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Synthetic cathinones are some of the most prevalent new psychoactive substances (NPSs) globally, with alpha-pyrrolidinoisohexanophenone (α-PiHP) being particularly noted for its widespread use in the United States, Europe, and Taiwan. However, the analysis of isomeric NPSs such as α-PiHP and alpha-pyrrolidinohexiophenone (α-PHP) is challenging owing to similarities in their retention times and mass spectra. This study proposes a dual strategy based on in vitro metabolic experiments and machine learning-based classification modelling for differentiating α-PHP and α-PiHP in urine samples: (1) in vitro metabolic experiments using pooled human liver microsomes and liquid chromatography tandem quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) were conducted to identify the key metabolites of α-PHP and α-PiHP from the high-resolution MS/MS spectra. After 5 h incubation, 71.4 % of α-PHP and 64.7 % of α-PiHP remained unmetabolised. Nine phase I metabolites were identified for each compound, including primary β-ketone reduction (M1) metabolites. Comparing the metabolites and retention times confirmed the efficacy of in vitro metabolic experiments for differentiating NPS isomers. Subsequently, analysis of seven real urine samples revealed the presence for various metabolites, including M1, that could be used as suitable detection markers at low concentrations. The aliphatic hydroxylation (M2) metabolite peak counts and metabolite retention times were used to determine α-PiHP use. (2) Classification models for the parent compounds and M1 metabolites were developed using principal component analysis for feature extraction and logistic regression for classification. The training and test sets were devised from the spectra of standard samples or supernatants from in vitro metabolism experiments with different incubation times. Both models had classification accuracies of 100 % and accurately identified α-PiHP and its M1 metabolite in seven real urine samples. The proposed methodology effectively distinguished between such isomers and confirmed their presence at low concentrations. Overall, this study introduces a novel concept that addresses the complexities in analysing isomeric NPSs and suggests a path towards enhancing the accuracy and reliability of NPS detection., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Immunoassays provide a rapid tool for the screening of drugs-of-abuse (DOA). However, results are presumptive and confirmatory testing is warranted. To reduce associated cost and delay, laboratories should employ assays with high positive and negative predictive values (PPVs and NPVs). Here, we compared the results of urine drug screens on cobas 6000 (cobas) and ARCHITECTi2000 (ARCHITECT) platforms for six drugs against LC-MS/MS to assess the analytical performance of these assays., Methods: Eighty nine residual urine specimens, which tested positive for amphetamine, THC-COOH, benzoylecgonine, EDDP, opiates and/or oxycodone during routine drug testing, were stored frozen until later confirmation by LC-MS/MS. Immunoassays were performed on cobas and ARCHITECT using a split sample. A third aliquot from these samples was tested by LC-MS/MS to assess the percentage of false positive, false negative, true positive and true negative results and calculate the PPVs and NPVs for each immunoassay., Results: The PPVs of THC-COOH and EDDP assays were 100% on both platforms. Suboptimal PPVs were achieved for oxycodone (cobas, 57.1% vs ARCHITECT, 66.7%), amphetamine (77.8 vs. 100%), opiates (80.0 vs. 84.6%) and benzoylecgonine (88.9 vs. 84.2%) assays. The NPV was 100% for cobas and ARCHITECT oxycodone assays. Lower NPVs were achieved for THC-COOH (cobas, 28.6% vs ARCHITECT, 25.0%), EDDP (72.7% for both assays), benzoylecgonine (74.4% vs 73.8%), amphetamine (83.3% vs 82.8%) and opiates (100% vs 85.3%)., Conclusion: Overall, cobas and ARCHITECT urine drug screens have comparable analytical performance. Confirmatory testing is warranted for positive test results especially for oxycodone, amphetamine, opiates and cocaine. Negative drug screen results must be interpreted with caution especially for THC-COOH, EDDP, benzoylecgonine, amphetamine and opiates., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Pyrrolidinophenones (PPs) are synthetic cathinones containing a pyrrolidine ring that are used recreationally worldwide. Recently, many studies on the metabolism and cytotoxicity of PPs have been published. Here, we focus on new designer drug containing an indan skeleton, 1-(2,3-dihydro-1H-inden-5-yl)-2-(pyrrolidine-1-yl)butan-1-one (5-PPDI), because there have been no reports to date regarding the metabolism of indan-type cathinones. The identification of 5-PPDI phase I metabolites in human urine enables us to determine whether a person has taken 5-PPDI. This metabolite detection approach plays a very important role in the field of forensic science. We synthesized analytical standards of 5-PPDI and four proposed metabolites. A urine sample was prepared by salting-out assisted liquid-liquid extraction with acetonitrile. Analyses of all standards and the urine sample were performed by liquid chromatography high resolution tandem mass spectrometry. As a result, we were able to detect 5-PPDI and its metabolites in the urine specimen. Two diastereomers of synthesized 1-OH metabolites were successfully separated, and only one diastereomer was observed in the urine specimen. To the best of our knowledge, this is the first report on the stereoselective reduction of PPs in humans. Further, we performed quantitative analyses of 5-PPDI and its metabolites in the urine. We identified three characteristic features of 5-PPDI phase I metabolism: (1) hydroxylation at the indan skeleton, (2) stereoselective reduction of the carbonyl group, and (3) hydroxylation of the indan skeleton possibly proceeding more preferentially than any other metabolization. In addition, several structural isomers and diastereomers of 2'-OH metabolites were detected. Based on these data, we propose phase I metabolic pathways of 5-PPDI, which will be essential in understanding the metabolism of other PPs with an indan skeleton., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Rationale: The presence of α-pyrrolidinovalerophenone (α-PVP) and its metabolites in urine is evidence of the administration of α-PVP. A toxicological challenge is that the metabolites of α-PVP exhibit amphoteric properties, which make them unsuitable for detection using gas chromatography-mass spectrometry (GC/MS). In the study reported, proper derivatization and sample extraction were essential for improving the sensitivity for GC/MS analysis., Methods: An automated solid-phase extraction (SPE) method has been developed and optimized. The derivatization efficiency was tested using longer reaction time and the addition of polar pyridine into a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane. Method validation, including linearity, limit of detection, precision, accuracy, and recovery, was evaluated using automatic SPE and GC/MS., Results: The results suggested that adding pyridine to BSTFA (1:1, v/v) significantly improved derivatization efficiency and precision. After optimization, the linear range was from 25 to 1000 ng mL -1 with R 2  > 0.9950. The limit of detection was 5 ng mL -1 for α-PVP and 25 ng mL -1 for OH-α-PVP. The recovery for SPE was over 88%. The inter-day and intra-day precisions were less than 15%. A forensic sample has been found containing α-PVP (67.3 ng mL -1 ) and OH-α-PVP (560.2 ng mL -1 )., Conclusions: This study is the first to validate an auto-SPE-GC/MS method for the quantification and qualification of α-PVP and OH-α-PVP in urine. We have successfully improved the derivatization efficiency and developed a sensitive and semi-automatic approach. This approach is desirable for the detection of synthetic cathinone at trace levels in biological samples., (© 2019 John Wiley & Sons, Ltd.)

LGD-4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD-4033 in the horse following oral administration, in order to identify the most appropriate analytical targets for doping control laboratories. LGD-4033 was orally administered to two Thoroughbred horses and urine, plasma and hair samples were collected and analysed for parent drug and metabolites. LC-HRMS was used for metabolite identification in urine and plasma. Eight metabolites were detected in urine, five of which were excreted only as phase II conjugates, with the longest detection time being observed for di- and tri-hydroxylated metabolites. The parent compound could only be detected in urine in the conjugated fraction. Seven metabolites were detected in plasma along with the parent compound where mono-hydroxylated metabolites provided the longest duration of detection. Preliminary investigations with hair samples using LC-MS/MS analysis indicated the presence of trace amounts of the parent compound and one of the mono-hydroxylated metabolites. In vitro incubation of LGD-4033 with equine liver microsomes was also performed for comparison, yielding 11 phase I metabolites. All of the metabolites observed in vivo were also observed in vitro., (© 2019 John Wiley & Sons, Ltd.)

