using CEDIA immunochemical screening and mass spectrometry confirmation. The exhaled breath was collected using the SensAbues device and analyzed by mass spectrometry for amphetamine, methamphetamine, diazepam, oxazepam, tetrahydrocannabinol, cocaine, benzoylecgonine, buprenorphine, methadone, morphine, codeine and 6-acetylmorphine., Results: A total of 122 cases with parallel urine and breath samples were collected; 34 of these were negative both in urine and breath. Out of 88 cases with positive urine samples 51 (58%) were also positive in breath. Among the patients on methadone treatment, all were positive for methadone in urine and 83% were positive in breath. Among patients in treatment with buprenorphine, 92% were positive in urine and among those 80% were also positive in breath. The questionnaire response documented that in general, patients accepted drug testing well and that the breath sampling procedure was preferred., Conclusion: Compliance testing for the intake of prescribed and unprescribed drugs among patients in treatment for dependence disorders using the exhaled breath sampling technique is a viable method and deserves future attention.

A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) has been developed for the determination of multiple drugs of abuse in biological samples by gas chromatography-triple quadrupole mass spectrometry (GC-QQQ-MS). A total of 100 µL of toluene as extraction solvent was dropped into the sample solution. Then the mixture was sonicated drastically in an ultrasonic bath for 3 min with occasional manual shaking to form a cloudy suspension. After centrifugation at 10,000 r/min for 3 min, the upper layer of low-density extractant was withdrawn and injected into the GC-QQQ-MS for analysis. The parameters affecting extraction efficiency have been investigated and optimized. Under the optimum conditions, good linearities were observed for all analytes with the correlation coefficients ranging from 0. 998 4 to 0. 999 4. The recoveries of 79.3%-100.3% with RSDs < 5.7% were obtained. The LODs (S/N = 3) were in the range from 0.05 to 0.40 µg/L. UA-LDS-DLLME technique has the advantages of less extraction time, suitable for batches of sample pretreatment simultaneously, and higher extraction efficiency. It was successfully applied to the analysis of amphetamines in real human urine samples.

Rapid component separation and robust surface-enhanced Raman scattering (SERS) identification of drugs in real human urine remain an attractive challenge because of the sample complexity, low molecular affinity for metal surface, and inefficient use of hotspots in one- or two-dimensional (2D) geometries. Here, we developed a 5 min strategy of cyclohexane (CYH) extraction for separating amphetamines from human urine. Simultaneously, an oil-in-water emulsion method is used to assemble monodisperse Ag nanoparticles in the CYH phase into spherical colloidal superstructures in the aqueous phase. These superstructures create three-dimensional (3D) SERS hotspots which exist between every two adjacent particles in 3D space, break the traditional 2D limitation, and extend the hotspots into the third dimension along the z-axis. In this platform, a conservative estimate of Raman enhancement factor is larger than 10(7), and the same CYH extraction processing results in a high acceptability and enrichment of drug molecules in 3D hotspots which demonstrates excellent stability and reproducibility and is suitable for the quantitative examination of amphetamines in both aqueous and organic phases. Parallel ultraperformance liquid chromatography (UPLC) examinations corroborate an excellent performance of our SERS platform for the quantitative analysis of methamphetamine (MA) in both aqueous solution and real human urine, of which the detection limits reach 1 and 10 ppb, respectively, with tolerable signal-to-noise ratios. Moreover, SERS examinations on different proportions of MA and 3,4-methylenedioxymethamphetamine (MDMA) in human urine demonstrate an excellent capability of multiplex quantification of ultratrace analytes. By virtue of a spectral classification algorithm, we realize the rapid and accurate recognition of weak Raman signals of amphetamines at trace levels and also clearly distinguish various proportions of multiplex components. Our platform for detecting drugs promises to be a great prospect for a rapid, reliable, and on-spot analyzer.

