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news.gifArticles - - Drug Testing and Analysis

Drug Testing and Analysis


Wiley Online Library : Drug Testing and Analysis


Semi‐quantitative Analysis of Tramadol, Dextromethorphan and Metabolites in Decomposed Skeletal Tissues by Ultra Performance Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry (UPLC‐qTOF‐MS)  Voir?

The use of filtration/pass‐through extraction (FPTE) and ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐qTOF‐MS) to detect tramadol (TRAM), dextromethorphan (DXM) and metabolites from skeletal remains is described. Rats (n=5) received 50 mg/kg tramadol and were euthanized by CO2 asphyxiation approximately 30 min post‐dose. Rats (n=4) received 75 mg/kg dextromethorphan and were euthanized by CO2 asphyxiation approximately 45 min post‐dose. Remains decomposed to skeleton outdoors and vertebral bones were collected. Bones were cleaned, dried and pulverized to a fine powder. Bone underwent dynamic methanolic extraction followed by FPTE before analysis using UPLC‐qTOF‐MS. Recovery was at least 90% of maximal value within the first 10 min of methanolic extraction for all samples assayed. Analytical response was measured over the concentration range of 1‐500 ng/mL, with precision and bias <20% in triplicate analyses of all calibrators, and a limit of detection of 1 ng/mL for TRAM, DXM and all metabolites. The vertebral bone analyzed using this method detected TRAM, DXM and their respective metabolites in all samples analyzed.

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Loop‐mediated isothermal amplification (LAMP) as an alternative to PCR: a rapid on‐site detection of gene doping  Voir?

Innovation in medical research has been diverted at multiple occasions to enhance human performance. The predicted great progress in gene therapy has raised some concerns regarding its misuse in the world of sports (gene doping) for several years now. Even though there is no evidence that gene doping has ever been used in sports, the continuous improvement of gene therapy techniques increases the likelihood of abuse. Therefore, since 2004, efforts have been invested by the anti‐doping community and WADA for the development of detection methods. Several nested PCR and qPCR‐based strategies exploiting the absence of introns in the transgenic DNA have been proposed for the long‐term detection of transgene in blood. Despite their great sensitivity, those protocols are hampered by limitations of the techniques that can be cumbersome and costly. The purpose of this perspective is to describe a new approach based on loop‐mediated isothermal amplification (LAMP) for the detection of gene doping. This protocol enables a rapid and simple method to amplify nucleic acids with a high sensitivity and specificity and with a simple visual detection of the results. LAMP is already being used in clinical application for the detection of viruses or mutations. Therefore, this technique has the potential to be further developed for the detection of foreign genetic material in elite athletes.

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Fatality involving ocfentanil documented by identification of metabolites  Voir?

New psychoactive substances (NPS) use has rapidly increased over the last decade, and in the last 4 years producers increasingly appear to be targeting non‐controlled synthetic opioids, involving fentanyl derivatives such as ocfentanil (OcF). Identification of metabolites is of major importance in the context of NPS use as it could improve detection window in biological matrices in clinical and forensic intoxication cases. Hence, this work aims to report a fatality involving OcF documented by the identification of metabolites. A 30‐year‐old woman was found dead at home: an unidentified powder was found near her body and some injection sites were found at the autopsy. Toxicological analyses allowed to determine the presence of OcF in the powder, blood (3.7/3.9 ÎŒg/L, peripheral/cardiac) and in other post‐mortem samples. The most relevant potential CYP‐ and UGT‐dependent metabolites of OcF were investigated in vitro using human liver microsome incubation and liquid chromatography coupled with high resolution mass spectrometry, and subsequently confirmed in post‐mortem samples. Four OcF metabolites were produced in vitro (a mono‐hydroxylated OcF, O‐desmethylOcF, a hydroxylated desmethylOcF and a glucuronidated form of the O‐desmethylOcF), and all except the glucuronide were observed in blood and bile post‐mortem samples. Considering the relative intensity of the chromatographic peak areas, O‐desmethylOcF can be suggested to be an abundant metabolite of OcF. Nevertheless, the relevance of O‐desmethylOcF as being a complementary analytical target of OcF for OcF use detection needs further in vivo confirmation, especially through analysis of urines from users.

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A simple validated multi‐method for detecting drugs in oral fluid by Ultra Performance Liquid Chromatography‐Tandem Mass Spectrometry (UPLC‐MS/MS)  Voir?

Background Oral fluid sampling offers several advantages compared to other methods of detecting drugs in biological matrices due to its easy sampling procedure and simple supervision. Hence, the use of oral fluid for drug testing is increasing in workplaces and in roadside testing. As a result, more laboratories are required to perform analyses of drugs in oral fluid. Methods A multi‐method using Ultra Performance Liquid Chromatography‐Tandem Mass Spectrometry (UPLC‐MS/MS) for tracing drugs in oral fluid was developed. Oral fluid was collected using the collection device Oral‐Eze. Samples were prepared by dilution with acetonitrile and methanol followed by centrifugation prior to UPLC‐MS/MS analysis. The UPLC separation was achieved by using a BEH‐C18 column. Mass detection was performed in positive ion mode, and the most common drugs/metabolites were detected for amphetamines, benzodiazepines, cocaine, benzoylecgonine, opiates, opioids, phencyclidine, Δ 9‐ tetrahydrocannabinol. Results 37 analytes were validated using the developed UPLC‐MS/MS multi‐method with a total run time of 5 minutes. Calibration curves were linear for all analytes over the concentration range 1.5‐600 ng/mL. Within‐ and between‐day CVs varied from 1.2% to 20% and from 0.7% to 20%, respectively, for most substances. Extraction recoveries from the oral fluid device were >70%, a few had a yield of 50%. Conclusion The developed multi‐method was successfully validated and applied in a clinical routine laboratory to detect drugs in oral fluid samples that were sent from different workplaces and clinics. High sensitivity, simple sample pretreatment and short analysis time are advantages of this method.

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Detection of Autologous Blood Transfusions using a Novel Dried Blood Spot Method  Voir?

In doping control laboratories, autologous blood transfusions are currently detected using an indirect method that monitors changes in an athlete's hemoglobin concentration [Hb] and reticulocyte percent (Ret%) over time. The method is limited by the need for a phlebotomist to collect venous blood and the limited blood stability which requires temperature‐controlled shipment and analysis within 72 hours. These limitations significantly reduce the number of samples collected from each athlete and thus the utility of the method. We have recently developed a method to measure immature reticulocytes (IRC) and red blood cells (RBC) in dried blood spots, which could replace the current venous blood method. In the DBS method, cell‐specific proteins are digested with trypsin and measured by mass spectrometry. Two proteins, CD71 and Band3, are measured to count IRC and RBC, respectively. The method was tested in an autologous transfusion study consisting of 15 subjects who received blood and 11 subjects who received saline. After transfusion, the average CD71/Band3 ratio in the blood group was statistically different from the saline group at days 5, 6, 13, and 20. The average CD71/Band3 ratio decreased to a minimum of 61 ± 8% of baseline, while Ret% decreased to 75 ± 5% of baseline. Based on experimentally defined criteria, the CD71/Band3 ratio could detect 7 out of 10 blood transfusion subjects, while Ret% could detect 3 out of 10. Thus, the DBS method could improve detection of autologous transfusion and allow increased sample collection.

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Fast IRMS screening of pseudoendogenous steroids in doping analyses.  Voir?

The detection of the abuse of pseudoendogenous steroids (testosterone and/or its precursors) is currently based, when possible, on the application of the steroid module of the World Antidoping Agency (WADA), Athletes' Biological Passport (ABP), implemented through ADAMS. When a suspicious sample is detected, the confirmation by isotope ratio mass spectrometry (IRMS) is required. It is well known that this confirmation procedure is time consuming, expensive and can be only applied on a reduced number of samples. In previous studies we have demonstrated that the longitudinal evaluation of the IRMS data is able to detect positive samples that otherwise will be evaluated as negative, improving the efficacy of the fight against doping in sport. This would require the analysis of a much larger volume of samples by IRMS. The aim of the present work is to describe an IRMS screening method allowing to increase the throughput of samples that can be analyzed by IRMS. The detection efficacy of the method is compared with the confirmation method in use, and to assess its robustness and applicability, all the samples of a major cycling stage competition were analyzed, with the agreement of the testing authority, under routine conditions and response times. The results obtained permit to conclude that the IRMS screening method here proposed has adequate selectivity and produces results that overlap with the already validated method currently in use permitting to analyze a much higher volume of samples even during a major event without compromising the detection capacity.

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Implementation of AICAR analysis by GC‐C‐IRMS for anti‐doping purposes  Voir?

AICAR (5‐aminoimidazole‐4‐carboxamide‐1‐ÎČ‐D‐ribofuranoside), is a naturally‐occurring substance which is part to the prohibited list of WADA. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the GC‐C‐IRMS analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC‐C‐IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC‐C‐IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3‐TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to HPLC problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a “wash step” before the HPLC purification and a HPLC purification step for the ERC fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances.

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Range of therapeutic prothipendyl and prothipendyl sulfoxide concentrations in clinical blood samples  Voir?

Due to a lack of reference blood concentrations in the literature, the forensic evaluation of prothipendyl findings in blood samples is difficult. Interpretations with regard to the assessment of blood concentrations as well as an estimation of the ingested prothipendyl amounts were often vague. To describe a concentration range in clinical samples, prothipendyl and prothipendyl sulfoxide concentrations were determined in serum samples of 50 psychiatric patients receiving 40 mg, 80 mg or 160 mg doses of prothipendyl. The analyses of prothipendyl and prothipendyl sulfoxide were carried out using validated methods of high performance liquid chromatography coupled to triple quadrupole mass spectrometry (LC‐QQQ‐MS), respectively. 40 mg doses caused average prothipendyl serum concentrations of 18.0 ng/mL (1 h after intake) and 7.9 ng/ml (10.5 h after intake), while 80 mg doses caused averages of 42.6 ng/mL and 15.2 ng/mL at the mentioned times of sampling. Irrespective of the given dose, prothipendyl concentrations below 30 ng/ml were observed in 80% of the patient samples taken 1 h after ingestion as well as in 90% of the samples collected 10.5 h after the administration. Serum concentrations of the phase I metabolite prothipendyl sulfoxide averaged 4.3 ng/mL (1 h after intake) and 3.6 ng/mL (10.5 h after intake). Possible drug‐drug interactions regarding absorption and metabolism of prothipendyl are discussed. Results of the herein presented study are useful for the interpretation of analytical prothipendyl findings in forensic toxicology. The utility of the described concentration range is demonstrated by discussing two death cases involving prothipendyl findings.

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DETERMINATION OF HYDROXY METABOLITES OF COCAINE IN HAIR SAMPLES FOR PROOF OF CONSUMPTION  Voir?

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination; this especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC‐positive samples for these compounds was performed by LC‐QTOF‐MS. Once identified, mass transitions for o‐, p‐ and m‐isomers of hydroxy COC as well as p‐ and m‐isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by LC‐MS/MS which was applied to 576 hair samples. Any hydroxy metabolites were present in 92.2 % of COC positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p‐OH‐BE, m‐OH‐BE as well as p‐OH‐NCOC and m‐OH‐NCOC have been identified for the first time in COC positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC positive hair samples.

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Immunomagnetic beads‐based isolation of erythropoietins from urine and blood for sports anti‐doping control.  Voir?

According to World Anti‐Doping Agency (WADA) technical document for erythropoiesis stimulating agents (ESA) analysis (TD2014EPO), double‐ blotting of serum/plasma samples is mandatory for all analysis by isoelectric focusing (IEF) and for the confirmation procedures (CP) performed by SDS‐PAGE or SAR‐PAGE. The goal is to prevent potential cross‐reactions of the secondary antibody with remaining proteins in the purified samples. To this end, we have developed an immunopurification method of ESA in serum/plasma samples using a combination of streptavidin‐coated immunomagnetic beads and biotinylated anti‐EPO polyclonal antibodies. Here we report that this immunomagnetic bead‐ based purification allows the analysis of serum/plasma samples by single‐ blotting. Serum and plasma samples, either intact or spiked with different ESAs, were immunopurified and analyzed by single‐blotting, after SAR‐ PAGE or IEF using a cross‐reaction minimized secondary antibody coupled to HRP. The results show that when samples are immunopurified according to this strategy, there is no non‐specific binding when single‐blotting is performed after SAR‐PAGE. With IEF we observe a faint smearing however in the pH gradient outside the ESA detection region. These interferences did not alter ESA profiles of spiked urinary samples or of samples received for routine testing. This approach was compared to the MAIIA monoliths purification or to the isolation of ESAs with other combinations of immunomagnetic reagents (i.e. anti‐Mouse IgG‐coated magnetic beads and anti‐EPO mAb). The recovery of ESAs was shown to be significant for serum/plasma samples. Our results suggest that single‐blotting could be performed on serum/plasma samples without non‐specific interferences.

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A novel and innovative hair test to determine glucocorticoid levels in racing camels for use in assessment of doping, health and disease  Voir?

Background The aim of this project was to develop and validate a new test for the analysis of glucocorticoids in camel hair and to use the new test to analyse hair samples from a variety of camel breeds in sports and racing applications. These findings could be of importance when evaluating racing camels for suspected doping offenses or for injury and disease control. Methods Camel hair samples were collected from 30 non‐racing dromedary camels along with 3 racing camels in Al Ain, UAE and were decontaminated, pulverized, sonicated and extracted prior to analysis. A liquid chromatographic‐mass spectrometric method was employed to determine the levels of glucocorticoids in the hair samples. Results The four drugs of interest, namely hydrocortisone, dexamethasone, flumethasone and methylprednisolone, and an internal standard were quantified in camel hair samples. All four of the glucocorticoids were detected in camel hair samples with concentrations ranging between 31‐935 pg/mg for hydrocortisone, 8‐59 pg/mg for dexamethasone, 0.7‐1034 pg/mg for flumethasone and 5‐66 pg/mg for methylprednisolone in non‐racing camels. One of the racing camels displayed high concentrations of hydrocortisone (1130pg/mg), flumethasone (2576 pg/mg), methylprednisone (1156 pg/mg) and dexamethasone (29 pg/mg). Conclusions The authors believe this is the first report of a test for corticosteroids in camel hair. The new test has been validated according to FDA guidelines. This new hair test could be useful for further studies in doping control, toxicological studies, pharmacological studies and other clinical applications in camel health, injury and disease.

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Sample Preparation Method for the Combined Extraction of Ethyl Glucuronide and Drugs of Abuse in Hair  Voir?