We report the analysis of the synthetic cathinones α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinopentiophenone (α-PVP), both pyrovalerone derivatives, in blood, urine, gastric contents, main tissues and hair of a deceased person. Qualitative and quantitative analyses were performed by LC-MS-MS. All the biological samples were collected during autopsy and extracted/purified onto a solid phase extraction cartridge before instrumental analysis. The method was validated for blood and urine and proved to be highly sensitive and specific for both the synthetic cathinones (limit of detection: 0.2 ng/mL and limit of quantification: 0.5 ng/mL). Analyses provided negative results for α-PVP in all biological samples except for the 2-cm proximal hair segment, confirming that the young man had consumed in the last 2 months this compound; instead hair analysis proved that the man was a heavy α-PHP user. α-PHP was identified and quantified in biological fluids and tissues. Interestingly, bile and urine concentrations (1.2 and 5.6 ng/mL, respectively) were fairly lower than blood collected into the thoracic cavity (15.3 ng/mL). The highest concentrations were measured for lung (71.1 ng/mL) and spleen (83.8 ng/mL). Concentrations of 3.5, 7.9, 4.7 and 23.6 ng/mL were measured in liver, kidney, brain and heart, respectively. Even if it is not possible to evaluate the presence of this drug in biological samples as a cause of death, to our knowledge, this is the first case of α-PHP finding in postmortem samples, and its potential toxic effects should be elucidated in the future.

Aim: To assess the effect of liver damage on methadone metabolism in opiate addicts undergoing methadone maintenance treatment (MMT)., Methods: This cross-sectional study recruited 74 patients treated at the outpatient clinic of Public Health Institute of Split-Dalmatia County from 2013-2016. Concentrations of methadone and its main inactive metabolite were measured in participants' biological samples on regular check-ups. Urine samples obtained before oral methadone intake, and blood and urine samples obtained 90 minutes after methadone intake were analyzed using gas chromatography/mass spectrometry. Participants were divided into groups according to liver damage criteria: hepatitis C virus status (positive, negative, or clinical remission); aspartate aminotransferase to platelet ratio (APRI) index (<0.7 and ≥0.7); and fibrosis-4 score (<1.45, 1.45-3.25, >3.25)., Results: Metabolic ratio and methadone metabolite concentration in plasma decreased linearly with HCV infection status by the factor of 1.67 (P=0.001) and 2.2 (P=0.043), respectively. Metabolic ratio in plasma decreased in patients with APRI index ≥0.7 by the average factor of 2.12 (P=0.01) and methadone metabolite concentration in plasma decreased by the factor of 6.16 (P=0.009). Metabolic ratio in urine decreased with the severity of fibrosis-4 score by the average factor of 1.63 (P=0.008), whereas methadone metabolite concentration decreased by the factor of 3.53 (P=0.007)., Conclusion: Liver damage decreases methadone metabolism. Indices of liver function should be calculated regularly during MMT for methadone dose titration.

Selective androgen receptor modulators (SARMs) are an emerging class of therapeutics targeted to cachexia, sarcopenia, and hypogonadism treatment. LGD-4033 is a SARM which has been included on the Prohibited List annually released by the World Anti-Doping Agency (WADA). The aim of the present work was the investigation of the metabolism of LGD-4033 in a human excretion study after administration of an LGD-4033 supplement, the determination of the metabolites' excretion profiles with special interest in the determination of its long-term metabolites, and the comparison of the excretion time of the phase I and phase II metabolites. The results were also compared to those derived from previous LGD-4033 studies concerning both in vitro and in vivo experiments. Supplement containing LGD-4033 was administered to one human male volunteer and urine samples were collected up to almost 21 days. Analysis of the hydrolyzed (with β-glucuronidase) as well as of the non-hydrolyzed samples was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in negative ionization mode and revealed that, in both cases, the two isomers of the dihydroxylated metabolite (M5) were preferred target metabolites. The gluco-conjugated parent LGD-4033 and its gluco-conjugated metabolites M1 and M2 can be also considered as useful target analytes in non-hydrolyzed samples. The study also presents two trihydroxylated metabolites (M6) identified for the first time in human urine; one of them was recently reported in an LGD-4033 metabolism study in horse urine and plasma., (© 2018 John Wiley & Sons, Ltd.)

The reactions involved in the metabolic pathways of SR9009 were characterized by liquid chromatography-mass spectrometry (LC-MS) to identify the most appropriate marker(s) of use. The effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009 were also evaluated. In vitro approaches were based on the use of human liver microsomes and cytochrome P450 isoforms. Sample preparation included an enzymatic hydrolysis (performed only for the phase II investigation) followed by liquid-liquid extraction. The chromatographic separation was carried out using a reverse-phase column; detection was performed by either a triple-quadrupole or a time-of-flight system in positive electrospray ionization and different acquisition modes. In the presence of human liver microsomes, SR9009 was biotransformed to 13 metabolites by CYP3A4, CYP3A5, CYP2C19, and CYP2D6 isoenzymes. The reactions included hydroxylation, de-alkylation, oxidation, and combinations thereof, the de-alkylated metabolites being the most abundant. Once formed the mentioned metabolites underwent glucuronidation. Concerning the effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009, our observation have shown the following: (a) No significant alterations were measured between female and male, (b) significant differences were registered using either the CYP2D6 or CYP2C19 allelic variants, and finally (c) significant alterations were registered in the presence of ketoconazole, miconazole, fluoxetine, nefazodone and paroxetine; moderate variation were instead registered with fluconazole, itraconazole, gestodene, and levonorgestrel. This observation put in evidence the importance to take into account both genetic polymorphism and drug-drug interaction to select the most appropriate marker(s) of use in doping analysis., (© 2018 John Wiley & Sons, Ltd.)

Gas chromatography (GC) hyphenated with nitrogen chemiluminescence detection (NCD) and quadrupole time-of-flight mass spectrometry (QTOFMS) was applied for the first time to the quantitative analysis of new psychoactive substances (NPS) in urine, based on the N-equimolar response of NCD. A method was developed and validated to estimate the concentrations of three metabolites of the common stimulant NPS α-pyrrolidinovalerophenone (α-PVP) in spiked urine samples, simulating an analysis having no authentic reference standards for the metabolites and using the parent drug instead for quantitative calibration. The metabolites studied were OH-α-PVP (M1), 2″-oxo-α-PVP (M3), and N,N-bis-dealkyl-PVP (2-amino-1-phenylpentan-1-one; M5). Sample preparation involved liquid-liquid extraction with a mixture of ethyl acetate and butyl chloride at a basic pH and subsequent silylation of the sec-hydroxyl and prim-amino groups of M1 and M5, respectively. Simultaneous compound identification was based on the accurate masses of the protonated molecules for each compound by QTOFMS following atmospheric pressure chemical ionization. The accuracy of quantification of the parent-calibrated NCD method was compared with that of the corresponding parent-calibrated QTOFMS method, as well as with a reference QTOFMS method calibrated with the authentic reference standards. The NCD method produced an equally good accuracy to the reference method for α-PVP, M3 and M5, while a higher negative bias (25%) was obtained for M1, best explainable by recovery and stability issues. The performance of the parent-calibrated QTOFMS method was inferior to the reference method with an especially high negative bias (60%) for M1. The NCD method enabled better quantitative precision than the QTOFMS methods To evaluate the novel approach in casework, twenty post- mortem urine samples previously found positive for α-PVP were analyzed by the parent calibrated NCD method and the reference QTOFMS method. The highest difference in the quantitative results between the two methods was only 33%, and the NCD method's precision as the coefficient of variation was better than 13%. The limit of quantification for the NCD method was approximately 0.25μg/mL in urine, which generally allowed the analysis of α-PVP and the main metabolite M1. However, the sensitivity was not sufficient for the low concentrations of M3 and M5. Consequently, while having potential for instant analysis of NPS and metabolites in moderate concentrations without reference standards, the NCD method should be further developed for improved sensitivity to be more generally applicable., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Porous silicon based surface-assisted laser desorption ionization mass spectrometry (pSi SALDI-MS) is an analytical technique well suited for high throughput analysis of low molecular weight compounds from biological samples. A potential application of this technology is the compliance monitoring of opioid addiction programmes, where methadone is used as a pharmacological treatment for drugs such as heroin. Here, we present the detection and quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) from water and clinical samples (saliva, urine, and plasma) from opioid dependent participants using pSi SALDI-MS. A one-step solvent phase extraction using chloroform was developed for the detection of methadone from clinical samples for analysis by pSi SALDI-MS. Liquid chromatography-mass spectrometry (LC-MS) was used as a comparative technique for the quantification of methadone from clinical saliva and plasma samples. In all cases, we obtained a good correlation of pSi SALDI-MS and LC-MS results, suggesting that pSi SALDI-MS may be an alternative procedure for high-throughput screening and quantification for application in opioid compliance testing. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