Objective: To investigate whether the use of the stimulants amphetamines and cocaine by truck drivers in Brazil was related to travel length., Methods: Truck drivers were randomly stopped by the Federal Highway Police on interstate roads in Sao Paulo State during morning hours from 2008 to 2011 and invited to participate in the project "Comandos de Saúde nas Rodovias" (Health Commands on the Roads). Participants were asked about the use of drugs, travel distance, and age, and gender was recorded. Samples of urine were collected and analyzed for amphetamine, benzoylecgonine (a metabolite of cocaine), and carboxytetrahydrocannabinol (THC-COOH; a metabolite of cannabis) by immunological screening and quantification by gas chromatography-mass spectroscopy., Results: Current use of amphetamine, cocaine, and cannabis was reported by 5.7%, 0.7%, and 0.3% of the truck drivers, respectively. Amphetamine, benzoylecgonine, and THC-COOH were found in urine samples from 5.4%, 2.6,% and in 1.0% of the drivers, respectively. There was a significant association between the positive cases for amphetamine and reported travel length; 9.9% of urine samples from drivers who reported travel length of more than 270 km were positive for amphetamine, and 10.9% of those drivers reported current use of amphetamines. In most cases, appetite suppressants containing amphetamines had been used, but the purpose was most often to stay awake and alert while driving. Truck drivers with travel length of more than 270 km had significantly higher odds ratio (OR) for having a urine sample that was positive for amphetamine when adjusted for age as confounding factor (OR = 9.41, 95% confidence interval [CI], 3.97-22.26). No significant association was found between the use of cocaine or cannabis and travel length., Conclusion: Truck drivers who reported driving more than 270 km had significantly higher frequencies of urine samples positive for amphetamine and reported significantly more frequent current use of amphetamines than those who reported shorter driving distances.

Exertional heat stroke incidence is on the rise and has become the third leading cause of death in high school athletes. It is entirely preventable, yet this is a case of a 15-year-old, 97-kg male football player who presented unresponsive and hyperthermic after an August football practice. His blood pressure was 80/30, and his pulse was 180. He had a rectal temperature of 107.3°F, and upon entering the emergency department, he was rapidly cooled in 40 minutes. As he progressed, he developed metabolic acidosis, elevated liver enzymes, a prolapsed mitral valve with elevated troponin levels, and worsening hypotension even with extracorporeal membrane oxygenation support. After 3 days in the hospital, this young man was pronounced dead as a result of complications from exertional heat stroke. We address not only the complications of his hospital course relative to his positive blood cultures but also the complications that can result from attention-deficit/hyperactivity disorder medication our patient was taking. As the population of young adults becomes more obese and more highly medicated for attention-deficit/hyperactivity disorder, we sought out these growing trends in correlation with the increase in incidence of heat-related illness. We also address the predisposing factors that make young high school athletes more likely to experience heat illness and propose further steps to educate this susceptible population.

Aim: Products for on-site urine drug testing offer the possibility to perform screening for drugs of abuse directly at the point-of-care. This is a well-established routine in emergency and dependency clinics but further evaluation of performance is needed due to inherent limitations with the available products., Methods: Urine drug testing by an on-site product was compared with routine laboratory methods. First, on-site testing was performed at the laboratory in addition to the routine method. Second, the on-site testing was performed at a dependency clinic and urine samples were subsequently sent to the laboratory for additional analytical investigation., Results: The on-site testing products did not perform with assigned cut-off levels. The subjective reading between the presence of a spot (i.e. negative test result) being present or no spot (positive result) was difficult in 3.2% of the cases, and occurred for all parameters. The tests performed more accurately in drug negative samples (specificity 96%) but less accurately for detecting positives (sensitivity 79%). Of all incorrect results by the on-site test the proportion of false negatives was 42%. The overall agreement between on-site and laboratory testing was 95% in the laboratory study and 98% in the clinical study., Conclusion: Although a high degree of agreement was observed between on-site and routine laboratory urine drug testing, the performance of on-site testing was not acceptable due to significant number of false negative results. The limited sensitivity of on-site testing compared to laboratory testing reduces the applicability of these tests.