In hair analysis often little hair sample is available while the analysis of a multitude of structurally diverse substances with different concentration ranges is demanded. Often, the analysis of the different substances requires different sample preparation methods, increasing the amount of required hair sample. When segmental hair analysis is necessary, the amount of needed hair sample is further increased. Therefore, the required sample amount for a full analysis can quickly exceed the available hair sample. To combat this problem, a method for the combined hair sample preparation using a single extraction procedure for analysis of ethyl glucuronide with LC‐MS3/MRM and common drugs of abuse with LC‐MRM was developed. The combined sample preparation is achieved by separating ethyl glucuronide from the drugs of abuse into separate extracts by fractionation in the solid phase extraction step during sample cleanup. A full validation for all substances for the parameters selectivity, linearity, limit of detection, limit of quantification, accuracy, precision, matrix effects and recovery was successfully completed. The following drugs of abuse were included in the method: Amphetamine; methamphetamine; 3,4‐methylenedioxy‐N‐methylamphetamine; 3,4‐methylenedioxyamphetamine; 3,4‐methylenedioxy‐N‐ethylamphetamine; morphine; 6‐monoacetylmorphine; codeine; acetylcodeine; cocaine; benzoylecgonine; norcocaine; cocaethylene; methadone; 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine and methylphenidate. In conclusion, as only one sample preparation is needed with one aliquot of hair, the presented sample preparation allows an optimal analysis of both ethyl glucuronide and of the drugs of abuse even when the sample amount is a limiting factor.

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Is zebrafish (Danio rerio) a tool for human‐like metabolism study?  Voir?

One of the greatest challenges in anti‐doping science is the large number of substances available and the difficulty in finding the best analytical targets for detecting their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non‐approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with well‐known metabolism that are widely used as doping agents in sports, was evaluated. They represent two of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid phase extraction and analysed by liquid chromatography with high resolution mass spectrometry (LC‐HRMS). Adult zebrafish were able to produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dihydroxylated and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidize and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human‐like metabolism when aiming to find analytical targets for doping control.

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LC‐MS/MS method for quantification of baclofen in hair: a useful tool to assess compliance in alcohol dependent patients?  Voir?

In order to evaluate adherence to treatment, we developed and validated a novel LC‐MS/MS method for baclofen quantification in hair. 20 mg were washed twice with dichloromethane, incubated in phosphate buffer (pH 5) for 10 min at 95°C, then extracted by liquid‐liquid extraction in alkaline condition. Baclofen‐d4 was used as internal standard. This method was applied to assess compliance in four treated alcohol dependent patients (3 dead and one alive). Blood quantification of baclofen and ethanol were performed in the 4 cases. Hair ethylglucuronide (ethanol metabolite, EtG) measurement (2 x 3 cm) was associated in one patient. Baclofen quantification in hair was validated over the range 10–5.000 pg/mg. The accuracy was within 96.0–110.9% and the precision was less than 9.3%. Baclofen segmental (3 x 2cm) hair concentrations found in the living patient were 4420, 4260 and 4380 pg/mg, reflecting a regular exposure over the last 6 months and suggesting patient's compliance. However, the high EtG level found in this patient in the analyzed segments (225 pg/mg and 215 pg/mg) showed excessive alcohol consumption during the same period, suggesting therapeutic failure. In the three deceased patients, the non‐segmental analysis of hair showed baclofen concentrations of 15, 545 and 2475 pg/mg. The low concentrations in the two first cases are compatible either with a poor compliance or to a beginning of a treatment. This is the first measurement of baclofen in hair of alcohol dependent patients. It could be used as a monitoring biomarker to assess patient's compliance.

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Pharmacokinetic Properties of the synthetic cannabinoid JWH‐018 in oral fluid after inhalation  Voir?

Background Each year, synthetic cannabinoids are occurring in high numbers on the illicit drug market, but data on their detectability are rarely available. Therefore, a pilot study was performed to assess adverse effects of JWH‐018, which is one of the oldest and best known synthetic cannabinoids. Oral fluid has been evaluated as a specimen for drug monitoring. Methods Six subjects inhaled smoke derived from 2 and 3 mg JWH‐018. The drug and ten of its metabolites were analyzed in oral fluid samples collected during the following 12 hours using the Quantisal collection device by liquid chromatography‐mass spectrometry (LC‐MS/MS). Results Maximum concentrations of JWH‐018 reached 2.2‐2036 (median 25.7) ng/ml after inhalation and decreased during the next hour to only 0.08‐8.42 (median 0.89) ng/ml. Metabolites were not found. During the elimination phase (median half‐life1.69 h), detection of the drug over 6‐12 (median 8) h after inhalation was achieved (0.024 ng/ml limit of quantification). Oral fluid/serum ratios varied considerably intra‐ and inter‐individually in a range of 0.05‐555 (median 1.38). Conclusion The detection of JWH‐018 in oral fluid requires high analytical sensitivity already one hour after inhalation. The pharmacokinetic properties of inhaled JWH‐018 are similar to those of THC. Times for detection are typically less than 12 h. High variability of the oral fluid/serum ratio precludes extrapolation of oral fluid concentrations to blood.

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High Volume Steroid Isotopic Standards Developed as Working Standards for Gas Chromatography‐Combustion Isotope Ratio Mass Spectrometry  Voir?

High precision carbon isotope ratio analysis of urinary steroids by gas chromatography combustion isotope ratio mass spectrometry (GCC‐IRMS) is the official test to detect illicit doping of synthetic versions of endogenous steroids such as testosterone. Our group created the first steroid isotopic standards (SIS) specifically for the World Anti‐Doping Agency (WADA) accredited laboratories. The standards contain mixtures of steroids as acetates or free steroids at ~400 ÎŒg each per ampoule and have been widely distributed to anti‐doping laboratories to facilitate comparability of inter‐laboratory results. Here we report on creation and characterization of three new high volume single component SIS suitable for use as working standards. They contain ~50 times more steroid mass per ampoule than previous SIS. The new SIS, coded CU/PCC 40‐1, CU/PCC 41‐1, & CU/PCC 42‐1, contain ~20 mg of androsterone, androsterone‐AC, and 5α‐cholestane, with determined isotopic values of ‐27.09 ± 0.07 mUr, ‐32.82 ± 0.01 mUr, ‐25.03 ± 0.01 mUr, respectively. We used our previously developed protocol to calibrate the isotopically uniform steroids against the isotopic standard gases methane and ethane in NIST RM 8559 that are traceable to the international standard Vienna PeeDee Belemnite (VPDB). Two sets of data, acquired 7 months apart, of absolute ÎŽ13CVPDB and ∆ή13CVPDB values from 8 randomly selected ampoules of all three SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(ÎŽ13C)<0.2 mUr Our results show that protocols for SIS extend to creation of high volume working standards that can also be used as internal standards under appropriate GC conditions.

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Detection of In Utero Cannabis Exposure by Umbilical Cord Analysis  Voir?

According to 2014 National Survey on Drug Use and Health, 5.3% of pregnant women smoked marijuana in the past month. This prevalence is expected to increase as a growing number of states and countries are now considering legalization. Although umbilical cord is becoming a useful objective tool to detect in utero drug exposure, currently data about analytical methods and its utility to detect cannabis exposure are scarce. The objective of this work was to develop a method for the determination of Δ9‐tetrahydrocannabinol (THC), 11‐hydroxyTHC (THC‐OH), 11‐nor‐9‐carboxy‐THC (THCCOOH), 8‐ÎČ‐11‐dihydroxyTHC (THC‐diOH), THC and THCCOOH glucuronides, and cannabidiol (CBD) in umbilical cord by liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS) with dual ionization source. Umbilical cord samples (0.5g) were homogenized in methanol and extracted by solid phase extraction. Reversed‐phase chromatographic separation was performed in 14 min, and two transitions per analyte were monitored in multiple reaction monitoring mode. Method validation included linearity (1‐10 to 20‐200ng/g), precision (4.1‐23.4%), accuracy (87.5‐111.4%), matrix effect (‐54.8 to ‐5.8%), extraction efficiency (25‐45.6%), limits of detection and quantification (1‐10ng/g), and endogenous (n=5) or exogenous interferences (not detected). The method was applied to 13 authentic samples from cannabis‐exposed newborns, which meconium samples had tested positive for cannabis. Twelve cord specimens tested positive for THCCOOH‐Glucuronide (1.6‐19.1ng/g). We developed and validated a specific and sensitive method for the simultaneous determination of THC, its metabolites, including THC and THCCOOH glucuronides, and CBD in umbilical cord samples by LC‐MS/MS. The analysis of authentic samples showed the usefulness of umbilical cord to detect cannabis in utero exposure.

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Characterization of in‐vitro generated metabolites of selected peptides < 2 kDa prohibited in sports  Voir?

With an increasing number of prohibited substances in doping controls, knowledge about their metabolism is crucial for efficient analysis. While for low molecular mass molecules, standard protocols for in‐vitro metabolism experiments are well established, the situation with peptidic drugs has been shown to be substantially more heterogeneous and complex. Two principle strategies aiming at simulating the metabolism of lower molecular mass peptides in‐vitro are presented within this study. The prohibited peptides ARA‐290, GHRP‐3 and Peforelin, with a to date unknown metabolism, were chosen as model compounds for these experiments and metabolism after incubation with different blood specimens (EDTA‐, heparin‐, citrate‐plasma and serum) and exposure to recombinant amidase were investigated. The characterization of in‐ vitro generated drug‐derived peptidic analytes was accomplished by means of liquid chromatography coupled to high resolution mass spectrometry. Identification of the generated metabolites was ensured by dedicated high resolution product ion experiments after liquid chromatographic separation. While extensive exopeptidase‐driven metabolism was observed for ARA‐290 (with one main metabolite PyrEQLERALN), GHRP‐3 and Peforelin were found to exhibit a considerable metabolic stability with a low tendency for deamidation only.

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PTCA (1H‐pyrrole‐2,3,5‐tricarboxylic acid) as a marker for oxidative hair treatment  Voir?

Hair analysis for the assessment of alcohol or drug abstinence became a routine procedure in forensic toxicology. Hair coloration leading to loss of incorporated xenobiotics and to false negative results turned out to be a major problem. Currently only colored extracts provide hints of manipulations but not bleaching. A liquid chromatographic‐mass spectrometric (LC‐MSMS) method was developed and validated to determine 1H‐pyrrole‐2,3,5‐tricarboxylic acid (PTCA), a major oxidation product of melanin. PTCA was determined in natural hair samples (n=21) after treatment with 3% hydrogen peroxide (H2O2) for 30 or 40 min with concentrations up to 12% for 40 min. In another series, 12 natural hair samples were submitted to different coloration procedures (henna, tinting, semi‐permanent and permanent dyeing, bleaching) and the changes in PTCA content were determined. A significant increase in the PTCA content was found for both incubation times and increasing H2O2 concentrations. Coloration with henna or tinting had no influence on PTCA levels detected, but a significant increase was observed after semi‐permanent and permanent dyeing and bleaching. As PTCA concentrations in natural hair were found to be in a range of <2.1 ‐ 16.4 ng/mg (8.4±3.8 ng/mg, mean±SD, n=33), a cut‐off of 20 ng/mg is recommended for the distinction between natural vs. excessively oxidized hair. In case of naturally low melanin content (lightblond or white hair), no marked increase in PTCA may occur. The present study demonstrated that PTCA is formed during oxidative treatment of melanin in hair, which can be used to detect previous hair coloration including oxidation.

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Antibody‐based strategies for the detection of Luspatercept (ACE‐536) in human serum  Voir?

Luspatercept (ACE‐536, ACVR2B‐Fc), a fusion protein consisting of the extracellular domain of ActRIIB receptor and the Fc‐part of human immunoglobulin G1 (IgG1), is currently under clinical development (Phase III). It stimulates the formation of red blood cells and hence may be misused by athletes for doping purposes in the future. Several antibody‐based strategies for the detection of Luspatercept and other ACVR2B‐Fc fusion proteins in human serum were evaluated (ELISA; IEF‐, SDS‐, and SAR‐PAGE followed by Western blotting; immunoprecipitation). Two methods led to useful results: a commercial “soluble” ACTR‐IIB ELISA, which also detected Luspatercept and other ACVR2B‐Fc's, but showed no cross‐reactivity with Sotatercept/ACVR2A‐Fc's. The ELISA might be applied as fast screening tool (100 ÎŒL serum; LOD ca 15.6 ng/mL). The second method uses a polyclonal ACVR2B‐antibody for immunoprecipitation followed by SAR‐PAGE and Western blotting with a monoclonal detection antibody (50 ÎŒL serum; LOD ca 1.0 ng/mL). It can be used for initial as well as for confirmatory testing. Due to the high doses (mg/kg) and long serum half‐life of Luspatercept, both strategies may be useful in anti‐doping control in the future.

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Influence of combined iron supplementation and simulated hypoxia on the haematological module of the Athlete Biological Passport  Voir?

The integrity of Athlete Biological Passport (ABP) is underpinned by understanding normal fluctuations of its biomarkers to environmental or medical conditions, e.g. altitude training or iron deficiency. The combined impact of altitude and iron supplementation on the ABP was evaluated in endurance‐trained athletes (n=34) undertaking 3‐weeks of simulated live‐high: train‐low (14 h.d‐1, 3000m). Athletes received either oral, intravenous (IV) or placebo iron supplementation, commencing two weeks prior and continuing throughout hypoxic exposure. Venous blood was sampled twice prior, weekly during, and up to 6‐weeks after altitude. Individual ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage (%retic), and OFF score were calculated using the adaptive model and assessed at 99% and 99.9% specificity. Eleven athletes returned values outside of the calculated reference ranges at 99%, with 8 at 99.9%. The percentage of athletes exceeding the thresholds in each group was similar, but IV returned the most individual occurrences. A similar frequency of abnormalities occurred across the three biomarkers, with abnormal [Hb] and OFF score values arising mainly during‐, and %retic values mainly post‐ altitude. Removing samples collected during altitude from the model resulted in ten athletes returning abnormal values at 99% specificity, two of whom had not triggered the model previously. In summary, the abnormalities observed in response to iron supplementation and hypoxia were not systematic and mostly in line with expected physiological adaptations. They do not represent a uniform weakness in the ABP. Nevertheless, altitude training and iron supplementation should be carefully considered by experts evaluating abnormal ABP profiles.

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A review of chemical ‘spot' tests: a presumptive illicit drug identification technique  Voir?