3,4-methylenedioxypyrovalerone (MDPV) is a synthetic cathinone belonging to the class of α-pyrrolidinophenones that become increasingly popular as a designer psychostimulant. Here, we report a comprehensive collection of MDPV exposure with quantitative serum level confirmation in Germany. During the years 2014-2016, we could proof consumption of MDPV in 23 cases where urine and blood samples were submitted to our laboratory by the police of Lower Saxony. Most of the samples underwent systematic toxicological analysis by gas chromatography-mass spectrometry (GC-MS), where MDPV could be detected in urine and/or serum samples. The determined concentrations of MDPV in serum showed a high variability, ranging from traces (<10ng/mL) up to 576ng/mL with a mean concentration of 118ng/mL and median of 47ng/mL. The majority of MDPV users were men (87%) and the age ranged from 23 to 49 years (mean 35.9, median 37 years). For most of the analytically confirmed MDPV cases we could prove co-consumption of other psychotropic drugs with frequent occurrence of opiates and cannabinoids in 22% of the cases, followed by benzodiazepines and cocaine in 17%. Analysis of urine samples by GC-MS disclosed the presence of MDPV and its metabolites 2'-oxo-MDPV, demethylenyl-MDPV, demethylenyl-methyl-MDPV, demethylenyl-oxo-MDPV, demethylenyl-methyl-oxo-MDPV and demethylenyl-methyl-N,N-bisdealkyl-MDPV. The metabolite pattern substantiates previous suggestions for principle metabolic pathways of MDPV in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

CEP-26401 is a novel orally active, brain-penetrant, high-affinity histamine H3 receptor (H3R) antagonist, with potential therapeutic utility in cognition enhancement. Two randomized, double-blind, placebo-controlled dose escalation studies with single (0.02 to 5 mg) or multiple administration (0.02 to 0.5 mg once daily) of CEP-26401 were conducted in healthy subjects. Plasma and urine samples were collected to investigate CEP-26401 pharmacokinetics. Pharmacodynamic endpoints included a subset of tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and nocturnal polysomnography. Population pharmacokinetic-pharmacodynamic modeling was conducted on one CANTAB and one polysomnography parameter of interest. CEP-26401 was slowly absorbed (median tmax range 3-6 hours) and the mean terminal elimination half-life ranged from 24-60 hours. Steady-state plasma concentrations were achieved within six days of dosing. CEP-26401 exhibits dose- and time-independent pharmacokinetics, and renal excretion is a major elimination pathway. CEP-26401 had a dose-dependent negative effect on sleep, with some positive effects on certain CANTAB cognitive parameters seen at lower concentrations. The derived three compartment population pharmacokinetic model, with first-order absorption and elimination, accurately described the available pharmacokinetic data. CEP-26401 was generally well tolerated up to 0.5 mg/day with most common treatment related adverse events being headache and insomnia. Further clinical studies are required to establish the potential of low-dose CEP-26401 in cognition enhancement., (© The Author(s) 2016.)

Context: An increasing number of new psychoactive substances (NPS) of different chemical classes have become available through marketing and sale over the Internet. This report from the Swedish STRIDA project presents the prevalence, laboratory results, and clinical features in a series of intoxications involving the stimulant NPS α-pyrrolidinovalerophenone (α-PVP), a potent dopamine re-uptake inhibitor, over a 4-year period., Study Design: Observational case series of consecutive patients with admitted or suspected intake of NPS presenting to hospitals in Sweden from 2012 to 2015., Patients and Methods: In the STRIDA project, blood and urine samples are collected from intoxicated patients with admitted or suspected intake of NPS or unknown drugs presenting to hospitals over the country. Analysis of NPS is performed by mass spectrometry multicomponent methods. Clinical data are collected when caregivers consult the Swedish Poisons Information Centre (PIC), and retrieved from medical records. The severity of poisoning is graded retrospectively using the Poisoning Severity Score (PSS). The inclusion criteria for this study included absence of other stimulants than α-PVP., Results: During the 4-year study period, 23 intoxications were originally coded as "α-PVP related" out of a total 3743 NPS-related inquiries (0.6%) at the PIC. The present study covered 42 analytically confirmed cases in which α-PVP was the only stimulant detected. The age range of patients was 20-58 (median 32) years, of which 79% were males. The α-PVP concentration in serum was 4.0-606 (median 64; n = 42) ng/mL and 2.0-41,294 (median 1782; n = 25) ng/mL in urine. There was no statistically significant association between the serum α-PVP concentration and urinary α-PVP/creatinine ratio in 25 cases, where both sets of data were available. In 14/42 (33%) cases, α-PVP was the only psychoactive substance identified. In the remaining cases, additional substances comprised opioids, benzodiazepines, and ethanol. The main clinical manifestations were tachycardia (80%), agitation (70%), hypertension (33%), hallucinations (20%), and delirium (18%). Classification of poisoning severity yielded 25 (60%) moderate (PSS 2), 7 (17%) severe (PSS 3), and 2 fatal cases (PSS 4)., Conclusions: In analytically confirmed α-PVP intoxication cases involving no other stimulant drugs, the urine and serum concentrations showed high variability. The clinical features were consistent with a severe sympathomimetic toxidrome. The results further demonstrated that α-PVP prevailed as a drug of abuse after being classified as a narcotic substance, and despite a high incidence of severe poisonings and fatalities. However, the low prevalence of α-PVP cases registered at the PIC suggested that many were unaware of the actual substance they had taken.