In order to achieve chromatographic separation, urine samples shown to be initially positive for amphetamines and methamphetamines in US Department of Defense immunoassays are derivatized with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-(-)-MTPA) prior to gas chromatography-electron impact-mass spectrometry (GC-EI-MS) analysis. Phentermine, a member of the phenethylamine class of drugs and a common appetite suppressant, interferes with GC-EI-MS assays of R-(-)-MTPA-derivatized d-amphetamine, degrading the chromatography of the internal standard and analyte ions and skewing concentration calculations. Additionally, when specimens with high concentrations of l-methamphetamine are derivatized with R-(-)-MTPA, signal peaks have the potential to be misidentified by integration software as d-methamphetamine. We have found that replacing R-(-) MTPA with (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride reduces phentermine interference problems related to internal standard chromatography, reduces the possibility of concentrated l-methamphetamine peaks being misidentified by integration software, improves resolution of d-methamphetamine in the presence of high l-methamphetamine concentrations, and is a cost-neutral change that can be applied to current amphetamines GC-EI-MS methods without the need for method modification., (Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Novel substances of expected doping activity are constantly introduced to the market. β-Methylphenethylamine (BMPEA) is classified as a doping agent by the World Anti-Doping Agency as it is a positional isomer of amphetamine. In this work, the development and application of a simple and rapid analytical procedure that enables discrimination between both isomers is described. The analytes of interest were extracted from urine by a two-step liquid-liquid extraction and then analyzed by UPLC/MS/MS under isocratic conditions. The entire analytical procedure was validated by evaluating its selectivity, discrimination capabilities, carry-over, sensitivity, and influence of matrix effects on its performance. Application of the method resulted in detection of BMPEA in eight anti-doping samples, including the first report of adverse analytical finding regarding its use. Further analysis showed that BMPEA may be eliminated unchanged along with its phase II conjugates, the hydrolysis of which may considerably improve detection capabilities of the method. Omission of the hydrolysis step may therefore, produce false-negative results. Testing laboratories should also carefully examine their LC/MS/MS-based amphetamine and BMPEA findings as both isomers fragment yielding comparable collision-induced dissociation spectra and their insufficient chromatographic separation may result in misidentification. This is of great importance in case of forensic analyses as BMPEA is not controlled by the public law, and its manufacturing, distribution, and use are legal.

A novel, rapid, simple and sensitive dispersive liquid-liquid microextraction method based on the solidification of floating organic drop (DLLME-SFO) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine amphetamine and methamphetamine in urine samples. The factors affecting the extraction efficiency of DLLME-SFO such as the kind and volume of the extraction and the disperser solvents, effect of concentration of K2CO3 and extraction time were investigated and the optimal extraction conditions were established. Under the optimum conditions (extraction solvent: 30.0μl 1-undecanol; disperser solvent: 300μl acetonitrile; buffer concentration: 2% (w/v) K2CO3 and extraction time: 1min), calibration curves are linear in the range of 10-3000μgl(-1) and limit of detections (LODs) are in the range of 2-8μgl(-1). The relative standard deviations (RSDs) for 100μgl(-1) of amphetamine and methamphetamine in diluted urine are in the range of 6.2-7.8% (n=7). The method was successfully applied for the determination of amphetamine and methamphetamine in the actual urine samples. The relative recoveries of urine samples spiked with amphetamine and methamphetamine are 87.8-113.2%. The obtained results show that DLLME-SFO combined with HPLC-UV is a fast and simple method for the determination of amphetamine and methamphetamine in urine., (Copyright © 2014 Elsevier B.V. All rights reserved.)