Chemical ‘spot' tests are a presumptive illicit drug identification technique commonly used by law enforcement, border security personnel, and forensic laboratories. The simplicity, low cost and rapid results afforded by these tests make them particularly attractive for presumptive identification globally. In this paper, we review the development of these long‐established methods and discuss color test recommendations and guidelines. A search of the scientific literature revealed the chemical reactions occurring in many color tests are either not actively investigated or reported as unknown. Today, color tests face many challenges, from the appearance of new psychoactive substances to concerns regarding selectivity, sensitivity, and safety. Advances in technology have seen color test reagents used in digital image color analysis, solid sensors and microfluidic devices for illicit drug detection. This review aims to summarize current research and suggest the future of presumptive color testing.

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Codeine influences the serum and urinary profile of endogenous androgens but does not interact with the excretion rate of administered testosterone  Voir?

Today's doping tests involve longitudinal monitoring of urinary steroids including the testosterone glucuronide and epitestosterone glucuronide ratio (T/E) in an athlete biological passport (ABP). The aim of this study was to investigate the possible influence of short term use of codeine on the urinary excretion of androgen metabolites included in the steroidal module of the passport prior to and after the co‐administration with testosterone. The study was designed as an open study with the subjects being their own control. Fifteen healthy male volunteers received therapeutic doses of codeine (Kodein Meda) for 6 days. On day three, 500 mg or 125 mg of testosterone enanthate (Testovironźâ€Depot) was administered. Spot urine samples were collected for 17 days, and blood samples were collected at baseline, 3, 6 and 14 days after codeine intake. The circulatory concentration of total testosterone decreased significantly by 20 % after three days use of codeine (p=0.0002) and an atypical ABP result was noted in one of the subjects. On the other hand, the concomitant use of codeine and testosterone did not affect the elevated urinary T/E ratio. In 75 % of the individuals, the concentration of urinary morphine (a metabolite of codeine) was above the decision limit for morphine. One of the participants displayed a morphine/codeine ratio of 1.7 after codeine treatment, indicative of morphine abuse. In conclusion our study shows that codeine interferes with the endogenous testosterone concentration. As a result, the urinary steroid profile may lead to atypical findings in the doping test.

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In Vitro Metabolism of the Synthetic Cannabinoids CUMYL‐PINACA, 5F‐CUMYL‐PINACA, CUMYL‐4CN‐BINACA, 5F‐CUMYL‐P7AICA and CUMYL‐4CN‐B7AICA  Voir?

Synthetic cannabinoid consumption trends underlie fast changes and provide several challenges to clinical and forensic toxicologists. Due to extensive metabolism parent compounds are hardly detectable in urine. Therefore, knowledge on the metabolism of synthetic cannabinoids is essential to allow their detection in biological matrices. The aim of the present study was the elucidation of the metabolism of CUMYL‐PINACA, 5F‐CUMYL‐PINACA, CUMYL‐4CN‐BINACA, 5F‐CUMYL‐P7AICA, CUMYL‐4CN‐B7AICA with focus on the analytical and interpretational differentiation of the compounds. Microsomal assay mixtures containing co‐substrates, 10 ÎŒg/ml substrate and 1 mg/mL pooled human liver microsomes were incubated for 1 h at 37 °C. Investigation of the metabolites was performed on a Thermo Fischer Ultimate 3000 UHPLC system coupled to a Sciex 6600 QTOF System. Hydroxylation was observed to be a major biotransformation step for all five cumyl‐derivatives, followed by dihydroxylation. For CUMYL‐PINACA, a major metabolic pathway was hydroxylation at the pentyl moiety, followed by a second hydroxylation at that pentyl moiety or oxidation to ketone. A major metabolic pathway for the compounds containing a nitrile function was nitrile hydrolysis followed by carboxylation and further hydroxylation. For the fluorinated compounds, oxidative defluorination and carboxylation were abundant metabolic steps. Some of the metabolic transformations lead to structurally identical metabolites, which should not be used as marker for the intake of a particular parent compound. In addition, several constitutional isomers containing either an indazole or azaindole core structure were detected, which should be differentiated by retention time rather than by their mass spectra alone.

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N‐Acetylcysteine as a chemical scavenger for sulfur mustard: New insights by mass spectrometry  Voir?

The vesicant sulfur mustard (SM) is a banned chemical warfare agent. Although, SM was used in combat since WWI there is still no causal therapy currently available. Accordingly, development and investigation of antidotes and scavengers targeting SM are of high clinical relevance. N‐acetylcysteine (NAC) was shown to mitigate symptoms of SM intoxications in vitro and in vivo. However, it is still unclear whether the beneficial effects of NAC are only due to physiological processes or also due to chemical scavenging of SM. Therefore, in this study we examined the scavenging potential of NAC towards SM. Co‐incubations of SM and different NAC concentrations in human serum were performed to monitor diverse adducts (covalent reaction products) of human serum albumin (HSA), NAC, and SM. After proteolytic cleavage of HSA with proteinase K the alkylated tripeptide hydroxyethylthioethyl‐CysProPhe (HETE‐CPF) and the disulfide bridged tripeptide NAC‐CPF were detected. Samples were analyzed by microbore liquid chromatography‐electrospray ionization high‐resolution tandem‐mass spectrometry (ÎŒLC‐ESI MS/HR MS). Furthermore, degradation kinetics of SM in phosphate buffered saline were measured in the presence and absence of NAC. Although NAC‐CPF was identified and characterized for the first time by mass spectrometry and reaction products of NAC and SM were detected and identified by MS/HR MS, analyses clearly documented minor reactivity not significantly contributing to reduction of SM concentrations. Therefore, we conclude that chemical scavenging of SM by NAC does not play the key role in NAC therapy of SM poisoning.

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Road‐Side Drug Testing: An Evaluation of the Alere DDSź2 Mobile Test System  Voir?

The number of drivers using drugs has increased over the last few years, and is likely to continue its upward trend. Testing drivers for alcohol use is routine and standardized, but the same is not true for the identification of driving under the influence of drugs (DUID). The Drug Evaluation and Classification Program (DECP) was developed to train police officers to recognize the signs and symptoms of recent drug use and remains an invaluable program; however there are insufficient numbers of these highly trained drug recognition experts (DRE's) who are available to attend every potential drug involved traffic incident. While blood and urine samples are used to test for drugs in a driver, both have disadvantages particularly as it pertains to the length of time required after a traffic stop to sample collection. Therefore, the development of oral fluid testing devices which can be operated at the roadside and have the potential to assist officers in the identification of drug use is a major advancement in DUID cases. This project evaluated the performance of one instrumental oral fluid roadside testing device (Alere DDSź2) compared to DRE opinion, oral fluid laboratory based analysis and routine blood testing. The results showed that there was a good correlation with DRE observations and the device performance was >80% in all drug categories compared to laboratory‐based analytical testing; both in oral fluid and blood, with few exceptions. The instrument can be considered a useful tool to assist law enforcement in identifying a drugged driver. Because the device does not test for all potentially impairing drugs, the opinion of the police officer regarding the condition of a driver should still be considered the most important aspect for arrest and further action.

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Identification of specific markers for amphetamine synthesised from the pre‐precursor APAAN following the Leuckart route and retrospective search for APAAN markers in profiling databases from Germany and the Netherlands  Voir?

α‐Phenylacetoacetonitrile (APAAN) is one of the most important pre‐precursors for the amphetamine production in recent years. This assumption is based on seizure data but up to now there is little analytical data available showing how much amphetamine really originated from APAAN. In this study several syntheses of amphetamine following the Leuckart route were performed starting from different organic compounds including APAAN. The organic phases were analysed using GC/MS to search for signals caused by possible APAAN markers. Three compounds were discovered, isolated and based on the performed syntheses it was found that they are highly specific for the use of APAAN. Using mass spectra, high resolution MS and NMR data the compounds were characterised and identified as 2‐phenyl‐2‐butenenitrile, 3‐amino‐2‐phenyl‐2‐butenenitrile and 4‐amino‐6‐methyl‐5‐phenylpyrimidine. To investigate their significance they were searched in data from seized amphetamine samples to determine to what extent they were present in illicitly produced amphetamine. Data of more than 580 cases from amphetamine profiling databases in Germany and the Netherlands were used for this purpose. These databases allowed to analyse the yearly occurrence of the markers going back to 2009. The markers revealed a trend that was in agreement with seizure reports and reflected an increasing use of APAAN from 2010 on. This paper presents experimental prove that APAAN is indeed the most important pre‐precursor of amphetamine in recent years. It also illustrates how important it is to look for new ways to identify current trends in drug production since such trends can change within a few years.

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RESOLUTION OF R‐(‐) AND S‐(+)‐ ENANTIOMERS OF CLENBUTEROL IN PHARMACEUTICAL PREPARATIONS AND BLACK MARKET PRODUCTS USING LIQUID CHROMATOGRAPHY‐TANDEM MASS SPECTROMETRY  Voir?

Several banned substances are illegally used by athletes in racemic mixtures for performance enhancement. These include clenbuterol, methyl hexaneamine, methamphetamines, amphetamines. Clenbuterol is present in a large number of doping samples from Olympic and non‐Olympic athletes that have adverse analytical findings. In some cases, the presence of these substance could be the result of consumption of meat contaminated with clenbuterol. In other cases, the origin is not clear. In this study, 27 products with racemic clenbuterol were evaluated using a new analytical methodology for the resolution of R‐(‐) and S‐(+)‐enantiomers of clenbuterol by Liquid Chromatography‐Tandem Mass Spectrometry LC‐MS/MS using a chiral column in 15 minutes with good separation. The method here developed can also be used for the analysis of other biological matrix as urine, serum, meat. The resolution between two peaks (Rs) value obtained using chromatographic data was 1.03. Both clenbuterol enantiomers were present in all products analyzed and the ratio was nearly 1. The origin of the product was not important for determining the presence of one or both enantiomers. All products displayed a 50:50 ratio of clenbuterol enantiomers. To the best of our knowledge, clenbuterol ratio determination of a large number of pharmaceutical preparations and black market products have not been reported previously. The information here shown could be use by the National Anti‐Doping Organizations and the athletes with AAF attributed to clenbuterol.

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Decrease of ethyl glucuronide concentrations in hair after exposure to chlorinated swimming pool water.  Voir?

The direct alcohol marker ethyl glucuronide (EtG) is widely used for the assessment of alcohol consumption behavior and abstinence monitoring by hair analysis. We investigated the influence of chlorinated swimming pool water on EtG concentrations in hair in comparison to deionized water (Milli‐Q) containing no chlorine. EtG concentrations were measured with a validated online‐SPE‐LC‐MS/MS method. EtG positive hair samples were obtained from three regular drinkers and incubated for 0, 2, 4, 6, 8, and 10 hours at room temperature. EtG concentrations in hair were reduced after two hours of incubation in chlorinated water by 20±12% (range:4‐33%), in deionized water by 24±5% (range:18‐29%). Incubation for 10 hours resulted in a decrease in EtG concentrations of 57±6% (range:52‐65%) for chlorinated water and 47±11% (range:32‐60%) for deionized water. To demonstrate washout in forensic hair samples, 20 samples from subjects with known alcohol consumption behavior were investigated additionally. The samples were divided into two strands and analyzed with incubation in chlorinated water for 10 hours and for comparison without any incubation. A mean decrease of 53±18% (range:26‐88%) was observed. These results clearly demonstrate that washout effects are caused by water and have a significant impact on EtG concentrations in hair. For people with hair that are regularly exposed to water for a longer period of time (e.g. swimmers), washout effects may lead to a significant decrease of EtG concentrations in hair. Concentrations may fall below threshold concentrations used for the interpretation of consumption habits (7 pg/mg for social consumption, 30 pg/mg for excessive consumption).

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MDA, MDMA and other mescaline‐like substances in the US military's search for a truth drug (1940s to 1960s)  Voir?

This article describes the broader context in which 3,4‐methylenedioxyamphetamine (MDA), 3,4‐methylenedioxymethamphetamine (MDMA) and other mescaline‐like compounds were explored as hallucinogens for military and intelligence purposes during the 1940s to the 1960s. Germans first tested mescaline as a "truth drug" in a military context. Since the 1940s, the United States military tested hallucinogenic drugs as "truth drugs" for the purpose of interrogation and behavior manipulation. After tests carried out using mescaline and other drugs in 1950, some derivatives of mescaline were synthesized by the Army for the exploration of possible „speech‐inducing“ effects. After insufficient animal testing, the substances were given to patients at the New York State Psychiatric Institute (NYSPI). 3,4‐Methylenedioxy‐N‐ethylamphetamine (MDE), a compound almost identical to MDMA, was among the mescaline derivatives delivered for testing at the NYSPI. During tests with other derivatives (3,4‐dimethoxyphenethylamine (DMA), 3,4‐methylenedioxyphenethylamine (MDPEA), MDA) in 1952‐53, an unwitting patient died in these tests, which was kept secret from the public. Research was interrupted and toxicological animal testing procedures were initiated. The secret animal studies run in 1953/54 revealed that some of the "mescaline derivatives" tested (e.g. MDA, MDE, DMA, 3,4,5‐trimethoxyamphetamine (TMA), MDMA) were considered for further testing in humans. Since 1955, the military changed focus to LSD, but some interest in mescaline‐like compounds remained for their ability to change mood and habit without interefing with cognition and sensory perception. Based on the known documents, it remains unclear (but probable) wether any of the mescaline derivatives tested were being used operationally.

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Steroid Profile and IRMS Analysis of Musk Administration for Doping Control  Voir?

Musk, the dried secretion of the musk pod (sac) of adult male musk deer, has been used as traditional Chinese Medicine (TCM) in China and south‐east Asian countries for thousands of years. Due to the anabolic steroid component in this TCM, musk preparations have been included in the list of medical products containing prohibited substances employed for doping by the State Food and Drug Administration of China. The application of musk pod formulation was claimed to be responsible for some adverse analytical findings (AAF) in the 2011 FIFA Women's World Cup. Our preliminary study has suggested that musk ingestion did not lead to AAF of doping control with the single dosage of 100 mg. However, the influences of musk administration in large and multi dosage are still unclear. The aim of this study is to further investigate the influences of musk administration for doping control. Wild and domestic deer musk samples were collected. The concentrations and ÎŽ13C‐values of steroids in musk were analyzed. In an excretion study, 200 and 100 mg of wild and domestic deer musk samples were administrated by 29 subjects respectively. Fluctuations in steroid profile could be observed, and the ratio of 5α‐androstane‐3α,17ÎČ‐diol to 5ÎČ‐androstane‐3α,17ÎČ‐diol was more sensitive than other parameters. In the IRMS test, the ∆ή13C‐value between endogenous reference compound and etiocholanolone was a sensitive parameter, and AAFs were obtained. It is the first time to confirm with excretion study that musk administration could lead to positive result of doping control.