α-Pyrrolidinovalerophenone (α-PVP) is a synthetic cathinone belonging to the group of "second generation" pyrrolidinophenones that becomes more and more popular as a designer psychostimulant. Here we provide toxicological analytical support for a severe poisoning with α-PVP. Serum and urine samples that were sent to our laboratory were subjected to a general unknown screening procedure. The procedure includes immunoassay-based screening of drugs of abuse in serum and systematic toxicological analysis of urine and serum after neutral and basic liquid-liquid extraction followed by gas chromatography-mass spectrometry (GC-MS). Whereas the immunoassay delivered negative results, analyzing the urine sample by GC-MS in full scan mode disclosed the presence of α-PVP and its metabolites α-(2″-oxo-pyrrolidino)valerophenone (2″-oxo-α-PVP) and 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol (OH-α-PVP). In the acetylated urine sample we found additionally N,N-bis-dealkyl-PVP. In serum, α-PVP could be detected after solid phase extraction and a concentration of 29ng/mL was determined. Other forensic relevant substances were not detected. The presented data can explain the psychotic symptoms and behavioural pattern of the subject after abuse of α-PVP, leading to a clinical condition similar to excited delirium syndrome., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The potential of high-resolution mass spectrometry (HRMS) for the investigation of human in vivo metabolism of 3,4-methylenedioxypyrovalerone (MDPV) using urine collected from a consumer (this is, in non-controlled experiments) has been investigated. As a control sample was not available, the common approach based on the comparison of a control/blank sample and samples collected after drug intake could not be used. Alternatively, an investigation based on common fragmentation pathways was applied, assuming that most metabolites share some fragments with the parent drug. An extension of this approach was also applied based on the fragmentation pathway of those metabolites identified in urine samples in the first step. The use of MS(E) experiments (sequential acquisition of mass spectra at low and high collision energy) has been crucial to this aim as it allowed promoting fragmentation in the collision cell without any previous precursor ion selection. MDPV belongs to the group of new psychoactive substances (NPS), being known as the "cannibal drug". This substance is being abused more and more and is associated with dangerous side effects. The human metabolites (both phase I and phase II) were detected and tentatively identified by accurate mass full-spectrum measurements using ultra-high performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Following this strategy, up to 10 phase I metabolites, together with some glucuronides and sulphates, were detected and tentative structures were proposed. Several compounds identified in this work have not been previously reported in the literature.

Background: Little or no pharmacological or toxicological data are available for novel psychoactive substances when they first emerge, making their identification and interpretation in biological matrices challenging., Materials & Methods: A new synthetic cathinone, α-pyrrolidinopentiothiophenone (α-PVT), was incubated with hepatocytes and samples were analyzed using liquid chromatography coupled to a Q Exactive™ Orbitrap mass spectrometer. Authentic urine specimens from suspected α-PVT cases were also analyzed. Scans were data mined with Compound Discoverer™ for identification and structural elucidation of metabolites., Results/conclusion: Seven α-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity. α-PVT dihydroxypyrrolidinyl, α-PVT 2-ketopyrrolidinyl, α-PVT hydroxythiophenyl and α-PVT thiophenol had the most intense in vivo signals.

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.)

Pyrrolidinophenones represent one emerging class of newly encountered drugs of abuse, also known as 'new psychoactive substances', with stimulating psychoactive effects. In this work, we report on the detection of the new designer drug α-pyrrolidinohexiophenone (α-PHP) and its phase I and II metabolites in a human urine sample of a drug abuser. Determination and structural elucidation of these metabolites have been achieved by liquid chromatography electrospray ionisation quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS). By tentative identification, the exact and approximate structures of 19 phase I metabolites and nine phase II glucuronides were elucidated. Major metabolic pathways revealed the reduction of the ß-keto moieties to their corresponding alcohols, didesalkylation of the pyrrolidine ring, hydroxylation and oxidation of the aliphatic side chain leading to n-hydroxy, aldehyde and carboxylate metabolites, and oxidation of the pyrrolidine ring to its lactam followed by ring cleavage and additional hydroxylation, reduction and oxidation steps and combinations thereof. The most abundant phase II metabolites were glucuronidated ß-keto-reduced alcohols. Besides the great number of metabolites detected in this sample, α-PHP is still one of the most abundant ions together with its ß-keto-reduced alcoholic dihydro metabolite. Monitoring of these metabolites in clinical and forensic toxicology may unambiguously prove the abuse of the new designer drug α-PHP., (Copyright © 2015 John Wiley & Sons, Ltd.)

Context: In the recent years, there have been an increasing number of new psychoactive substances (NPS) available through marketing and sale on the Internet. The stimulant 3,4-methylenedioxypyrovalerone (MDPV) is a potent dopamine reuptake inhibitor, which can cause serious intoxications requiring intensive care and even fatality. This report from the STRIDA project presents the prevalence, laboratory results, and clinical features in a series of intoxications involving MDPV over a 5-year period., Study Design: Observational case series of consecutive patients with admitted or suspected intake of NPS presented at hospitals in Sweden from 2010 to 2014., Patients and Methods: Blood and/or urine samples were collected from intoxicated patients with admitted or suspected intake of NPS presenting at hospitals over the country. Analysis of NPS was performed by a liquid chromatography-tandem mass spectrometry multicomponent method. Clinical data were collected when caregivers consulted the Swedish Poisons Information Centre and also retrieved from medical records. The severity of poisoning was graded retrospectively using the poisoning severity score., Results: During the 5-year study period, the number of MDPV-related inquiries to the Poisons Information Centre was 662 out of a total ∼4500 suspected NPS-related inquiries (∼15%), and 201 analytically confirmed MDPV intoxications were enrolled in the study. The study period covered the period when the use of MDPV in Sweden was at its peak and also the decline to an almost zero level. The age range of patients was 18-68 (mean 36, median 35) years, and 71% were males. The MDPV concentrations in serum ranged between 1.0 ng/mL and 1509 ng/mL (mean 63.6, median 20) and between 1.0 ng/mL and 81 000 ng/mL (mean 3880, median 1160) in urine. The urinary values were also creatinine corrected for variation in urine dilution, and the MDPV/creatinine ratio ranged between 0.10 ng/mmol and 2480 ng/mmol (mean 247, median 92.6). There was a statistically significant association between the serum MDPV concentration and the urinary MDPV/creatinine ratio, for 118 cases where both data were available (r = 0.764; p < 0.0001, Spearman's rank correlation). In 30 (15%) cases, MDPV was the single psychoactive substance identified in the serum or urine specimens. In the other 171 cases, other psychoactive substances were detected together with MDPV. The additional substances (n = 61) comprised of both conventional drugs of abuse, other NPS (n = 39), pharmaceuticals, and ethanol. The cathinone-derivative alpha-pyrrolidinovalerophenone (α-PVP) was the most frequent other NPS, and was detected in 58 (29%) cases, followed by methylone in 14 (7%) cases. The main clinical manifestations reported in patients testing positive for MDPV included agitation, tachycardia (≥100/min), and hypertension (systolic blood pressure ≥140 mmHg), which were observed in 130 (67%), 106 (56%), and 65 (34%) cases, respectively. Other symptoms included hallucinations (n = 31, 16%), delirium (n = 29, 15%), hyperthermia (>39°C/102.4°F; n = 18, 10%), and rhabdomyolysis (n = 16, 8%). In MDPV intoxications with serum levels >100 ng/mL, the cases were graded as more severe and hyperthermia was less common., Conclusions: In a large number of analytically confirmed MDPV intoxications from mostly polydrug users, the urine and serum MDPV concentrations showed a high variability. The clinical features were consistent with a severe sympathomimetic toxidrome. The results also demonstrated that MDPV prevailed as a drug of abuse for a long time, after its classification as a narcotic substance and despite a high incidence of severe poisonings.

Urinary phase I metabolites of α-pyrrolidinobutiophenone (α-PBP) in humans were investigated by analyzing urine specimens obtained from drug abusers. Unequivocal identification and accurate quantification of major metabolites were realized using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry with newly synthesized authentic standards. Two major phase I metabolic pathways were revealed: (1) reduction of the ketone group to 1-phenyl-2-(pyrrolidin-1-yl)butan-1-ol (OH-α-PBP, diastereomers) partly followed by conjugation to its glucuronide and (2) oxidation at the 2″-position of the pyrrolidine ring to α-(2″-oxo-pyrrolidino)butiophenone (2″-oxo-α-PBP) via the putative intermediate α-(2″-hydroxypyrrolidino)butiophenone (2″-OH-α-PBP). Of the phase I metabolites retaining the structural characteristics of the parent drug, OH-α-PBP was the most abundant in all specimens examined. Comparison of the phase I metabolism of α-PBP and α-pyrrolidinovalerophenone (α-PVP) suggested a relationship between the aliphatic side chain length and the metabolic pathways in α-pyrrolidinophenones: the shorter aliphatic side chain (1) led to more extensive metabolism via reduction of the ketone group than via the oxidation at the 2″-position of the pyrrolidine ring and (2) influenced the isomeric ratio of a pair of diastereomers., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

In February 2015, an outbreak of recently acquired HIV infections among people who inject drugs (PWID) was identified in Dublin, following similar outbreaks in Greece and Romania in 2011. We compared drug and risk behaviours among 15 HIV cases and 39 controls. Injecting a synthetic cathinone, snow blow, was associated with recent HIV infection (AOR: 49; p=0.003). Prevention and control efforts are underway among PWID in Dublin, but may also be needed elsewhere in Europe.