4-Methyl-amphetamine (1-(4-methylphenyl)propane-2-amine; 4-MA) and its isomers 2-methyl-amphetamine (2-MA) and 3-methyl-amphetamine (3-MA) belong to the group of amphetamine-type stimulants and of new psychoactive substances. Several studies showed similar potencies in releasing noradrenalin and dopamine, but higher potencies in releasing serotonin than amphetamine. In March 2013, the EU Council decided on an EU-wide control based on the European Monitoring Centre for Drugs and Drug Addiction risk assessment report documenting that 4-MA was sold as amphetamine on the illicit market and detected in several fatal cases. Therefore, 4-MA and its isomers should be covered by drug testing in clinical and forensic toxicology. The aims of the presented work were to study the metabolism and detectability of each isomer in urine samples. For metabolism studies, rat urine samples were isolated by solid-phase extraction without and after enzymatic cleavage of conjugates. The phase I metabolites were separated and identified after acetylation by gas chromatography-mass spectrometry (GC-MS) and/or liquid chromatography-high resolution-linear ion trap mass spectrometry (LC-HR-MS(n)) and the phase II metabolites by LC-HR-MS(n). From the identified phase I and II metabolites, the following main metabolic pathways were deduced: aromatic hydroxylation, hydroxylation of the phenylmethyl group followed by oxidation to the corresponding carboxylic acid, hydroxylation of the side chain, and glucuronidation and/or sulfation of the hydroxy and carboxy groups. CYP2D6 was involved in the aromatic hydroxylation. Finally, the intake of a commonly used dose of the MAs could be confirmed in rat urine using the authors' GC-MS and the LC-MS(n) standard urine screening approaches. Differentiation of the isomers to confirm the intake of a specific isomer was possible with an additional workup in rat urine.

Objective: The purpose of this study was to estimate the prevalence of recent use of illicit drugs among truck drivers who had parked their vehicles at the terminal port in Paranaguá City at Paraná State, southern Brazil., Methods: This cross-sectional study was part of a larger research project conducted among drivers at a regional Brazilian port. Data on professional characteristics, involvement in road traffic injuries, sleep, and use of alcohol and illicit drugs were collected using a questionnaire. Urine samples were collected and analyzed for amphetamines, cocaine, and cannabis using gas chromatography with mass spectrometric detection., Results: Sixty-two drivers were included in the study. Toxicological analyses showed that 8.1 percent (95% confidence interval [CI], 2.7-17.8%) of the urine samples were positive for drugs (4.8% for cocaine, 1.6% for amphetamine, and 1.6% for both); 8.1 percent reported drug use during the preceding 30 days in the questionnaire and only one tested positive for the drug in the urine sample. No sample was positive for cannabinoids. In total, at least 14.5 percent (95% CI, 6.9-25.8%) had used illicit drugs during the preceding 30 days based on self-reports and urine testing. Drivers who reported involvement in traffic injuries the year before more often tested positive for drugs in biological samples (P <.05)., Conclusions: This research provides preliminary evidence that the use of illicit stimulants was common among professional truck drivers transporting grain loads. Thus, actions are needed to reduce drug use among truck drivers in order to prevent drug-related road traffic injuries.

Background: The purpose of this study was to determine whether the act of in vivo urine dilution is a random phenomenon., Methods: The sample population was divided into 4 groups for each of the drugs tested. The groups are precreatinine normalization laboratory positive, precreatinine normalization laboratory negative, and postcreatinine normalization laboratory drug positive, and postcreatinine normalization laboratory drug negative. The nonparametric Kruskal-Wallis 1-way ANOVA by ranks with post hoc Mann-Whitney U testing Bonferroni adjustment was used to compare the mean urine creatinine concentration of each group., Results: The Kruskal-Wallis test revealed that each drug had a statistically significant difference in group comparisons. Post hoc testing found that cocaine demonstrated no statistically significant difference between the prenormalization drug positive and negative groups. Amphetamines, marijuana, and opiates did confirm statistically significant differences between the prenormalization drug positive and negative groups. The examination of phencyclidine was limited by the lack of a prenormalization drug positive group; therefore, no definitive findings can be presented. Statistically significant differences were found between the postnormalization drug positive and negative groups for each drug tested., Conclusions: This study supports the theory that urine dilution is not a random happening and it may adversely affect the integrity of the drug-testing process.