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Epiandrosterone sulfate prolongs the detectability of testosterone, 4‐androstenedione and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry  Voir?

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow‐up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4‐androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T‐related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted.

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Application of a screening method for fentanyl and its analogues using UHPLC‐QTOF‐MS with data‐independent acquisition (DIA) in MSE mode and retrospective analysis of authentic forensic blood samples  Voir?

The steady appearance of new fentanyl analogues and the associated overdose deaths require the development of sensitive screening approaches to detect these compounds in biological samples and seizures. We developed a targeted screening method to detect 50 4‐anilidopiperidine‐related fentanyl analogues in whole blood using ultrahigh performance liquid chromatography quadrupole time‐of‐flight mass spectrometry in data‐independent acquisition mode. Sample preparation was performed using protein precipitation on a fully automated robotic setup. Thirteen analogues were selected to validate the method. A small matrix ion enhancement effect (110–123%) was observed for all of the compounds; the recovery ranged from 67–81% and the process efficiency from 81–98%. Limit of detection was within 0.0005–0.001 mg/kg and limit of identification ranged from 0.001–0.005 mg/kg. In the retrospective analysis of 2,339 forensic blood samples, the major finding was fentanyl (n = 56), followed by alfentanil (n = 5) and remifentanil (n = 1). Identification of 34 fentanyl analogues was based on the predicted product ions resulting from common fentanyl‐specific collision‐induced cleavages, particularly on the product ion result of the fragmentation on the C‐N bond between the phenylamide moiety and the piperidine ring. The proposed hypothesis was supported by the targeted analysis of 16 fentanyl analogues using this method and available published mass spectral data sources for fentanyl analogues. A targeted screening method for 50 fentanyl analogues was successfully validated and implemented to analyse authentic blood samples, where identifying targeted fentanyl analogues was tentatively achieved without using reference standards.

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Development of a quantitative method for the analysis of cocaine analogue impregnated into textiles by Raman spectroscopy  Voir?

Cocaine trafficking in the form of textile impregnation is routinely encountered as a concealment method. Raman spectroscopy has been a popular and successful testing method used for in situ screening of cocaine in textiles and other matrices. Quantitative analysis of cocaine in these matrices using Raman spectroscopy has not been reported to date. This study aimed to develop a simple Raman method for quantifying cocaine using atropine as the model analogue in various types of textiles. Textiles were impregnated with solutions of atropine in methanol. The impregnated atropine was extracted using less hazardous acidified water with the addition of potassium thiocyanate (KSCN) as an internal standard for Raman analysis. Despite the presence of background matrix signals arising from the textiles, the cocaine analogue could easily be identified by its characteristic Raman bands. The successful use of KSCN normalised the analyte signal response due to different textile matrix background interferences and thus removed the need for a matrix‐matched calibration. The method was linear over a concentration range of 6.25‐37.5 mg/cm2 with a coefficient of determination (R2) at 0.975 and acceptable precision and accuracy. A simple and accurate Raman spectroscopy method for the analysis and quantification of a cocaine analogue impregnated in textiles has been developed and validated for the first time. This proof‐of‐concept study has demonstrated that atropine can act as an ideal model compound to study the problem of cocaine impregnation in textile. The method has the potential to be further developed and implemented in real world forensic cases.

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The Analysis of Illicit 25X‐NBOMe Seizures in Western Australia  Voir?

The NBOMe class of compounds have gained prominence in recent years in the illicit drug scene and are marketed as an alternative to LSD, or in some cases sold under the pretense of being LSD. Publications reporting the identification of a novel NBOMe compound are common however, there is no scientific data reporting the amounts of these compounds found in seizures that have been submitted to forensic science laboratories for analysis. This publication reports the quantitation of three different NBOMe compounds, 25C‐NBOMe, 25I‐NBOMe and 25B‐NBOMe in seizures by Western Australia Police of blotter papers and tablets.

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European Guidelines for Workplace Drug Testing in Oral Fluid  Voir?

These guidelines for Legally Defensible Workplace Drug Testing have been prepared and updated by the European Workplace Drug Testing Society (EWDTS). The European Guidelines are designed to establish best practice procedures whilst allowing individual countries to operate within the requirements of national customs and legislation. The EWDTS recommends that all European laboratories that undertake legally defensible workplace drug testing should use these guidelines as a template for accreditation. These guidelines are relevant to laboratory‐based testing only. These guidelines follow current best practices and are constantly under review.

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Different localizations of drugs simultaneously administered in a strand of hair by micro‐segmental analysis  Voir?

Segmental hair analysis is used to estimate the time of drug intake at monthly precision in drug‐related crimes. Previously, we advanced this analytical method to specify the day of drug intake by cutting a strand of hair into 0.4‐mm segments, which correspond to daily hair growth. Herein, we investigated the distributions of 7 compounds in a strand of hair using micro‐segmental analysis. Several strands of hair were collected 33.1−229.4 days after subjects were administered 4 pharmaceutical products that contained 10 drugs in single doses within 32 hours. The administered drugs and resulting metabolites were extracted from 0.4‐mm hair segments and quantified using liquid chromatography–tandem mass spectrometry. Acidic and neutral compounds were detected at low amounts in any of the hair segments analyzed. Epinastine, fexofenadine, dihydrocodeine, chlorpheniramine, and the chlorpheniramine metabolite, desmethylchlorpheniramine each was localized to 2 regions within a strand of hair. By contrast, methylephedrine and its metabolite, ephedrine, each was localized to only a region. Among 20 individual strands of hair associated with different subjects and head regions, few differences in the shapes of drug concentration–hair segment curves for each compound were detected. Our data indicated that 2 mechanisms for drug uptake into hair can operate depending on drug properties and that co‐administered drugs can be localized to different regions in a strand of hair. Micro‐segmental analysis may aid in the identification of the day of drug intake and help to elucidate the mechanisms of drug uptake into hair. Micro‐segmental analysis of a strand of hair visualized the distributions of 7 compounds in 22 strands of hair. The spatial resolution in the analysis corresponds to 1‐day hair growth length. The differences in localizations between co‐administered drugs were first founded.

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The identification and analytical characterization of 2,2â€Č‐difluorofentanyl  Voir?

New psychoactive substances (NPS) have expanded their distribution and become widely available in the global market in recent years. The illicit use of fentanyl and its analogs has become an important worldwide concern linked to their high potency and risk of fatal overdose. This study describes the analytical characterization of a new fentanyl derivative N‐(1‐(2‐fluorophenethyl)‐4‐piperidinyl)‐N‐(2‐fluorophenyl)propionamide (2,2â€Č‐difluorofentanyl). Identification was based on ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight–mass spectrometry (UHPLC–QTOF–MS), gas chromatography–mass spectrometry (GC–MS), Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. To our knowledge, this study is the first to report on analytical data for this compound. The most abundant fragment ion in the electrospray ionization (ESI) mass spectrum under collision‐induced dissociation (CID) mode was formed by the cleavage between the piperidine ring and the N‐phenyl‐amide moiety of the protonated molecule. Two diagnostic ions in the electron ionization (EI) mass spectrum were formed by the loss of a tropylium ion (M‐91), and by the degradation of the piperidine ring and dissociate of the COC2H5 moiety altogether, respectively. A fentanyl derivative N‐(1‐(2‐fluorophenethyl)piperidin‐4‐yl)‐N‐(2‐fluorophenyl)propionamide (2,2â€Č‐difluorofentanyl) was identified using UHPLC–QTOF–MS, GC–MS, FTIR, and NMR.

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Determination of GnRH and its synthetic analogues' abuse in doping control: Small bioactive peptide UPLC–MS/MS method extension by addition of in vitro and in vivo metabolism data; evaluation of LH and steroid profile parameter fluctuations as suitable biomarkers  Voir?

Gonadotropin‐releasing hormone (GnRH) and its small peptide synthetic analogues are included in Section S2 of the World Anti‐Doping Agency (WADA) Prohibited List as they stimulate pituitary luteinizing hormone (LH) and testicular testosterone (T) secretion. Both the following approaches can be applied for determination of abuse of these peptides: direct identification of intact compounds and their metabolites in athletes' biofluids and evaluation of LH and T concentrations as mediate markers of drug intake. To develop an effective concept for GnRH and its analogues determination in anti‐doping control, in vitro and in vivo studies were conducted. A new method was applied to the evaluation of the slow‐release profile of buserelin, goserelin, and leuprolide biodegradable microspheres after the intramuscular injection in male volunteers. Eight metabolites of 10 GnRH analogues were identified after incubation with human kidney microsomes, most of them were leuprolide degradation products. Obtained data were added into ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for GnRH analogues determination. The detection time windows for administered peptides and their metabolites in urine samples were evaluated with 2 sample preparation techniques: dilute‐and‐shoot and solid‐phase extraction. To support the second hypothesis, the measurement of LH and the main parameters of the steroid profile were performed in urine samples. Just 1 compound among those investigated resulted in the LH concentration dropping to non‐physiological levels. Thus, for doping‐control purposes, monitoring of hormone levels fluctuations could be applied only together with longitudinal passport steroid profile data. Approaches to detect GnRH and its analogues' abuse based on direct intact compounds along with their metabolites' identification and evaluation of biomarkers concentrations.

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Effect of alcohol dehydrogenase‐1B and ‐7 polymorphisms on blood ethanol and acetaldehyde concentrations in healthy subjects with a history of moderate alcohol consumption  Voir?

This study aims to evaluate the effect of ADH1B and ADH7 genotypes on blood acetaldehyde and ethanol levels after alcohol ingestion, and to measure the genotoxic effect of smoking and ethanol on the buccal cells, also controlling for ADH variants. We recruited healthy Italian subjects with at least a moderate history of alcohol consumption. All subjects were given an alcoholic drink of 0.4 g ethanol /kg of body weight. Blood venous samples were collected at baseline, and 30, 60, 90, and 120 minutes after ingestion. Buccal cells were collected before ethanol ingestion. Sixty subjects were enrolled in the study. Individuals with the ADH1B GG genotype had median ethanol levels of 5.0mM (IQR 3.4–7.2), and those with the ADH1B GT/TT genotype had 4.7mM (IQR 4.2–4.8). Corresponding acetaldehyde levels were 1.5ÎŒM (IQR 0.7–2.6) for ADH1B GG genotype and 1.6ÎŒM (IQR 1.5–1.7) for ADH1B CG/GG genotype. Individuals with the ADH7 CC genotype had median ethanol levels of 5.0mM (IQR 3.3–7.2), while 5.0mM (IQR 4.7–5.6) was in those with the ADH7 CG/GG genotype. Corresponding acetaldehyde levels were 1.5 ÎŒM (IQR 0.7–2.6) for ADH7 CC genotype and 1.5 ÎŒM (IQR 1.4–1.6) for ADH7 CG/GG genotypes. A non‐significant increase in the frequency of karyolitic and pyknotic cells was found in the group of heavy drinkers and current smokers, when compared to the moderate drinkers and the non‐smokers. Our study does not support the hypothesis that ADH1B and ADH7 genotypes affect blood ethanol and acetaldehyde concentration. This study aims to evaluate the effect of ADH1B and ADH7 genotypes on blood acetaldehyde and ethanol levels after alcohol ingestion, and to measure the genotoxic effect of smoking and ethanol on the buccal cells, also controlling for ADH variants. We recruited healthy Italian subjects with at least a moderate history of alcohol consumption. Our study does not support the hypothesis that ADH1B and ADH7 genotypes affect blood ethanol and acetaldehyde concentration.

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Recent advances in analytical methods for the therapeutic drug monitoring of immunosuppressive drugs  Voir?

Therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs) with a narrow therapeutic index is an increasingly popular tool for minimizing drug toxicity while maximizing the prevention of graft loss and organ rejection. This review focuses on trends regarding analytical methods for the TDM of ISDs since 2011. The five most commonly prescribed immunosuppressive medications are critically reviewed: cyclosporine A, tacrolimus, sirolimus (rapamycin), everolimus, and mycophenolic acid. This review introduces the general background of TDM and ISDs and presents the recent developments in using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and immunoassays for the TDM of ISDs. Finally, a future perspective for these analytical methods is briefly discussed. Therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs) with a narrow therapeutic index is an increasingly popular tool for minimizing drug toxicity while maximizing the prevention of graft loss and organ rejection. This review focuses on trends regarding analytical methods for theTDMof ISDs since 2011.

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Kinetic and metabolic profiles of synthetic cannabinoids NNEI and MN‐18  Voir?

In 2014 and 2015, synthetic cannabinoid receptor agonists NNEI (N‐1‐naphthalenyl‐1‐pentyl‐1H‐indole‐3‐carboxamide) and MN‐18 (N‐1‐naphthalenyl‐1‐pentyl‐1H‐indazole‐3‐carboxamide) were detected in recreationally used and abused products in multiple countries, and were implicated in episodes of poisoning and toxicity. Despite this, the pharmacokinetic profiles of NNEI and MN‐18 have not been characterized. In the present study NNEI and MN‐18 were incubated in rat and human liver microsomes and hepatocytes, to estimate kinetic parameters and to identify potential metabolic pathways, respectively. These parameters and pathways were then examined in vivo, via analysis of blood and urine samples from catheterized male rats following intraperitoneal (3 mg/kg) administration of NNEI and MN‐18. Both NNEI and MN‐18 were rapidly cleared by rat and human liver microsomes, and underwent a range of oxidative transformations during incubation with rat and human hepatocytes. Several unique metabolites were identified for the forensic identification of NNEI and MN‐18 intake. Interestingly, NNEI underwent a greater number of biotransformations (20 NNEI metabolites versus 10 MN‐18 metabolites), yet parent MN‐18 was eliminated at a faster rate than NNEI in vivo. Additionally, in vivo elimination was more rapid than in vitro estimates. These data highlight that even closely related synthetic cannabinoids can possess markedly distinct pharmacokinetic profiles, which can vary substantially between in vitro and in vivo models. Synthetic cannabinoids NNEI and MN‐18 have been implicated in episodes of poisoning and toxicity, but the pharmacokinetic profiles of these compounds have not been determined. The metabolic profiles of NNEI and MN‐18 are evaluated and compared, revealing marked differences in pharmacokinetics between these structurally related compounds.

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Determination of higenamine and coclaurine levels in human urine after the administration of a throat lozenge containing Nandina domestica fruit  Voir?