Gas chromatography-mass spectrometry quantitative method was developed to monitor concentrations of methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in plasma and urine of patients. The developed method was simple, accurate and reproducible to quantify methadone and EDDP in plasma and urine samples in the concentration range of 15-1,000 and 50-2,000 ng/mL, respectively. The proposed analytical method was applied to plasma and urine samples obtained from 96 patients undergoing methadone maintenance treatment (MMT) with daily methadone doses of 2-120 mg/day. Urinary methadone excretion was observed to be significantly affected by pH, in which the ratio of methadone to EDDP was two times higher in acidic urine (P = 0.029). The findings of this study further enhance the guidelines for monitoring of methadone treatment among outpatients. Methadone-to-EDDP ratio in urine was found to be consistent at 24 and 4 h, hence suggesting the possibility that outpatients may be monitored with single urine sample in order to check for compliance. This study which provides data on peak concentrations of methadone and EDDP as well as the ratio of both compounds has added to the body of knowledge regarding pharmacokinetic properties of methadone among heroin-dependent patients under MMT., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We report on a case of repeated MDPV consumptions that resulted in severe psychosis and agitation prompting the concomitant abuse of benzodiazepines. A 27-year-old man was found irresponsive in his apartment and was brought to the emergency department (ED) of a local hospital. When in ED, he rapidly recovered and self-reported to have recently injected some doses of MDPV that he had bought in the Internet. He left the hospital without medical cares. 15 days after, he was again admitted to the same ED due to severe agitation, delirium and hallucinations, and reported the use of MDPV and pharmaceutical drugs during the preceding week. He was sedated with diazepam and chlorpromazine. Urine samples collected in both occasions were sent for testing using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-high resolution multiple mass spectrometry (LC-HRMS/MS) on an Orbitrap. The LC-HRMS analysis revealed the presence of MDPV and its phase I and phase II metabolites (demethylenyl-MDPV, demethylenyl-methyl-MDPV, demethylenyl-methyl-oxo-MDPV, demethylenyl-hydroxy-alkyl-MDPV, demethylenyl-methyl-hydroxy alkyl-MDPV, demethylenyl-oxo-MDPV and their corresponding glucuronides), alprazolam and alprazolam metabolite at the first ED admission; at the time of the second ED access, the same MDPV metabolites, alprazolam, temazepam, and chlordiazepoxide were detected together with diazepam and metabolites. LC-HRMS/MS was use to determine the following concentrations, respectively on his first and second admission: MDPV 55ng/mL, alprazolam 114ng/mL, α-hydroxyalprazolam 104ng/mL; MDPV 35ng/mL, alprazolam 10.4ng/mL, α -hydroxyalprazolam 13ng/mL; chlordiazepoxide 13ng/mL, temazepam 170ng/mL, diazepam 1.3ng/mL, nordiazepam 61.5, oxazepam 115ng/mL. The toxicological findings corroborated the referred concomitant use of multiple pharmaceutical drugs and benzodiazepines. Confirmation of previous hypothesis on human metabolism of MDPV could be inferred by the analysis of urine., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The development of a liquid chromatography high-resolution mass spectrometry quadrupole-time-of-flight (LC-HRMS-QTOF) method for the analysis of new stimulant designer drugs (e.g. phenethylamine, amphetamine, cathinone and piperazine derivatives) and common drugs of abuse (e.g. ketamine and ritalinic acid) in urine is reported. Sample preparation was carried out by a fast and convenient salting-out liquid-liquid extraction (SALLE) procedure. The data was generated by a preferred target list combined with untargeted data-dependent acquisition recording additional sample information (i.e. not listed metabolites of target compounds or not database-stored drugs). The identification is realised by a fully automated data extraction algorithm, taking into account accurate mass spectra, fragment masses and retention times. Method validation comprised selectivity, linearity, accuracy, stability, determination of the limit of detection (LOD) and limit of quantification (LOQ) and evaluation of matrix effects and recoveries for a total set of 39 compounds. Acceptable quantitative results were obtained for 35 of the 39 analytes. Exemplarily, application of the additional untargeted data-dependent acquisition mode enabled the identification of metabolites of the preferred target list compounds ketamine and methylenedioxypyrovalerone (MDPV) without use of reference standards. Therefore, improvement of the database is feasible with every positive library hit. The approach presented here provides a very useful tool for the combined targeted and untargeted analysis of drugs of abuse in biological matrices such as urine.

3',4'-Methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a designer drug of the pyrrolidinophenone-type, was first seized in Germany in 2009. It was also identified in 'legal high' samples investigated in the UK. Therefore, the aim of the presented work was to identify its in vivo and in vitro phase I and II metabolites using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ion trap mass spectrometry (LC-MS(n) ). Furthermore, detectability of MDPBP in rat and human urine using standard urine screening approaches (SUSA) by GC-MS and LC-MS(n) was studied. The metabolites were isolated either directly or after enzymatic cleavage of conjugates by solid-phase extraction (C18, HCX). The metabolites were then analyzed and structures proposed after GC-MS (phase I) and LC-MS(n) (phase II). Based on these identified metabolites, the following main metabolic steps could be proposed: demethylenation followed by methylation of one hydroxy group, aromatic and side chain hydroxylation, oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Furthermore, in rat urine after a typical user's dose as well as in human urine, mainly the metabolites could be detected using the authors' SUSA by GC-MS and LC-MS(n) . Thus, it should be possible to monitor an application of MDPBP assuming similar toxicokinetics in humans. Finally, CYP2C19 and CYP2D6 could be identified as the isoenzymes mainly responsible for demethylenation., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: We evaluated a new EDDP [2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine] enzyme immunoassay (EDDPI; Lin-Zhi International, Inc., Sunnyvale, CA) for the detection of this primary methadone urinary metabolite., Methods: All specimens were tested with two different cutoff calibrators at 150 and 300 ng/ml EDDP on an ADVIA 1200 Chemistry System auto-analyzer. Controls containing 0, -25% (negative control), and +25% (positive control) of the cutoff calibrators (Lin-Zhi) were analyzed with each batch. All urine specimens were then analyzed by high-pressure liquid chromatography/ mass spectrometry/mass spectrometry (HPLC-MS/MS) for EDDP., Results: Approximately, 42% (151) of the 362 specimens yielded positive results by the EDDP assay at 150 and/or 300 ng/ml cutoff values. Of these specimens, HPLC-MS/MS confirmed the presence of EDDP > 25 ng/ml in all 151 specimens. No specimen yielding negative EDDPI results contained EDDP by HPLC/MS/MS. At 150 ng/ml cutoff, the EDDPI demonstrated a sensitivity of 1.00, a specificity of 0.986, and an overall agreement of HPLC/MS/MS of >99%. At 300 ng/ml cutoff, the EDDPI demonstrated a sensitivity of 1.00, a specificity of 0.959, and an overall agreement of HPLC/MS/MS results of 97.5%., Conclusion: The Lin-Zhi EDDPI provides a precise, reliable method for the routine detection of methadone metabolite in urine specimens, particularly in pain management compliance testing., (© 2014 Wiley Periodicals, Inc.)