This study developed a selective and rapid high-performance liquid chromatography-diode array detection method for the confirmation of different cathinone derivates in human urine. Samples were prepared by solid-phase extraction (SPE) using procaine hydrochloride as the internal standard. The chromatographic separation was performed on a Kinetex PFP column using isocratic elution. The mobile phase was composed of a mixture of acetonitrile (33%, v/v) and 0.005M ammonium trifluoroacetate buffer (67%, v/v; pH 4.93 ± 0.03) with a flow rate of 0.350 mL/min. The diode array detection was performed at 262 nm. The method was linear over the concentration range of 25-2,400 ng/mL. Intra-day and inter-day precision values for cathinones were less than 1.26% (relative standard deviation). The limit of detection for any compounds extracted from human urine by the optimized SPE method was 40 ng/mL and the limit of quantification was 100 ng/mL in the urine. The recovery rate of SPE was between 71 and 82% with a lower relative standard deviation than 2.35%.

A novel analytical toxicology method has been developed for the analysis of drugs of abuse in human urine by using a high resolution and high mass accuracy hybrid linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap-MS). This method allows for the detection of different drugs of abuse, including amphetamines, cocaine, opiate alkaloids, cannabinoids, hallucinogens and their metabolites. After solid-phase extraction with Oasis HLB cartridges, spiked urine samples were analysed by HPLC/LTQ-Orbitrap-MS using an electrospray interface in positive ionisation mode, with resolving power of 30,000 full width at half maximum (FWHM). Gradient elution off of a Hypersil Gold PFP column (50mm×2.1mm) allowed to resolve 65 target compounds and 3 internal standards in a total chromatographic run time of 20min. Validation of this method consisted of confirmation of identity, selectivity, linearity, limit of detection (LOD), lowest limits of quantification (LLOQ), accuracy, precision, extraction recovery and matrix effect. The regression coefficients (r(2)) for the calibration curves (LLOQ - 100ng/mL) in the study were ≥0.99. The LODs for 65 validated compounds were better than 5ng/ml except for 4 compounds. The relative standard deviation (RSD), which was used to estimate repeatability at three concentrations, was always less than 15%. The recovery of extraction and matrix effects were above 50 and 70%, respectively. Mass accuracy was always better than 2ppm, corresponding to a maximum mass error of 0.8 millimass units (mmu). The accurate masses of characteristic fragments were obtained by collisional experiments for a more reliable identification of the analytes. Automated data analysis and reporting were performed using ToxID software with an exact mass database. This procedure was then successfully applied to analyse drugs of abuse in a real urine sample from subject who was assumed to be drug addict., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

The rise in amphetamine, ketamine and opiates abuse in Taiwan has created a need for a reliable confirmatory assay. A method that combines superficially porous liquid chromatography tandem mass spectrometry (LC-MS/MS) with solid-phase extraction (SPE) was developed for the simultaneous quantification of amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), ketamine, opiates, and their corresponding metabolites in urine. The total run time of the method was 6.7min including equilibration time. The method was validated in accordance with the European Commission (EC) Decision 2002/642/EC. The within- and between-day precision was below 13.6% and the accuracy ranged from -17.1% to +9.9% for all analytes. Ion suppression was observed but compensated by using deuterated internal standards. No carryover was detected and the analytes were stable at room temperature for 16h, and for 72h at 4°C, and three-thaw cycles. The method was further validated by comparison with a reference gas chromatography-mass spectrometry (GC-MS) method, using 52 authentic urine samples. The results indicated that for the target analytes studied, the LC-MS/MS analysis was as precise, accurate, and specific as the GC-MS method. In conclusion, the present LC-MS/MS method is robust and reliable, and suitable for use as a confirmation assay in the simultaneous urine drug testing and quantification of amphetamines, ketamines, and opiates., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The performance of the previously validated LUCIO(®)-Direct-enzyme linked immunosorbent assay (direct ELISA) screening tests according to forensic guidelines is compared to that of cloned enzyme donor immunoassays (CEDIA) test for drugs of abuse in urine as defined in the new re-licensing German medical and psychological assessment (MPA) guidelines. The MPA screening cut-offs correspond to 10 ng/ml 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), 50 ng/ml amphetamine and designer amphetamines, 25 ng/ml morphine, codeine and dihydrocodeine, 30 ng/ml benzoylecgonine, 50 ng/ml methadone metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and metabolites of diazepam, oxazepam, bromazepam, alprazolam, flunitrazepam and lorazepam at 50 ng/ml. Average relative sensitivities and relative specificities were 99.7 % and 98.4 % for direct ELISA and 66 % and 91.4 % for CEDIA, respectively., (Copyright © 2013 John Wiley & Sons, Ltd.)