Higenamine is a key component of traditional Chinese herbal medicine. The fruit of Nandina domestica (which contains this component) is available as an ingredient in the so‐called Nanten‐nodo‐ame throat lozenge found on the Japanese market, which is an over‐the‐counter pharmaceutical and is easy to purchase for Japanese athletes. However, higenamine is a non‐selective ÎČ2‐agonist, which is exemplified in the prohibited list of the World Anti‐Doping Agency (WADA). Therefore, some have raised a concern regarding the potential cause of increased unintentional higenamine doping cases in the Asian region. This study aimed to investigate components of throat lozenges and develop a mass‐spectrometry method for the quantification of higenamine and coclaurine in human urine. Moreover, a population study of Japanese subjects (n = 246) and an excretion study (n = 4) of the corresponding throat‐lozenge recipients were performed to test the applicability of the current reporting threshold (i.e., 10 ng/mL) of higenamine set by WADA. The estimates of higenamine and coclaurine were 2.2 ± 0.1 ÎŒg/drop (mean of n = 12) and 0.5 ± 0.01 ÎŒg/drop (mean of n = 12), respectively. The maximum concentrations of higenamine and coclaurine were 0.2–0.4 and 0.3–1.0 ng/mL, respectively, at 10–12 h after administration of higenamine (nine drops); however, the concentrations in all four volunteers did not reach the positivity criterion of 10 ng/mL. No higenamine and coclaurine could be detected in the Japanese subjects. Therefore, there is no risk of detecting unintentional higenamine doping when the WADA reporting threshold is used. Higenamine is a banned ÎČ2‐agonist in sports. The fruit of Nandina domestica (which contains this component) is available as an ingredient in a throat lozenge found on the market. The maximum concentrations of higenamine were 0.2–0.4 ng/mL after administration of the throat lozenge, which did not reach the positivity criterion of 10 ng/mL. There is no risk of detecting unintentional higenamine doping when the WADA reporting threshold of 10 ng/mL is used.

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Potential impact of the sewer system on the applicability of alcohol and tobacco biomarkers in wastewater‐based epidemiology  Voir?

Understanding the actual consumption of alcohol and tobacco in the population is important for forming public health policy. For this purpose, wastewater‐based epidemiology has been applied as a complementary method to estimate the overall alcohol and tobacco consumption in different communities. However, the stability of their consumption biomarkers – ethyl sulfate, ethyl glucuronide, cotinine, and trans‐3â€Č‐hydroxycotinine – in the sewer system has not yet been assessed. This study aimed to conduct such assessment using sewer reactors mimicking conditions of rising main, gravity sewer, and wastewater alone, over a 12‐hour period. The results show that cotinine and trans‐3â€Č‐hydroxycotinine are relatively stable under all sewer conditions while ethyl sulfate was only stable in wastewater alone and gradually degraded in rising main and gravity sewer conditions. Ethyl glucuronide quickly degraded in all reactors. These findings suggest that cotinine and trans‐3â€Č‐hydroxycotinine are good biomarkers to estimate tobacco consumption; ethyl sulfate may be used as a biomarker to estimate alcohol consumption, but its in‐sewer loss should be accounted for in the calculation of consumption estimates. Ethyl glucuronide, and probably most of glucuronide compounds, are not suitable biomarkers to be used in wastewater‐based epidemiology due to their in‐sewer instability. Tobacco consumption can be measured with high confidence through wastewater‐based epidemiology, as its biomarkers are stable in the sewer system while monitoring alcohol consumption is more difficult because its biomarkers degrade rapidly in‐sewer.

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Confirmation of recent heroin abuse: Accepting the challenge  Voir?

Confirmation or exclusion of recent heroin consumption is still one of the major challenges for forensic and clinical toxicologists. A great variety of biomarkers is available for heroin abuse confirmation, including various opium alkaloids (eg, morphine, codeine), street heroin impurities (eg, 6‐acetylcodeine [6‐AC], noscapine, papaverine) as well as associated metabolites (eg, 6‐monoacetylmorphine [6‐MAM], morphine glucuronides). However, the presence of most of these biomarkers cannot solely be attributed to a previous heroin administration but can, among other things, also be due to consumption of poppy seed products (‘poppy seed defense'), opium preparations or specific medications, respectively. A reliable allocation is of great importance in different contexts, for instance in the case of DUID (driving under the influence of drugs) investigations, in driving licence re‐granting processes, in workplace drug testing (WDT), as well as in post‐mortem identification of illicit opiate use. Additionally, differentiation between illicit street heroin abuse and pharmaceutical heroin administration is also important, especially within the frame of heroin‐assisted treatments. Therefore, analysis of multiple biomarkers is recommended when illicit opiate consumption is assumed to obtain the most reliable results possible. Beyond that, interpretation of positive opiate test results requires a profound insight into the great variety of biomarkers available and their validity regarding the alleged consumption. This paper aims to provide an overview of the wide variety of heroin abuse biomarkers described in the literature and to review them regarding their utility and reliability in daily routine analysis. Confirmation or exclusion of recent heroin consumption is still one of the major challenges for forensic and clinical toxicologists. A great variety of biomarkers is available for heroin abuse confirmation, including various opium alkaloids, street heroin impurities, as well as associated metabolites. This paper aims to provide an overview of the wide variety of heroin abuse biomarkers described in the literature and to review them regarding their utility and reliability in daily routine analysis.

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Alkaloid profiling of herbal drugs using high resolution mass spectrometry  Voir?

Herbal infusions are consumed worldwide thanks to their “natural” beneficial effects, also due to the presence of alkaloids, although these compounds can have poisonous effects. A method combining online solid‐phase purification with high resolution mass spectrometry was used to define the alkaloid profiles of 117 herbs and 7 commercial blends. Forty‐one alkaloids were quantified in reference to analytical standards, while the presence of a further 116 was confirmed based on accurate mass, retention time, and fragmentation profile. The targeted study showed that 52% of herbs and 42% of commercial blends contained at least one alkaloid. Pyrrolizidines were the most commonly present (26% of samples), with concentrations generally ranging from the quantification limit to roughly 100 ÎŒg kg−1. Moreover, a homemade infusion was studied, finding on average 45% and 6% lower extraction for pyrrolizidine and steroidal alkaloids, respectively. Nevertheless, the migration of pyrrolizidines was confirmed. The study confirmed the frequent presence, natural or accidental, of alkaloids in commercial infusion herbs, highlighting the urgent need for routine and accurate controls. In this work we screened more than 120 commercial herbal teas, studying 41 targeted and 116 untargeted alkaloids, including acridinones, acridone, benzophenantridines, glycosteroidals, indoles, isoquinolines, piperidines, piridines, protoalkaloids, purines, pyrrolidines, pyrrolizidines, quinolines, quinolizidines, steroidals, terpenoids and tropanes. The study, performed using liquid chromatography coupled with high resolution mass spectrometry, aimed to evaluate alkaloid migration in homemade hot water infusions.

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Power of Orbitrap‐based LC‐high resolution‐MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSn or GC–MS procedures  Voir?

Reliable, sensitive, and comprehensive urine screening procedures by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) with low or high resolution (HR) are of high importance for drug testing, adherence monitoring, or detection of toxic compounds. Besides conventional urine sampling, dried urine spots are of increasing interest. In the present study, the power of LC–HR–MS/MS was investigated for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSn or GC–MS procedures. Authentic human urine samples (n = 103) were split in 4 parts. One aliquot was prepared by precipitation (UP), one by UP with conjugate cleavage (UglucP), one spot on filter paper cards and prepared by on‐spot cleavage followed by liquid extraction (DUSglucE), and one worked‐up by acid hydrolysis, liquid–liquid extraction, and acetylation for GC–MS analysis. The 3 series of LC–HR–MS/MS results were compared among themselves, to corresponding published LC–MSn data, and to screening results obtained by conventional GC–MS. The reference libraries used for the 3 techniques contained over 4500 spectra of parent compounds and their metabolites. The number of all detected hits (770 drug intakes) was set to 100%. The LC–HR–MS/MS approach detected 80% of the hits after UP, 89% after UglucP, and 77% after DUSglucE, which meant over one‐third more hits in comparison to the corresponding published LC–MSn results with ≀49% detected hits. The GC–MS approach identified 56% of all detected hits. In conclusion, LC–HR–MS/MS provided the best screening results after conjugate cleavage and precipitation. This study investigated the screening power of a new LC‐HR‐MS/MS approach for comprehensive urine drug testing in comparison to established LC‐MSn or GC‐MS procedures using over 100 authentic human urine samples. In conclusion, the new approach provided the best screening results after conjugate cleavage and simple precipitation.

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Study of the in vitro and in vivo metabolism of the tryptamine 5‐MeO‐MiPT using human liver microsomes and real case samples  Voir?

The synthetic tryptamine 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (5‐MeO‐MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5‐MeO‐MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5‐MeO‐MiPT using liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC–MS), LC‐HRMS/MS, and nuclear magnetic resonance (NMR). 5‐MeO‐MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5‐MeO‐MiPT in urine. Seven different in vitro phase I metabolites of 5‐MeO‐MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5‐MeO‐MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5‐methoxy‐N‐isopropyltryptamine (5‐MeO‐NiPT), 5‐hydroxy‐N‐methyl‐N‐isopropyltryptamine (5‐OH‐MiPT), 5‐methoxy‐N‐methyl‐N‐isopropyltryptamine‐N‐oxide (5‐MeO‐MiPT‐N‐oxide), and hydroxy‐5‐methoxy‐N‐methyl‐N‐isopropyltryptamine (OH‐5‐MeO‐MiPT) as biomarkers for the development of new methods for the detection of 5‐MeO‐MiPT consumption, as they have been present in both blood and urine samples. The present study investegates the in vitro and in vivo metabolism of the synthetic tryptamine 5‐MeO‐MiPT. In vitro seven different Phase |metabolites were detectable. In vivo results showed that in blood four different in urine seven phase | metabolites were present. Metabolic pathways were postulated and biomarkers proposed for the development of new methods for the detection of 5‐MeO‐MiPT in urine and blood samples.

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A multi‐analyte approach to help in assessing the severity of acute poisonings – Development and validation of a fast LC–MS/MS quantification approach for 45 drugs and their relevant metabolites with one‐point calibration  Voir?

Diagnosis, monitoring of the efficiency of detoxification, and estimating the prognosis of acute poisonings are important tasks in emergency toxicology. Comprehensive screening and quantification of relevant substances by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) help in assessing the severity of most acute poisonings. Turnaround time for such analyses must be short enough to impact on clinical decisions. Therefore, a multi‐analyte LC–MS/MS approach with a 5‐minute gradient was developed and validated for 45 drugs and their active metabolites as a complement to an existing GC–MS approach using the same liquid–liquid extraction. The determination ranges were defined by quality control samples of low and high, representing concentrations from low therapeutic to highly toxic levels. To shorten the turnaround time, one‐point calibration was used. Validation showed low matrix effects and ionization effects of co‐eluting analytes thanks to APCI source as well as sufficient recoveries, precisions, and selectivities. For accuracy, 32 of the 45 compounds fulfilled the criteria for quantification in lower therapeutic and 41 in overdosed and toxic concentrations, considering limits of ±30% deviation. The reuse of the processed calibrator for a period of 30 days was possible for 32 compounds, showing sufficient stability at 8°C. In addition, analysis of authentic blood samples showed the applicability and yielded drug levels, which were comparable to those determined by fully validated therapeutic drug monitoring methods. In conclusion, the present approach in combination with the GC–MS approach should provide sufficient support for clinical assessment of the severity of poisonings with 68 compounds in an acceptable turnaround time. In emergency toxicology, fast toxicological screening and blood levels of relevant drugs can support diagnosis of poisonings, monitoring efficiency of detoxification, and assessing prognosis. This study presents a validated LC‐MS/MS approach for quantification of 45 drugs and active metabolites with one‐point calibration. In conclusion, it should be sufficient to help in assessing the severity of corresponding poisonings in context of emergency toxicology.

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Validation of an LC–MS/MS method for analysis of anti‐diabetic drugs in botanical dietary supplements labeled for blood sugar management  Voir?

We developed and validated a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to detect and quantitate 14 anti‐diabetic, 2 anti‐obesity, and 3 cholesterol‐lowering drugs in botanical dietary supplements marketed for blood sugar management. Many botanical dietary supplements which carry label statements related to blood sugar management are available over the Internet. Potential adulteration of such dietary supplements with anti‐diabetic and other prescription drugs, some of which have been removed from the market due to adverse events, is of concern. No significant matrix effects were observed and mean recoveries of all 19 analytes from a single product matrix were 88 to 113% at spiking concentrations from 500 to 2000 ÎŒg/g. Mean recoveries of metformin, phenformin, and sibutramine from matrices prepared from multiple product composites ranged from 93 to 115% at a spiking concentration of 100 ÎŒg/g. The relative standard deviations (RSD) (%) of intra‐day analyses ranged from 0.2 to 13 for all recovery studies. Eighty dietary supplements obtained in the USA and carrying label statements related to blood sugar management were analyzed using this method and none were found to be adulterated with the above 19 drugs. Two products obtained outside of the USA and known to be adulterated were also analyzed by this method and found to contain phenformin, glibenclamide, and sibutramine. This method provided satisfactory selectivity, linearity, accuracy, precision, and sensitivity for rapid determination of 19 drugs and has broad applicability for the analysis of dietary supplements for possible adulteration with these compounds. Eighty (80) dietary supplements carrying label statements for blood sugar management were analyzed by a validated LC–MS/MS method for the presence of 14 anti‐diabetic, 2 anti‐obesity and 3 cholesterol‐lowering drugs. None of the potential adulterants were found in any of the products. Two additional products known to be adulterated with pharmaceuticals were also analyzed and found to contain mg/g quantities of the analytes of interest. The method is applicable to the determination of potential adulterants in dietary supplements.

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In‐source collision‐induced dissociation (IS‐CID): Applications, issues and structure elucidation with single‐stage mass analyzers  Voir?

A discussion of the definition, advantages, and issues with the formation of ions in the transition region between an electrospray ionization (ESI) source and the ion optics of a mass analyzer is presented. The various types of ions formed in the so‐called in‐source collision‐induced dissociation (IS‐CID) process are illustrated. Applications of IS‐CID with single‐stage mass analyzers, such as structure elucidation and quantitation, are demonstrated. The discussion is illustrated by examples of the in‐source fragmentation of ginkgolides, which are marker compounds found only in Ginkgo biloba. Supercritical fluid chromatography (SFC) with non‐aqueous eluents was used to achieve a fast resolution of the ginkgolides without the hydrolysis reactions possible with aqueous high‐performance liquid chromatography (HPLC) eluents. In‐source ion generation occurs at relatively high pressures (ca. 1–3 torr) compared to the low pressure normally observed in collision chambers of tandem mass spectrometry (MS/MS). As a result, the fragmentation process is complex and often generates ions other than the fragments observed with classic CID or the same ions at different intensities. The objective of the current tutorial is to illustrate the conditions under which single‐stage, quadrupole or time‐of‐flight mass analyzers with electrospray or in‐air (direct analysis in real time; DART) ionization can be used for quantitation and structure elucidation in a manner similar to that observed with MS/MS. While the low m/z (≀ [M±H]±) ions formed in‐source often duplicate the ions observed in MS/MS systems, it is the focus of this discussion to illustrate the utility of in‐source generated fragment ions that may not be observed or observed at different intensities than in the collision cells of MS/MS instruments. A discussion of the definition, advantages and issues with the generation of ions in the transition region between an ESI source and the ion optics of a mass analyzer is presented. The various types of ions formed in the so‐called in‐source collision induced dissociation (IS‐CID) process are illustrated. Applications of IS‐CID ions with single‐stage mass analyzers, such as structure elucidation and quantitation, are demonstrated.