Background: Methadone blood concentrations in fatal cases are highly variable and there is an appreciable overlap between therapeutic methadone concentrations and the concentrations detected in fatalities. As with other opioids, the background of these methadone-related deaths is unclear. The aim of this study was to investigate if short-time abstinence was contributing to the cause of death in methadone-related deaths by evaluation of the EDDP/methadone ratio in blood and urine., Methods: Samples of blood and urine were collected from 103 autopsy cases and analysed for the concentrations of methadone and its main metabolite EDDP. The cases were divided into three groups according to the cause of death: cases where methadone was the cause of death (N=67), cases where poly-drug poisoning including methadone was the cause of death (N=24) and cases where death were caused by other factors (N=12). Urine samples from 11 living persons receiving methadone were also included., Results: In general, a substantial overlap of the methadone concentrations in blood and urine was seen between the groups. There was a tendency of lower median EDDP/methadone urinary ratios in the methadone poisoning group (median: 1.0), poly-drug poisoning group (median: 0.94) and in the fatalities not related to methadone (median: 1.1) compared to the living subjects in methadone treatment (median: 1.6), although the differences were not significant., Conclusion: It was not possible to reveal a possible abstinence period prior to death by using the EDDP/methadone ratio in blood and urine in methadone-related deaths., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Naphyrone (1-naphthalen-2-yl-2-pyrrolidin-1-yl-pentan-1-one; naphthylpyrovalerone, β-naphyrone) is a cathinone designer drug and was marketed as replacement for the synthetic cathinone derivative mephedrone. Meanwhile, naphyrone is also classified as a controlled drug in several countries. Therefore, the aim of this study was to identify the metabolites of naphyrone in rat urine using gas chromatography-mass spectrometry techniques and to show its detectability in urine samples. The following metabolic steps could be detected in rat urine: oxidation of the pyrrolidine ring to the corresponding lactam, hydroxylation of the propyl side chain and the naphthyl ring, degradation to the primary amines after opening of the pyrrolidine ring, and combinations of these steps. Assuming similar kinetics, an intake of naphyrone should be detectable in human urine mainly via its metabolites., (Copyright © 2013 John Wiley & Sons, Ltd.)

The analysis of designer drugs, including those in the synthetic cathinone and piperazine classes, may be complicated by the poor stability of these compounds in biological specimens. The stability of four of these compounds was investigated: 3,4-methylenedioxypyrovalerone, 4-methyl-N-methylcathinone (mephedrone), N-benzylpiperazine and 1-[3-(trifluoromethyl)phenyl]piperazine. Compound stability was monitored in three different biological matrices when each matrix was stored under three different conditions. These matrices and conditions included human whole blood, human serum and human urine, each stored at -20, 4 and 22°C for a period of 14 days in the dark in a sealed glass container. Analysis by liquid chromatography-tandem mass spectrometry was performed on Day 1 to establish the initial concentration for each drug in each specimen type, and then the samples were divided into three parts for storage under the various conditions. Analysis was performed in triplicate on Days 2, 4, 7 and 14 for each specimen type under each storage condition and the results were compared to those obtained on Day 1. Following analysis of the data, it became clear that mephedrone was not stable, and that care must be taken following specimen receipt to ensure minimal degradation.

In the last years, the number of new psychoactive substances, so-called 'legal highs', has enormously increased. They are sold via online shops often with inaccurate and false information about the content. The aim of this work was to study the metabolism and the detectability of the drug of abuse diphenyl-2-pyrrolidinemethanol (D2PM) in rat urine using gas chromatography-mass spectrometry and liquid chromatography-high resolution-tandem mass spectrometry. Five phase I and two phase II metabolites were identified suggesting hydroxylation at the pyrrolidine and diphenyl part as the main metabolic steps. Assuming similar kinetics, an intake of D2PM should be detectable in human urine mainly via its metabolites., (Copyright © 2013 John Wiley & Sons, Ltd.)

Introduction: The use of designer drugs commonly marketed as bath salts or plant food has risen dramatically in recent years. Several different synthetic cathinones have been indentified in these products, including mephedrone, 3,4-methylenedioxypyrovalerone (MDPV), and 4-fluoromethcathinone (flephedrone). We report a case of bath salt intoxication with quantitative MDPV and flephedrone levels in a patient's serum and urine, and from the bath salt product., Case Report: A 23-year-old male with a prior psychiatric history arrived via EMS for bizarre behavior, suicidality, and hallucinations after reportedly insufflating a bath salt. He was found to have MDPV levels of 186 and 136 ng/mL in his serum and urine, respectively, and flephedrone levels of 346 and 257 ng/mL in the serum and urine, respectively. The white powder in question was found to contain 143 μg MDPV and 142 μg flephedrone per milligram powder. His psychosis and agitation resolved with lorazepam, droperidol, and observation in the emergency department., Discussion: Agitation, psychosis, movement disorders, tachycardia, and hypertension have all been attributed to the use of MDPV; there are no prior reports detailing clinical experience with flephedrone. Considering that our patient's serum flephedrone levels were twofold higher than his MDPV level, it is likely flephedrone contributed to his clinical toxicity. This case suggests the possibility that fluorinated cathinones, such as flephedrone, may have altered metabolism and/or elimination which may affect their course of clinical toxicity. This case highlights the evolving composition of synthetic cathinones found in bath salt products.

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.

Methadone is used to treat moderate to severe pain in patients not responsive to non-narcotic analgesics and for maintenance treatment of opioid addiction. Methadone is primarily metabolized by N-demethylation to an inactive metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidene (EDDP) by CYP3A4 and CYP2B6. Establishing expected concentrations for metabolism of methadone to EDDP using urine excretion data may be useful for monitoring "medications" and toxicity. Urine specimens from chronic pain patients were collected during routine clinic visits. Methadone and EDDP were quantified by liquid chromatography-tandem mass spectrometry. Approximately 8,000 subjects who reported taking methadone had creatinine concentrations ≥20 mg/dL, and excreted concentrations of methadone and EDDP above ≥100 ng/mL were selected. The median methadone urine concentration was 3.03 mg/g cr. Ninety-five percent of the population had concentrations between 0.175 and 20.9 mg/g cr. EDDP was, on average, twice the methadone concentration. The wide variance in relationship of methadone to its metabolite was not concentration-dependent. Variability between subjects was larger than variability within subjects. As the urinary pH increased, the proportion of excreted EDDP increased, implying a preferred excretion of EDDP.

In-line solid-phase extraction-capillary electrophoresis coupled with mass spectrometric detection (SPE-CE-MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE-CE-MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L(-1) ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 μL min(-1) was isopropanol-water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08-10 ng mL(-1). Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL(-1). Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.

Purpose: There is increasing interest in the use of pipradrol and pipradrol derivatives, such as diphenylproplinol [diphenyl-2-pyrrolidinemethanol (D2PM)] and desoxypipradrol [2-diphenylmethylpiperidine (2-DPMP)], as recreational drugs. There is limited information on the acute toxicity related to both D2PM and 2-DPMP. We report here a case series of five individuals with acute toxicity related to the use of D2PM., Case Series: Five patients aged between 21 and 33 years old presented to the Emergency Department (ED) on unrelated occasions having used a range of different novel psychoactive substances; none had actually purchased D2PM. They presented with ongoing prolonged neuropsychiatric symptoms of agitation, anxiety and insomnia lasting 24-96 h post-ingestion. None had evidence of sympathomimetic toxicity on presentation to the ED. All were reassured and discharged home after review. TOXICOLOGICAL SCREENING: Urine collection at the time of presentation to the ED was subsequently analysed by gas chromatography-mass spectrometry (GC-MS). All of the urine samples tested positive for D2PM and benzophenone. Additional screening by liquid chromatography tandem mass-spectrometry (LC-MS-MS) demonstrated that the benzophenone detected was an analytical artefact due to the high-injection temperature of the GC-MS analysis., Conclusions: This descriptive case series provides more detailed information on the acute toxicity related to the use of D2PM. This information is useful for clinical pharmacologists and clinicians managing these individuals to be able to provide more appropriate advice on the acute toxicity associated with the use of D2PM, particularly in relation to the prolonged neuropsychiatric symptoms seen.