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL(-1) for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10-1000 ng mL(-1) and 21-1000 ng mL(-1), respectively., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cocaine is a potent sympathomimetic drug that is associated with cardiotoxicity, including ventricular arrhythmia, systemic hypertension, acute myocardial infarction and left ventricular hypertrophy. The use of cocaine in Australia has risen steadily since the late 1990s. What remains unclear in the literature is whether cocaine-associated death can occur in the absence of other contributing factors, such as concomitant drug use or natural disease. A search was conducted on the National Coroners Information System database, to identify all deaths occurring in Victoria, Australia, between January 2000 and December 2011, where cocaine or its metabolites were detected by post-mortem toxicological analysis. All cases were closed by the Coroner. These cases were examined with regards to case circumstances, pathology and toxicology results, and coronial findings, to determine the prevalence of cardiotoxicity and the involvement of cocaine in the deaths compared with other contributing factors. There were 49 cases where cocaine, benzoylecgonine, ecgonine methyl ester, methylecgonine or cocaethylene, were detected in the 11-year period. The individuals ranged in age from 16 to 70 years (median 30). There were 36 males. In 22 cases the cause of death was determined to be drug toxicity, 22 were external injury and 5 were attributed to natural disease. The concentration of cocaine in the cases was relatively low (range 0.01-3 mg/L, median 0.1 mg/L). Cocaine metabolites were detected frequently in blood and urine: benzoylecgonine (46 cases); ecgonine methyl ester (12 cases); cocaethylene (8 cases); and methylecgonine (9 cases). Opioids were commonly detected (23 cases), in addition to amphetamines (15 cases), ethanol (17 cases) and benzodiazepines (12 cases). Of the 43 cases receiving a full autopsy, there were 14 cases involving significant heart disease. This included coronary artery disease (11 cases), an enlarged heart (5 cases), myocarditis and contraction band necrosis. Cocaine is detected relatively infrequently in Victorian coronial cases. However it appears to be associated with a significant degree of cardiotoxicity, particularly coronary artery disease and ventricular hypertrophy, independent of cocaine concentration., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: This study examines the effect of creatinine normalization on urine drug concentrations of 5 substances (amphetamines, cocaine, marijuana, opiates, and phencyclidine) and how this affects the proportion of reported positives., Methods: The Wilcoxon matched-pairs signed-ranks test was used to compare the mean prenormalization urinary drug concentration with the mean postnormalization urinary drug concentration. Frequency analysis was performed on dichotomous drug test results and the information was used to complete McNemar testing for each drug to determine the difference of proportions for prenormalization positive drug tests to postnormalization positive drug test., Results: Each drug tested (N = 4460) was found to have a statistically significant increase in mean urinary drug concentration after creatinine normalization with effect sizes ranging from small to medium with cocaine having the largest effect size (r = 0.229) and phencyclidine having the lowest effect size (r = 0.121). The differences in proportion of dichotomous results between study and control groups for drugs tested were compared with the McNemar test. Each drug had a statistically significant (P = 0.0010) increase of positive drug tests., Conclusions: This result indicates that specimen dilution does affect the number of laboratory-positive results confirmed.