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Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography–high resolution mass spectrometry  Voir?

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography–high‐resolution tandem mass spectrometry (LC–HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC–HRMS/MS system consisted of a YMC‐UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using ÎČ‐glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono‐hydroxy metabolites, and mono‐hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono‐hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2–19‐fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites. This study investigated the human metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam, and suggested analytical targets for urine drug testing. Besides the parent compounds, main excretion products were parent glucuronides, mono‐hydroxy metabolites, and mono‐hydroxy glucuronides. Without hydrolysis, the most common flubromazolam metabolites were one mono‐hydroxy glucuronide and one parent glucuronide; for pyrazolam, a parent glucuronide was most common. With hydrolysis, the flubromazolam concentration increased 2‐19‐fold, and flubromazolam hydroxy metabolites were considered the primary targets for urine drug testing.

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Heroin in Malaysia and Singapore  Voir?

Clandestine heroin laboratories have been a feature of the Malaysian illicit drug scene since soon after the abuse of heroin emerged in 1972. The first few clandestine heroin laboratories which synthesised heroin via the acetylation of imported morphine were uncovered in 1973 and 1977. By the mid‐1980s, this type of laboratory was replaced by heroin‐cutting laboratories whereby imported high‐grade heroin was cut to street heroin. This was to meet the rising demand for the drug owing to the rapid escalation of the number of drug users. Over the years, the most significant change in the composition of the street heroin is the decrease in its purity from 30%–50% to 3%–5%. Caffeine has remained the major adulterant and chloroquine is detected in virtually all recent seizures. A mini‐review of the two types of clandestine heroin laboratories in Malaysia following the emergence of heroin abuse in Malaysia and Singapore in 1972, and the profile of street heroin most commonly encountered in both countries.

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Silibinin affects the pharmacokinetics of methadone in rats  Voir?

The aim of the present study was to investigate the pharmacokinetic effect of silibinin on methadone in rats. Twenty‐four male Sprague–Dawley rats were randomly divided into 4 groups: control group, single dose of 100 mg/kg group, multiple doses of 100 mg/kg group, and multiple doses of 30 mg/kg group. A single dose of 6 mg/kg methadone was administrated to rats orally without or with silibinin. Plasma samples were collected via tail vein at different time points and concentrations of methadone and its metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), were determined by ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Compared with the control group (without silibinin), both 30 and 100 mg/kg silibinin significantly increased the Cmax of methadone, but only 100 mg/kg silibinin significantly increased the AUC(0‐t) of methadone and decreased its clearance. Pharmacokinetics parameters of EDDP were not altered by 30 mg/kg silibinin; its Tmax was decreased by 100 mg/kg silibinin and the Cmax was increased by single dose of 100 mg/kg silibinin. It is concluded that silibinin significantly altered the pharmacokinetics of methadone in rats by increasing the exposure of methadone. Further investigations in human should be conducted. Therapeutic drug monitoring of methadone in individuals undergoing methadone maintenance therapy is recommended when silibinin is concomitant. Both 30 and 100 mg/kg silibinin significantly increased the Cmax of methadone, but only 100 mg/kg silibinin significantly increased the AUC(0‐t) of methadone and decreased its clearance. It's concluded that silibinin significantly altered the pharmacokinetics of methadone in rats by increasing the exposure of methadone. More attention should be paid to side effects of methadone and therapeutic drug monitoring was recommended when silibinin is concominant.

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Detection of metabolites of the new synthetic cannabinoid CUMYL‐4CN‐BINACA in authentic urine samples and human liver microsomes using high‐resolution mass spectrometry  Voir?

CUMYL‐4CN‐BINACA(1‐(4‐cyanobutyl)‐N‐(2‐phenylpropan‐2‐yl)‐1H–indazole‐3‐carboxamide) is a recently introduced indazole‐3‐carboxamide‐type synthetic cannabinoid (SC) that was detected in herbal incense seized by of the Council of Forensic Medicine, Istanbul Narcotics Department, in May 2016 in Turkey. Recently introduced SCs are not detected in routine toxicological analysis; therefore, analytical methods to measure these compounds are in demand. The present study aims to identify urinary marker metabolites of CUMYL‐4CN‐BINACA by investigating its metabolism in human liver microsomes and to confirm the results in authentic urine samples (n = 80). In this study, 5 ÎŒM CUMYL‐4CN‐BINACA was incubated with human liver microsomes (HLMs) for up to 3 hours, and metabolites were identified using liquid chromatography–high‐resolution mass spectrometry (LC–HRMS). Less than 21% of the CUMYL‐4CN‐BINACA parent compound remained after 3 hours of incubation. We identified 18 metabolites that were formed via monohydroxylation, dealkylation, oxidative decyanation to aldehyde, alcohol, and carboxylic acid formation, glucuronidation or reaction combinations. CUMYL‐4CN‐BINACA N‐butanoic acid (M16) was found to be major metabolite in HLMs. In urine samples CUMYL‐4CN‐BINACA was not detected; CUMYL‐4CN‐BINACA N‐butanoic acid (M16) was major metabolite after ÎČ‐glucuronidase hydrolysis. Based on these findings, we recommend using M16 (CUMYL‐4CN‐BINACA N‐butanoic acid), M8 and M11 (hydroxylcumyl CUMYL‐4CN‐BINACA) as urinary marker metabolites to confirm CUMYL‐4CN‐BINACA intake. CUMYL‐4CN‐BINACA was detected in herbal incense seized by of the Council of Forensic Medicine, in May 2016 in Turkey. Recently introduced SCs are not detected in routine toxicological analysis; therefore, analytical methods to measure these compounds are in demand. This study aims to identify urinary marker metabolites of CUMYL‐4CN‐BINACA by investigating its metabolism in human liver microsomes and authentic urine samples (n = 80). The study provides the first information about the metabolism of CUMYL‐4CN‐BINACA in vitro and in vivo.

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Development and validation of an optical biosensor for rapid monitoring of adalimumab in serum of patients with Crohn's disease  Voir?

Therapeutic drug monitoring of adalimumab is recommended to improve therapeutic outcome in patients with Crohn's disease. Performing an ELISA requires a rather long time‐to‐result and the necessity of collecting multiple samples to decrease the cost per adalimumab determination. In this study, we aim to develop and validate a rapid assay suitable for measuring a single adalimumab serum sample using a fiber‐optic surface plasmon resonance (FO‐SPR) based sensor. Therefore, we have immobilized MA‐ADM28B8 as capture antibody on an FO‐probe and conjugated MA‐ADM40D8 as detecting antibody to gold nanoparticles. A dose–response curve ranging from 2.5 to 40 ng/mL adalimumab was obtained in 1/400 diluted serum. Serum samples of patients with adalimumab concentrations between 1 and 16 ÎŒg/mL were measured whereas the negative control, a sample spiked with infliximab at a concentration of 16 ÎŒg/mL, showed no significant signal. Using a pre‐functionalized FO‐probe, the technology requires less than 45 minutes for measuring a single sample. Comparison of measurements between the biosensor and the ELISA revealed an excellent agreement with a Pearson r coefficient of 0.99 and an intra‐class coefficient of 0.99. The reduced assay time and the possibility of measuring a single sample are major advantages compared to the ELISA. The developed and validated optical adalimumab biosensor could be a valuable point‐of‐care diagnostic tool for adalimumab quantification in patients with Crohn's disease. An adalimumab optical biosensor was developed to detect adalimumab concentrations ranging from 1 to 16 ÎŒg/mL in single serum samples within 45 min. Validation of the adalimumab biosensor revealed an excellent agreement with a reference ELISA. As a potential point‐of‐care diagnostic tool, the adalimumab biosensor could facilitate therapeutic drug monitoring in daily clinical practice.

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UPLC–MS/MS method for therapeutic drug monitoring of 10 antibiotics used in intensive care units  Voir?

A large variation in the levels of different àŸâ€lactams and other antibiotics used in critically ill patients has been documented. The aim of this study is to establish and validate a fast, ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous analysis of ten antibiotics (Meropenem, Cefepime, Ceftazidime, Piperacillin, Benzylpenicillin, Ampicillin, Flucloxacillin, Linezolid, and Sulfamethoxazole/Trimethoprim) in human plasma according to European Medicines Agency (EMA) guidelines. Protein precipitation with ice‐cold methanol containing 9 isotopically labeled internal standards was used for sample clean up. Antibiotics were detected, following a 4‐minute gradient separation, in multiple reactions monitoring (MRM) using API 4000 instrument equipped with electrospray source operating in positive ion mode. The lower limit of quantification was 0.1 mg/L for Meropenem, Ceftazidime, Piperacillin, Ampicillin, Flucloxacillin, and Sulfamethoxazole; 0.05 mg/L for Cefepime, Benzylpenicillin, and Trimethoprim; and 0.02 mg/L for Linezolid. The method proved to be precise and accurate and applicable for therapeutic drug monitoring and other pharmacokinetic studies. A robust and fast ultra‐performance liquid chromatography tandem mass spectrometry method for the simultaneous analysis of Meropenem, Cefepime, Ceftazidime, Piperacillin, Benzylpenicillin, Ampicillin, Flucloxacillin, Linezolid, and Sulfomethoxazole/Trimethoprim is developed and validated. Sample preparation consists of a simple protein precipitation, using 9 isotopically labeled internal standards, followed by dilution in water. The method is fast, precise and accurate. The method is suitable for therapeutic drug monitoring as well as for pharmacokinetic studies.

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Discrimination between closely related synthetic cannabinoids by GC–Cold–EI–MS  Voir?

Gas chromatography thermal‐electron ionization mass spectrometry (GC–EI–MS) is an established method for the identification of mind‐altering substances and is routinely used by forensic laboratories. However, some commonly analyzed drugs of abuse, relating to the synthetic cannabinoids receptor agonist group (SCs), pose a challenge for this conventional technique. As the molecular cation radicals of many excited SCs are labile within the ion source, the relative abundance of the molecular ions obtained by the GC‐EI‐MS is often too small to allow discrimination of structurally related drugs. In contrast, the cold‐electron ionization (cold‐EI) method allows the enhancement and clear identification of the molecular ions, while maintaining the ability to compare unknown analytes with comprehensive mass spectrum libraries. This technique was explored for mass‐spectrometric identification and unambiguous differentiation of 15 emerging synthetic cannabinoids found on the drug market in Israel and elsewhere. The current method was demonstrated to discriminate pairs of closely related SCs: FUB‐PB‐22 and FDU‐PB‐22, and 5F–PB‐22 and NM‐2201. In addition, the dependence of the molecular ion enhancement on the cold‐EI parameters was examined. Finally, analysis of SCs from seized street samples provided by the Israeli police demonstrates the enhanced identification power of GC–cold–EI–MS. GC‐Cold‐EI‐MS method was applied to the analysis of 15 synthetic cannabinoids reported on the global drug market. Accurate identification and discrimination between closely related substances was achieved through enhancement of the molecular peaks. The ability to compare unknown analytes with comprehensive mass spectrum libraries was maintained in this method. Analysis of SCs from police seizures demonstrated the applicability of the cold‐EI‐MS.

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Determination of testosterone esters in the hair of male greyhound dogs using liquid chromatography–high resolution mass spectrometry  Voir?

The doping of greyhound dogs with testosterone is done in an attempt to improve their athletic performance, but such doping cannot easily be confirmed, especially in male dogs owing to the natural presence of endogenous testosterone. As testosterone is usually administered as its esters, their direct detection in hair would provide confirmatory evidence of the administration of a pharmaceutical product. This article demonstrates that the use of a liquid chromatography–high resolution mass spectrometry method with heated electrospray ionisation (HESI) combined with the use of amino solid‐phase extraction (SPE) cartridges for sample clean‐up, is suitable for the sensitive determination of propionate, phenyl propionate, isocaproate, decanoate, and enanthate esters of testosterone in greyhound hair. The method is linear over the range, 0.1 ÎŒg/kg–10 ÎŒg/kg, for all the testosterone esters analysed. The limits of detection (LOD) are 0.05 ÎŒg/kg for testosterone phenyl propionate, isocaproate, and decanoate, 0.025 ÎŒg/kg for testosterone propionate, and 0.25 ÎŒg/kg for testosterone enanthate. This method was applied to hair samples collected from male greyhounds before and after a single administration of a product containing several testosterone esters, each of which could be detected up to 100 days post‐administration. The study also demonstrates that tail hair is the specimen of choice for the analysis of testosterone in dog hair and that washing of dogs does not impact the analysis of testosterone esters in hair. This method may be useful in racing regulation for the detection of illegitimate use of testosterone in all species. The study demonstrates that the use of a liquid chromatography‐high resolution mass spectrometry method with heated electrospray ionisation (HESI) combined with the use of amino SPE cartridges for sample clean up, improves the selectivity and sensitivity of the method for the determination of propionate, phenyl propionate, isocaproate, decanoate and enanthate esters of testosterone in greyhound hair. This method was applied to hair samples collected from male greyhounds before and after a single administration of a product containing several testosterone esters each of which could be detected up to 100 days post‐administration.

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Optimization of recombinant ÎČ‐glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry  Voir?