1,3-Butadiene (BD) is an important industrial chemical that is classified as a human carcinogen. BD carcinogenicity has been attributed to its metabolism to several reactive epoxide metabolites and formation of the highly mutagenic 1,2:3,4-diepoxybutane (DEB) has been hypothesized to drive mutagenesis and carcinogenesis at exposures experienced in humans. We report herein the formation of DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in BD-exposed workers as a biomarker of DEB formation. pyr-Val was determined in BD monomer and polymer plant workers that had been previously analyzed for several other biomarkers of exposure and effect. pyr-Val was detected in 68 of 81 (84%) samples ranging from 0.08 to 0.86 pmol/g globin. Surprisingly, pyr-Val was observed in 19 of 23 administrative control subjects not known to be exposed to BD, suggesting exposure from environmental sources of BD. The mean ± SD amounts of pyr-Val were 0.11 ± 0.07, 0.16 ± 0.12, and 0.29 ± 0.20 pmol/g globin in the controls, monomer, and polymer workers, respectively, clearly demonstrating formation of DEB in humans. The amounts of pyr-Val found in this study suggest that humans are much less efficient in the formation of DEB than mice or rats at similar exposures. Formation of pyr-Val was more than 50-fold lower than has been associated with increased mutagenesis in rodents. The results further suggest that formation of DEB relative to other epoxides is significantly different in the highest exposed polymer workers compared with controls and BD monomer workers. Whether this is due to saturation of metabolic formation or increased GST-mediated detoxification could not be determined.

This paper presents an overview of a cross-species investigation of the metabolic fate of [(14)C]-zibotentan (ZD4054), with particular focus on the main analytical challenges encountered during the study. A combination of detection methods were used including HPLC coupled to UV, RAD and/or MS(MS), and (1)H NMR spectroscopy. The objective was to characterise and identify the major metabolites found in the circulation and excreta of rat and dog for comparison with those produced in human. Initial investigations in rat, using [(14)C]-labelled zibotentan positioned on the oxadiazole ring and HPLC-UV-RAD analysis, revealed seven labelled resolved metabolite peaks. Parallel analysis by HPLC-UV-MS (with in-source fragmentation) uncovered two additional metabolites, indicating loss of the radiolabel during biotransformation. Hence, in subsequent studies in rat, dog and human, dual-radiolabelled zibotentan was employed with the (14)C-label positioned on the pyridine ring, which was shown to be less prone to metabolism. A total of 12 metabolites were found in the excreta and plasma in all species. One of these metabolites was found in the circulation in humans, which warranted further investigations. Characterisation of the isolated human circulating metabolite by (1)H NMR was complicated by the co-extraction of a matrix component with a similar UV-chromophore to zibotentan, which was identified as daidzein, an isoflavone derived from the animal feed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Three phase 1 studies of eliglustat tartrate (Genz-112638), an oral inhibitor of glucosylceramide synthase under development for treating Gaucher disease type 1 (GD1), evaluated the safety, tolerability, and pharmacokinetics in healthy volunteers after escalating single doses (n = 99), escalating multiple doses (n = 36), and food (n = 24). Eliglustat tartrate was well tolerated at single doses ≤ 20 mg/kg and multiple doses ≤ 200 mg bid, with 50 mg bid producing plasma concentrations in the predicted therapeutic range. No serious adverse events occurred. Mild to moderate events of nausea, dizziness, and vomiting increased in frequency with escalating single and multiple doses. Single doses ≥ 10 mg/kg caused mild increases in electrocardiogram PR, QRS, and QT/QTc intervals. Single-dose pharmacokinetics showed dose linearity but not proportionality. Maximum plasma concentrations occurred at ~2 hours, followed by a monophasic decline with a ~6-hour terminal half-life. Unchanged drug in 8-hour urine collections was <1.5% of administered doses. Food did not significantly affect the rate or extent of absorption. Multiple-dose pharmacokinetics was nonlinear, showing higher than expected plasma drug concentrations. Steady state was reached ~60 hours after bid dosing. Higher drug exposure occurred in slower CYP2D6 metabolizers. Based on favorable results in healthy participants, a phase 2 trial of eliglustat tartrate was initiated in GD1 patients.

A gas chromatography-mass spectrometry (GCMS) procedure was developed for the quantitative analysis of the new designer drug methylenedioxypyrovalerone (MDPV) in urine together with the common stimulants amphetamine, methamphetamine, and methylenedioxymethamphetamine (MDMA). The procedure involved electron ionization (EI) GCMS in the selected ion monitoring (SIM) mode after liquid-liquid extraction with toluene and derivatization with heptafluorobutyric acid anhydride. All MDPV findings were confirmed by positive chemical ionization GCMS in SIM mode. Positive chemical ionization-GCMS allowed the protonated molecule M+H+ m/z 276 to be used as a target ion with 3 abundant fragments as qualifier ions. By electron ionization-GCMS, the limit of quantification (LOQ) for MDPV was 0.02 mg/L; and for amphetamine, methamphetamine, and MDMA, the LOQ was 0.05 mg/L. The method was applied to monitoring urine samples from opioid-dependent patients undergoing opioid substitution treatment. Nine of the 34 urine samples (26%) analyzed were MDPV positive by the GCMS procedure. The positive samples were obtained from 2 female and 7 male patients with a mean age of 31 years. The median (range) MDPV concentration was 0.16 mg/L (0.04-3.9 mg/L) based on the 7 samples for which a numeric value was obtained, whereas the concentration was below the LOQ but above the limit of detection in 2 samples. The method revealed amphetamine in approximately 40% of the cases, and there was no statistical difference between the MDPV-positive and MDPV-negative groups. Urine amphetamine concentrations were on average 10 times higher than those of MDPV. The opioid-dependent patients used MDPV mainly as a substitute for amphetamine, judging from the laboratory findings of this study and the information from our patients.

Since the late 1990s, many derivatives of the α-pyrrolidinophenone (PPP) drug class appeared on the drugs of abuse market. The latest compound was described in 2009 to be a classic PPP carrying a methylenedioxy moiety remembering the classic entactogens (ecstasy). Besides Germany, 3,4-methylene-dioxypyrovalerone (MDPV) has appeared in many countries in Europe and Asia, indicating its worldwide importance for forensic and clinical toxicology. The aim of the presented work was to identify the phase I and II metabolites of MDPV and the human cytochrome-P450 (CYP) isoenzymes responsible for its main metabolic step(s). Finally, the detectability of MDPV in urine by the authors' systematic toxicological analysis (STA) should be studied. The urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified after work-up by GC-MS and liquid chromatography (LC)-high-resolution MS (LC-HR-MS). The studies revealed the following phase I main metabolic steps in rat and human: demethylenation followed by methylation, aromatic and side chain hydroxylation and oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Using LC-HR-MS, most metabolite structures postulated according to GC-MS fragmentation could be confirmed and the phase II metabolites were identified. Finally, the formation of the initial metabolite demethylenyl-MDPV could be confirmed using incubation of human liver microsomes. Using recombinant human CYPs, CYP 2C19, CYP 2D6 and CYP 1A2 were found to catalyze this initial step. Finally, the STA allowed the detection of MDPV metabolites in the human urine samples., (Copyright © 2010 John Wiley & Sons, Ltd.)

A method for the toxicological screening of the new designer drug methylenedioxypyrovalerone (MDPV) is described; with an emphasis on its application for anti-doping analysis. The metabolism of MDPV was evaluated in vitro using human liver microsomes and S9 cellular fractions for CYP450 phase I and uridine 5'-diphosphoglucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II metabolism studies. The resulting metabolites were subsequently liquid/liquid extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) derivatives. The structures of the metabolites were further confirmed by accurate mass measurement using a liquid chromatography/quadrupole time-of-flight (LC/QTOF) mass spectrometer. The studies demonstrated that the main metabolites of MDPV are catechol and methyl catechol pyrovalerone, which are in turn sulfated and glucuronated. The method for the determination of MDPV in urine has been fully validated by assessing the limits of detection and quantification, linearity, repeatability, and accuracy. This validation demonstrates the suitability for screening of this stimulant substance for anti-doping and forensic toxicology purposes., (2010 John Wiley & Sons, Ltd.)

Methadone is a synthetic opioid used to relieve pain, treat opioid withdrawal, and wean heroin addicts. Measurement of methadone and its major metabolite EDDP in urine is useful for assessing compliance with addiction rehabilitation and pain management programs. This method quantitatively measures methadone and its metabolite EDDP in urine. Methadone and EDDP are recovered from urine by solid phase extraction at pH 6.0. Analysis is performed by gas chromatography with mass spectrometry detection, using selective ion monitoring.

Objective: To investigate the possibility of utilizing the ratio of the methadone metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), to urine creatinine to develop a regression model that would predict drug adherence in patients prescribed methadone for either pain management or drug addiction., Design: Retrospective study., Setting: Marshfield Clinic-Lakeland Center, one of 41 regional centers that make up Marshfield Clinic, a large, private, multi-specialty healthcare institution in central Wisconsin., Participants: Patients receiving methadone treatment for substance abuse or chronic pain. Group 1 was an initial pilot group consisting of 7 patients who were followed for a 4-month period. Group 2 consisted of 33 patients who were followed over a 28-month period., Methods: Age, gender, weight, height, methadone dosage, quantitative urine creatinine and EDDP levels, reported compliance/non-compliance, and relevant clinical cofactors were retrospectively abstracted from the patients' medical records. Log-log regression analyses were used to model EDDP and the EDDP/creatinine ratio from urine screening results as functions of methadone dose, and in the larger cohort (group 2), body size, gender and age. The coefficient of determination adjusted for the number of predictor terms (R(adj)(2)) was reported as a measure of model fit., Results: For group 1 data, there was a significant positive relation (P<0.001) but also substantial variability (R(adj)(2) = 0.49). Adjustment for creatinine through the EDDP/creatinine ratio provided a tighter relation (R(adj)(2) = 0.95). Similarly, for group 2 data, there was a significant positive relation (P=0.001) and substantial variability (R(adj)(2) = 0.53). Adjustment for creatinine through EDDP/creatinine ratios provided a substantially stronger relation (R(adj)(2) = 0.73). Gender and age showed no evidence of association with the EDDP/creatinine ratio (P=0.60 and P=0.51, respectively). Body size was significant in the model, both when measured by body surface area and by lean body weight, and improved the prediction when added to our model (R(adj)(2) = 0.80)., Conclusion: For the first time, urine analyses may be used to monitor methadone over- or under-use in a clinical setting, regardless of the state of patient hydration or the manipulation of a sample by addition of another substance, such as bleach, soap, or even methadone, which could render an appropriate sample inappropriate or an inappropriate sample appropriate. A similar approach may prove useful for other drug treatments, allowing for more accurate monitoring of commonly abused prescription medications.

The aim of the present study was to identify the metabolites of the new designer drug alpha-pyrrolidinovalerophenone (PVP) in rat urine using GC/MS techniques. Eleven metabolites of PVP could be identified suggesting the following metabolic steps: hydroxylation of the side chain followed by dehydrogenation to the corresponding ketone; hydroxylation of the 2''-position of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or followed by ring opening to the respective aliphatic aldehyde and further oxidation to the respective carboxylic acid; degradation of the pyrrolidine ring to the corresponding primary amine; and hydroxylation of the phenyl ring, most probably in the 4'-position. The authors' screening procedure for pyrrolidinophenones allowed the detection of PVP metabolites after application of a dose corresponding to a presumed user's dose. In addition, the involvement of nine different human cytochrome P450 (CYP) isoenzymes in the side chain hydroxylation of PVP was investigated and CYP 2B6, 2C19, 2D6, and 3A4 were found to catalyze this reaction., (Copyright 2009 John Wiley & Sons, Ltd.)

Disposition of lasofoxifene (LAS; 6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-naphthalen-2-ol. tartrate) was investigated in rats and monkeys after oral administration of a single oral dose of [(14)C]LAS. Total mean recoveries of the radiocarbon were 96.7 and 94.3% from rats and monkeys, respectively. The major route of excretion in both species was the feces, and based on a separate study in the bile duct-cannulated rat, this likely reflects excretion in bile rather than incomplete absorption. Whole-body autoradioluminography suggested that [(14)C]LAS radioequivalents distributed rapidly in the rat with most tissues achieving maximal concentrations at 1 h. Half-life of radioactivity was longest in the uvea (124 h) and shortest in the spleen ( approximately 3 h). LAS was extensively metabolized in both rats and monkeys because no unchanged drug was detected in urine and/or bile. Based on area under the curve((0-24)) values, >78% of the circulating radioactivity was due to the metabolites. A total of 22 metabolites were tentatively identified by liquid chromatography-tandem mass spectrometry. Based on the structures of the metabolites, six metabolic pathways of LAS were identified: hydroxylation at the tetraline ring, hydroxylation at the aromatic ring attached to tetraline, methylation of the catechol intermediates by catechol-O-methyl transferase, oxidation at the pyrrolidine ring, and direct conjugation with glucuronic acid and sulfuric acid. LAS and its glucuronide conjugate (M7) were the major circulating drug-related moieties in both rats and monkeys. However, there were notable species-related qualitative and quantitative differences in the metabolic profiles. The catechol (M21) and its sulfate conjugate (M10) were observed only in monkeys, whereas the glucuronide conjugate of the methylated catechol (M8) and hydroxy-LAS (M9) were detected only in rats.

Objective: To report a case of unusually low concentrations of methadone in a polydrug abuser during maintenance treatment with methadone., Case Summary: A 25-year-old man (weight 55 kg, height 165 cm) with a 12-year history of polydrug abuse was admitted to an opiates withdrawal methadone program. At the time of our observation, he was using both cannabinoids and heroin; no other medical conditions were discovered. Within the opiates withdrawal methadone program, under medical supervision, the patient started methadone therapy (20 mg/day). Two weeks later, an Abuscreen assay for methadone screening in the urine was negative and, to prevent the development of withdrawal symptoms, the dose of methadone was increased to 60 mg/day. One day later, the patient was asked to collect another urine sample in the presence of a nurse. The Abuscreen for methadone in urine remained negative. Evaluation of urinary samples collected over 24 hours documented low concentrations of methadone and high levels of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (the primary metabolite of methadone). Evaluation for the presence of the most common polymorphisms in the cytochrome P450 and P-glycoprotein genes showed that the patient was heterozygous for the CYP3A5(*)1 allele and for 2 single nucleotide polymorphisms in the P-glycoprotein gene (1236C/T and 3435C/T)., Discussion: In this patient, poor methadone adherence was ruled out because of the presence of physicians and nurses during both methadone maintenance treatment and Abuscreen screening. Moreover, because the patient reported only heroin and cannabis at the time of evaluation, drug interactions were ruled out as possible causes for the rapid clearance of methadone., Conclusions: In this case, CYP3A5 polymorphism may have played a role in the rapid methadone metabolism.