Prolonged urinary cannabinoid excretion in chronic frequent cannabis users confounds identification of recent cannabis intake that may be important in treatment, workplace, clinical, and forensic testing programs. In addition, differentiation of synthetic Δ9‐tetrahydrocannabinol (THC) intake from cannabis plant products might be an important interpretive issue. THC, 11‐hydroxy‐THC (11‐OH‐THC) and 11‐nor‐9‐carboxy‐THC (THCCOOH) urine concentrations were evaluated during previous controlled cannabis administration studies following tandem alkaline/E. coli ÎČ‐glucuronidase hydrolysis. We optimized recombinant ÎČ‐glucuronidase enzymatic urinary hydrolysis before simultaneous liquid chromatography tandem mass spectrometry (LC–MS/MS) quantification of THC, 11‐OH‐THC, THCCOOH, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabivarin (THCV) and 11‐nor‐9‐carboxy‐THCV (THCVCOOH) in urine. Enzyme amount, incubation time and temperature, buffer molarity and pH were optimized using pooled urine samples collected during a National Institute on Drug Abuse, Institutional Review Board‐approved clinical study. Optimized cannabinoid hydrolysis with recombinant ÎČ‐glucuronidase was achieved with 2000 IU enzyme, 2 M pH 6.8 sodium phosphate buffer, and 0.2 mL urine at 37°C for 16 h. The LC–MS/MS quantification method for hydrolyzed urinary cannabinoids was validated per the Scientific Working Group on Toxicology guidelines. Linear ranges were 1–250 ÎŒg/L for THC and CBG, 2–250 ÎŒg/L for 11‐OH‐THC, CBD, CBN, THCV and THCVCOOH, and 1–500 ÎŒg/L for THCCOOH. Inter‐batch analytical bias was 92.4–112.4%, imprecision 4.4–9.3% CV (n = 25), extraction efficiency 44.3–97.1% and matrix effect −29.6 to 1.8% (n = 10). The method was utilized to analyze urine specimens collected during our controlled smoked, vaporized, and edible cannabis administration study to improve interpretation of urine cannabinoid test results. The growing interest in multiple cannabinoids other than ∆9‐tetrahydrocannabinol emphasizes the need for analytical method to quantify a wide spectrum of urinary cannabinoids, and to optimize hydrolysis of glucuronide conjugates present in urine. We optimized recombinant ÎČ‐glucuronidase enzymatic urinary hydrolysis before simultaneous liquid chromatography tandem mass spectrometry quantification of eight cannabinoids and metabolites in urine.

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Investigation of drug products received for analysis in the Swedish STRIDA project on new psychoactive substances  Voir?

The web‐based open sale of unregulated new psychoactive substances (NPS) has shown a steady increase in recent years. Analysis of drug products sold as NPS is useful to confirm the true chemical contents, for comparison with the substances detected in corresponding body fluids, but also to study drug trends. This work describes the examination of 251 drug products that were randomly submitted for analysis in 173 cases of suspected NPS‐related intoxications in the Swedish STRIDA project in 2010–2015. Of the products, 39% were powders/crystals, 32% tablets/capsules, 16% herbal materials, 8% liquids, 1% blotters, and 4% others. The analysis involved tandem mass spectrometry and nuclear magnetic resonance spectroscopy. In 88 products (35%), classic psychoactive substances, prescription pharmaceuticals, dietary supplements, or doping agents were found; however, in none of these cases had an NPS‐related intoxication been indicated from product markings or patient self‐reports. Another 12 products tested negative for psychoactive substances. The remaining 151 products contained 86 different NPS (30% contained ≄2 substances). In 104 drug products, a specific NPS ingredient was indicated based on labelling (69%) or patient self‐report; in 92 cases this was also analytically confirmed to be correct. Overall, the NPS products submitted for analysis in the STRIDA project showed a high degree of consistency between suspected and actual content (88%). The results of related urine and/or blood analysis further demonstrated that the patients commonly (89%) tested positive for the indicated NPS, but also revealed that polysubstance intoxication was common (83%), indicating use of additional drug products. In 173 intoxications in the Swedish STRIDA project suspected to involve new psychoactive substances (NPS), 251 drug products were submitted for analysis of which 151 contained 86 different NPS. The content commonly (88%) agreed with that indicated from product markings or patient self‐reports. The results of related urine and/or blood analysis further demonstrated that the patients commonly (89%) tested positive for the indicated NPS, but also revealed that polysubstance intoxication was common (83%), indicating use of additional drugs.

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Structural characterization and pharmacological evaluation of the new synthetic cannabinoid CUMYL‐PEGACLONE  Voir?

The number of new psychoactive substances (NPS) that have emerged on the European market has been rapidly growing in recent years, with a particularly high number of new compounds from the group of synthetic cannabinoid receptor agonists. There have been various political efforts to control the trade and the use of NPS worldwide. In Germany, the Act to control the distribution of new psychoactive substances (NpSG) came into force in November 2016. In this new act, two groups of substances were defined, the group “cannabimimetics/synthetic cannabinoids” covering indole, indazole, and benzimidazole core structures, and a second group named “compounds derived from 2‐phenethylamine.” Shortly after, the first retailers of “herbal blends” promoted new products allegedly not violating the German NpSG. We describe the identification and structural elucidation of one of the first synthetic cannabinoids not being covered by the NpSG, 5‐pentyl‐2‐(2‐phenylpropan‐2‐yl)‐2,5‐dihydro‐1H‐pyrido[4,3‐b]indol‐1‐one. For isolation of the substance a flash chromatography separation was applied. The structure elucidation was performed using gas chromatography–mass spectrometry (GC–MS), gas chromatography‐solid state infrared spectroscopy (GC–sIR), liquid chromatography–electrospray ionization–quadrupole time of flight–mass spectrometry (LC–ESI–qToF–MS) and nuclear magnetic resonance (NMR) analysis. Additionally, binding affinity towards the cannabinoid receptors CB1 and CB2 and efficacy in a cAMP accumulation assay were measured, showing full agonistic activity and high potency at both receptors. The new compound bears a γ‐carboline core structure circumventing the German NpSG and the generic definitions in other national laws. As a semi‐systematic name for 2‐cumyl‐5‐pentyl‐gamma‐carbolin‐1‐one CUMYL‐PEGACLONE is suggested. This article describes the identification and structural elucidation of a new synthetic cannabinoid, 5‐pentyl‐2‐(2‐phenylpropan‐2‐yl)‐2,5‐dihydro‐1H‐pyrido[4,3‐b]indol‐1‐one. Additionally, the affinity of the new substance towards the cannabinoid receptors CB1 and CB2 and the efficacy in a cAMP accumulation assay were measured. The new compound bears a γ‐carboline core structure not covered by most generic legislative definitions of synthetic cannabinoids and presumably acts as a full agonist at the cannabinoid receptors. CUMYL‐PEGACLONE is suggested as a semi‐systematic name for the compound.

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Transcriptomic biomarkers of altered erythropoiesis to detect autologous blood transfusion  Voir?

Autologous blood transfusion is a powerful means of improving performance and remains one of the most challenging methods to detect. Recent investigations have identified 3 candidate reticulocytes genes whose expression was significantly influenced by blood transfusion. Using quantitative reverse transcription polymerase chain reaction as an alternative quantitative method, the present study supports that delta‐aminolevulinate synthase 2 (ALAS2), carbonic anhydrase (CA1), and solute carrier family 4 member 1 (SLC4A1) genes are down‐regulated post‐transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. Moreover, the repression of reticulocytes' gene expression was more pronounced than the diminution of immature reticulocyte fraction and reticulocyte percentage following blood transfusion. It suggests that the 3 candidate genes are reliable predictors of bone marrow's response to blood transfusion and that they represent potential biomarkers for the detection of this method prohibited in sports. Using quantitative reverse transcription polymerase chain reaction, the present study supports that ALAS2, CA1 and SLC4A1 genes are down‐regulated after an autologous blood transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. It suggests that the three candidate genes are reliable predictors of bone marrow's response to blood transfusion and that they represent potential biomarkers for the detection of this method prohibited in sports.

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The influence of small doses of ethanol on the urinary testosterone to epitestosterone ratio in men and women  Voir?

Endogenous steroid use can increase urinary testosterone/epitestosterone (T/E) values. In addition, ethanol in amounts >0.5 g per kg of body weight (g/kg) can also increase T/E values. However, the effect of smaller doses of ethanol on T/E values is unknown. The influence of 0.2 and 0.4 g/kg of ethanol on baseline T/E values in 20 men and 20 women with low and high baseline T/E values was investigated and correlated with ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations. T/E values for 7 of the women were excluded from the study because of undetectable T concentrations or for other reasons. One man and 1 woman with a high T/E baseline value had a significant increase in their T/E value after ingestion of 0.2 g/kg of ethanol. One man and 2 women with a high T/E baseline, and 1 woman with a low T/E baseline had significantly increased T/E values after ingestion of 0.4 g/kg of ethanol. There was wide variability in peak EtG concentrations and a lack of correlation between ethanol dose and EtG concentrations. Interestingly, 1 man and 2 women with increased T/E values following ethanol ingestion had EtG concentrations below the World Anti‐Doping Agency (WADA) cut‐off of 5000 ng/mL. These findings demonstrate that small amounts of ethanol can elevate T/E values, with women being more susceptible. In addition, consideration should be given to the lowering of the WADA EtG cut‐off to detect samples with elevated T/E values from ingestion of low doses of ethanol. The effect of low doses of ethanol on urinary T/E values in men and women with low and high baseline T/E values was investigated. Ethanol at 0.2 and 0.4 g/kg of body weight produced elevated T/E values in 18% of the participants, with women being more susceptible to this effect. There was wide variation in peak ethyl glucuronide concentrations and 50% of the participants with elevated T/E values had ethyl glucuronide concentrations <5 ÎŒg/mL.

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Profiling ephedrine prepared from N‐methylalanine via the Akabori‐Momotani reaction  Voir?

Novel methods for synthesising methylamphetamine precursors are appearing in clandestine laboratories within Australia. One such laboratory involved the synthesis of ephedrine from N‐methylalanine and benzaldehyde via the Akabori‐Momotani reaction. This article presents chiral and stable isotope ratios of ephedrine synthesised via this method, along with a chemical profile of methylamphetamine produced from this ephedrine. Based on the chiral results and the ή13C, ή15N, and ή2H values, it is possible to distinguish ephedrine made via the Akabori‐Momotani reaction from ephedrine of a “natural”, “semi‐synthetic”, or “fully‐synthetic” origin. Methylamphetamine and ephedrine samples synthesised from benzaldehyde having an enriched ή2H value (ie, > 0‰), via the Akabori‐Momotani reaction, had an isotopic profile which set them apart from all other methylamphetamine samples. It was noted, however, that using stable isotope ratios alone to determine the precursor of methylamphetamine is limited; they could not with confidence differentiate between methylamphetamine and ephedrine synthesised from benzaldehyde having a depleted ή2H value (ie, <0‰) from other ephedrine sources and phenyl‐2‐propanone based methylamphetamine samples profiled. Ephedrine made from N‐methylalanine and benzaldehyde via the Akabori‐Momotani reaction was chemically profiled to obtain carbon, nitrogen and hydrogen stable isotope ratios and chiral composition.

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The emergence of new psychoactive substance (NPS) benzodiazepines: A review  Voir?

The market for new psychoactive substances has increased markedly in recent years and there is now a steady stream of compounds appearing every year. Benzodiazepines consist of only a fraction of the total number of these compounds but their use and misuse has rapidly increased. Some of these benzodiazepines have only been patented, some of them have not been previously synthesised, and the majority have never undergone clinical trials or tests. Despite their structural and chemical similarity, large differences exist between the benzodiazepines in their pharmacokinetic parameters and metabolic pathways and so they are not easily comparable. As benzodiazepines have been clinically used since the 1960s, many analytical methods exist to quantify them in a variety of biological matrices and it is expected that these methods would also be suitable for the detection of benzodiazepines that are novel psychoactive substances. Illicitly obtained benzodiazepines have been found to contain a wide range of compounds such as opiates which presents a problem since the use of them in conjunction with each other can lead to respiratory depression and death. This review collates the available information on these benzodiazepines and provides a starting point for the further investigation of their pharmacokinetics which is clearly required. NPS benzodiazepines are increasingly being used and abused around the world. This paper critically reviews both the state of knowledge of the emerging NPS‐benzodiazepines and also the analytical methods that can be used to detect them in human body fluids.

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Non‐targeted acquisition strategy for screening doping compounds based on GC‐EI‐hybrid quadrupole‐Orbitrap mass spectrometry: A focus on exogenous anabolic steroids  Voir?

This is a first look at a non‐targeted screening method based on Orbitrap gas chromatography–mass spectrometry (GC–MS) technology for a large number of banned compounds in sports found in urine, including exogenous anabolic steroids, ÎČ‐agonists, narcotics, stimulants, hormone modulators, and diuretics. A simple sample preparation was processed in four steps: an enzymatic hydrolysis, liquid–liquid extraction, evaporation, and trimethylsilylation. All compounds were able to meet the World Anti‐Doping Agency's sensitivity criteria with mass accuracies less than 1 ppm and with sufficient points across the peak by running the Orbitrap GC–MS in full‐scan mode. In addition, we discuss our initial findings of using a full‐scan selected ion monitoring‐tandem mass spectrometry (SIM‐MS/MS) approach as a way to obtain lower detection limits and reach desirable selectivity for some exogenous anabolic steroids. By performing non‐targeted initial screening method in GC Orbitrap MS, the majority of compounds were able to meet WADA's sensitivity requirements with mass accuracies less than 1 ppm and sufficient points across the GC peaks. In addition, we will discuss our initial findings performing simultaneously full scan/MSMS/SIM analysis to overcome selectivity and sensitivivty issues.

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A step towards removing plasma volume variance from the Athlete's Biological Passport: The use of biomarkers to describe vascular volumes from a simple blood test  Voir?

The haematological module of the Athlete's Biological Passport (ABP) has significantly impacted the prevalence of blood manipulations in elite sports. However, the ABP relies on a number of concentration‐based markers of erythropoiesis, such as haemoglobin concentration ([Hb]), which are influenced by shifts in plasma volume (PV). Fluctuations in PV contribute to the majority of biological variance associated with volumetric ABP markers. Our laboratory recently identified a panel of common chemistry markers (from a simple blood test) capable of describing ca 67% of PV variance, presenting an applicable method to account for volume shifts within anti‐doping practices. Here, this novel PV marker was included into the ABP adaptive model. Over a six‐month period (one test per month), 33 healthy, active males provided blood samples and performed the CO‐rebreathing method to record PV (control). In the final month participants performed a single maximal exercise effort to promote a PV shift (mean PV decrease −17%, 95% CI −9.75 to −18.13%). Applying the ABP adaptive model, individualized reference limits for [Hb] and the OFF‐score were created, with and without the PV correction. With the PV correction, an average of 66% of [Hb] within‐subject variance is explained, narrowing the predicted reference limits, and reducing the number of atypical ABP findings post‐exercise. Despite an increase in sensitivity there was no observed loss of specificity with the addition of the PV correction. The novel PV marker presented here has the potential to improve the ABP's rate of correct doping detection by removing the confounding effects of PV variance. Plasma volume (PV) shifts represent a major confounding factor within the Athlete's Biological Passport (ABP) reference limit calculations for concentration‐based haematological markers, haemoglobin concentration ([Hb]) and the OFF‐score. A newly developed PV marker (comprising eight common chemistry variables, from a simple blood test) is capable of removing 66% of [Hb] within‐subject variance, narrowing the predicted reference limits for [Hb] and the OFF‐score and also allowing for PV fluctuations. This approach has the potential to improve the ABP's rate of correct doping detection by removing the confounding effects of PV variance.

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Prevalence of new psychoactive substances and prescription drugs in the Belgian driving under the influence of drugs population  Voir?

Driving under the influence of drugs (DUID) is a worldwide problem. Several countries have adopted DUID legislations which prove their deterrent effect and impact on road safety. However, the use of new psychoactive substances (NPS) and prescription drugs is not known, as the applied roadside screening tests have not yet been adapted for these compounds. In this study, 558 blood samples obtained during roadside controls in Belgium (January to August 2015) after a positive Drugwipe 5Sź test and 199 oral fluid (OF) samples obtained from negatively screened test pads were analyzed. The NPS positivity rate was 7% in blood, while it reached 11% in OF. NPS detected were: diphenidine, ketamine, 4‐fluoroamphetamine, 2‐amino‐indane, methoxetamine, α‐PVP, methiopropamine, a mix of 5‐MAPB/5‐EAPB, TH‐PVP, mephedrone, methedrone, 4‐methylethylcathinone, 5‐MeO‐DALT, 4‐Acetoxy‐DiPT, AB Fubinaca, FUB‐JWH018, JWH020, trifluoromethylphenylpiperazine, and ethylphenidate. Moreover, 17% of blood samples (and 5% of OF) contained an analgesic drug, 10% (0.5%) a benzodiazepine/hypnotic, 5% (2%) an antidepressant, 2% (3%) an antipsychotic, 2% an antiepileptic drug, and 1% methylphenidate. The presence of NPS in the young (and predominately male) DUID population is proven. Furthermore, a high level of poly‐drug use including combinations of NPS, licit, and drugs of abuse was observed. Further research concerning the development of on‐site NPS detection techniques should be established. Meanwhile, the effects of combined drug use on driving ability and the physical/psychological signs after NPS use should be performed to improve the on‐site DUID detection of NPS by police officers, so they can engage in blood sampling for a general unknown screening. Nearly 600 blood samples obtained during roadside controls in Belgium (2015) after a positive Drugwipe 5Sź test and 199 oral fluid (OF) samples obtained from negatively screened test pads were analyzed. The presence of poly‐drug use including combinations of NPS, licit, and classic illicit drugs in the young (and predominately male) driving population is proven with a positivity rate for NPS in 7% of the blood and 11% for OF samples collected from on‐site screening tests.

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A study of the origin of chloramphenicol isomers in honey  Voir?

Due to the unexpected detection of chloramphenicol isomer residues in honey, we have studied the hypothesis of unauthorized or unintended use of unregistered veterinary drug preparations. First, we have investigated honey samples in which a discrepancy was observed between the results of the immunological screening methods and the confirmatory liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. In all samples, previously identified to be contaminated with the banned antibiotic chloramphenicol according to LC–MS/MS only, the presence of dextramycin (SS‐para isomer of chloramphenicol) was detected by chiral LC–MS/MS. The source of dextramycin in honey was investigated by studying the preparations utilized in apiaries from which the above non‐compliant honey samples have been received. In all these preparations (beehive strips applied against the mite Varroa destructor) chloramphenicol was detected in the concentrations ranging from 33 to 34,400 ÎŒg kg−1. Chiral LC–MS/MS demonstrated the presence of chloramphenicol and dextramycin in different ratios, and it was concluded that these preparations can be the source of chloramphenicol and dextramycin residues in honey. These preparations were of foreign production and are not officially registered in accordance with current legislation. The Hypothesis of un authorized or unintended use of unregistered veterinary drug pre parations containing chloramphenicol isomer in apiaries has been studied. In honey samples, perviously identified to be contaminated with chloramphenicol according to LC–MS/MS only, the presence of dextramycin was detected by chiral LC–MS/MS. Chloramphenicol and dextramycin in different ratios have been found in beehive strips fo honey bees, and it was concluded that these preparations can be the source of chloramphenicol and dextramycin residues in honey.

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Return of the lysergamides. Part IV: Analytical and pharmacological characterization of lysergic acid morpholide (LSM‐775)  Voir?

Lysergic acid diethylamide (LSD) is perhaps one of the best‐known psychoactive substances and many structural modifications of this prototypical lysergamide have been investigated. Several lysergamides were recently encountered as ‘research chemicals' or new psychoactive substances (NPS). Although lysergic acid morpholide (LSM‐775) appeared on the NPS market in 2013, there is disagreement in the literature regarding the potency and psychoactive properties of LSM‐775 in humans. The present investigation attempts to address the gap of information that exists regarding the analytical profile and pharmacological effects of LSM‐775. A powdered sample of LSM‐775 was characterized by X‐ray crystallography, nuclear magnetic resonance spectroscopy (NMR), gas chromatography mass spectrometry (GC–MS), high mass accuracy electrospray MS/MS, high performance liquid chromatography (HPLC) diode array detection, HPLC quadrupole MS, and GC solid‐state infrared analysis. Screening for receptor affinity and functional efficacy revealed that LSM‐775 acts as a nonselective agonist at 5‐HT1A and 5‐HT2A receptors. Head twitch studies were conducted in C57BL/6J mice to determine whether LSM‐775 activates 5‐HT2A receptors and produces hallucinogen‐like effects in vivo. LSM‐775 did not induce the head twitch response unless 5‐HT1A receptors were blocked by pretreatment with the antagonist WAY‐100,635 (1 mg/kg, subcutaneous). These findings suggest that 5‐HT1A activation by LSM‐775 masks its ability to induce the head twitch response, which is potentially consistent with reports in the literature indicating that LSM‐775 is only capable of producing weak LSD‐like effects in humans. Lysergic acid morpholide (LSM‐775) appeared as a research chemical in 2013. An extensive analytical characterization is presented followed by pharmacological investigations. LSM‐775 acted as an agonist at the 5‐HT1A receptor and all three 5‐HT2 receptor subtypes. However, the head twitch response was not induced in C57BL/6J mice unless 5‐HT1A receptors were blocked by pretreatment with the antagonist WAY‐100,635. These findings are potentially consistent with reports indicating that LSM‐775 might only be capable of producing weak LSD‐like effects in humans.

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Metabolism of novel opioid agonists U‐47700 and U‐49900 using human liver microsomes with confirmation in authentic urine specimens from drug users  Voir?

Recently, the number of adverse events, including death, involving novel opioids has continued to increase, providing additional and sustained challenges for forensic and medical communities. Identification of emerging novel opioids can be challenging, compounded by detection windows and unknown metabolic profiles. In this study, human liver microsomes were used for the generation of in vitro metabolic profiles of U‐47700 and U‐49900. Generated metabolites were analyzed via a SCIEX TripleTOFź 5600+ quadrupole time‐of‐flight mass spectrometer and resulting data files were processing using MetabolitePilotℱ. Characterized metabolites were verified in vivo by analysis of authentic human urine specimens collected after analytically confirmed cases of overdose involving U‐47700 or U‐49900. In total, four metabolites were identified and present in urine specimens for U‐47700, and five metabolites for U‐49900. N‐Desmethyl‐U‐47700 was determined to be the primary metabolite of U‐47700. Parent U‐47700 was identified in all urine specimens. N‐Desmethyl‐U‐47700 and N,N‐didesmethyl‐U‐47700 were structurally confirmed for the first time during this study following acquisition of standard reference material. N‐Desethyl‐U‐49900 was determined to be the primary metabolite of U‐49900 following microsomal incubations, while N,N‐didesethyl‐N‐desmethyl‐U‐49900 was the most abundant in a urine specimen. Similarities in metabolic transformation were identified between U‐47700 and U‐49900, resulting in a common metabolite and isomeric species. These phenomena should be considered in cases involving U‐47700 or U‐49900. This study is the first to map the metabolic profiles of U‐47700 and U‐49900 using human liver microsomes, as well as the first to report any literature involving U‐49900 and analysis of case specimens. Recently, the number of adverse events, including death, involving novel opioids has continued to increase. In this study, human liver microsomes were used for the generation of in vitro metabolic profiles of U‐47700 and U‐49900. Characterized metabolites were verified in vivo by analysis of authentic human urine specimens collected after analytically confirmed cases of overdose involving U‐47700 or U‐49900.

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Syntheses, analytical and pharmacological characterizations of the ‘legal high' 4‐[1‐(3‐methoxyphenyl)cyclohexyl]morpholine (3‐MeO‐PCMo) and analogues  Voir?

New psychoactive substances (NPS) are commonly referred to as ‘research chemicals', ‘designer drugs' or ‘legal highs'. One NPS class is represented by dissociative anesthetics, which include analogues of the arylcyclohexylamine phencyclidine (PCP), ketamine and diphenidine. A recent addition to the NPS market was 4‐[1‐(3‐methoxyphenyl)cyclohexyl]morpholine (3‐MeO‐PCMo), a morpholine analogue of 3‐MeO‐PCP. Although suspected to have dissociative effects in users, information about its pharmacological profile is not available. From clinical and forensic perspectives, detailed analytical data are needed for identification, especially when facing the presence of positional isomers, as these are frequently unavailable commercially. This study presents the analytical and pharmacological characterization of 3‐MeO‐PCMo along with five additional analogues, namely the 2‐ and 4‐MeO‐PCMo isomers, 3,4‐methylenedioxy‐PCMo (3,4‐MD‐PCMo), 3‐Me‐PCMo and PCMo. All six arylcyclohexylmorpholines were synthesized and characterized using chromatographic, mass spectrometric and spectroscopic techniques. The three positional isomers could be differentiated and the identity of 3‐MeO‐PCMo obtained from an internet vendor was verified. All six compounds were also evaluated for affinity at 46 central nervous system receptors including the N‐methyl‐d‐aspartate receptor (NMDAR), an important target for dissociative anesthetics such as PCP and ketamine. In vitro binding studies using (+)‐[3‐3H]‐MK‐801 in rat forebrain preparations revealed moderate affinity for NMDAR in the rank order of 3‐Me >3‐MeO > PCMo >3,4‐MD > 2‐MeO > 4‐MeO‐PCMo. 3‐MeO‐PCMo was found to have moderate affinity for NMDAR comparable to that of ketamine, and had an approximate 12‐fold lower affinity than PCP. These results support the anecdotal reports of dissociative effects from 3‐MeO‐PCMo in humans. Syntheses, analytical and pharmacological characterizations of the ‘legal high' 4‐[1‐(3‐methoxyphenyl)cyclohexyl]morpholine (3‐MeO‐PCMo) and analogues are presented. 3‐MeO‐PCMo was differentiated from its 2‐MeO‐ and 4‐MeO‐PCMo positional isomers using multiple analytical techniques. All six arylcyclohexylmorpholines were found to be moderate‐affinity NMDA receptor antagonists, with some compounds having additional affinities for monoamine transporters including the dopamine and serotonin transporters.

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Assessing the toxicological significance of new psychoactive substances in fatalities  Voir?

The number of new psychoactive substances (NPS) has increased significantly, especially within the last 5 years. The EMCDDA conducts risks assessments of such substances, especially in relation to serious adverse events. Examination of the individual health risks of a substance is a fundamental requirement of the process. Based on a number of considerations, the Toxicological Significance Score has been developed to support the risk assessment of NPS by allowing the role of a substance in deaths to be better assessed and classified.

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An LC–MS/MS method for determination of bioactive components of liquorice and Semen Strychni in rat plasma: Application to a pharmacokinetics study  Voir?

Semen Strychni is known for its treatment of rheumatic arthritis with a low therapeutic index. Liquorice contributes a lot in herb detoxification according to the traditional Chinese medicine theory. A simple, rapid, and sensitive liquid chromatography–mass spectrometric method (LC–MS) was developed and validated for simultaneous determination of main bioactive ingredients in liquorice and Semen Strychni in rat plasma. Using moclobemide and cyproterone acetate as the internal standards, the analytes were pretreated via protein precipitation with methanol. An Ultimate AQ‐C18 column (3.0 ÎŒm, 3.0 à— 100 mm) was employed for chromatographic separation, combining with gradient elution. The mobile phase consisted of 0.07% formic acid and 0.12% ammonium acetate in aqueous phase (A) and acetonitrile in organic phase (B). The elution program was as follows: 0–0.5 min, 20% B; 0.5–1 min, 20–60% B; 1–7 min, 60–85% B; and 7–7.5 min, returned to 20% B, then continued to 12 min. Selected reaction monitoring was performed in both positive and negative ESI. Positive mode was adopted for detection of strychnine, brucine, and moclobemide, while negative mode was used for glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, isoliquiritigenin, liquiritin, and cyproterone acetate. The method was validated for specificity, linearity, matrix effect, recovery, precision, accuracy, and stability. The results show that this method is sensitive, accurate and robust for biological matrix analysis. Moreover, the proposed method was applied to a pharmacokinetic study in Sprague–Dawley rats for investigating the mechanism of which liquorice detoxifies Semen Strychni. A HPLC‐MS/MS method for the determination of three different types of compounds in rat plasma was developed and fully validated. The quantitative method was then successfully applied to a pharmacokinetic study after oral administration of brucine and subsequent (30 minutes later) licorice extracts for detoxification in Sprague‐Dawley rats. The findings would be beneficial for evaluating the ADME and underlying detoxification mechanism of licorice, as well as the safety clinical application of Semen Strychni.

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Fatal intoxication by 5F–ADB and diphenidine: Detection, quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q‐TOFMS  Voir?

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F–ADB (methyl 2‐[1‐(5‐fluoropentyl)‐1H–indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC/Q‐TOFMS) in the information‐dependent acquisition mode only detected diphenidine. Further urinary screening using an in‐house database containing NPS and metabolites detected not only diphenidine but also possible 5F–ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F–ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F–ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F–ADB. Mono‐ and di‐hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N‐alkyl position are known to show higher cannabinoid receptor affinity relative to their non‐fluorinated analogues; 5F–ADB is no exception with high CB1 receptor activity and much greater potency than Δ9‐THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F–ADB may be attributed to enzymatic and/or non‐enzymatic degradation, and further investigation into these possibilities is underway. We describe the detection and quantification of new psychoactive substances 5F–ADB and diphenidine in an acute intoxication case using LC/MS/MS and LC/Q‐TOFMS. This is the first report of postmortem blood concentration and the metabolic pathway of 5F–ADB in humans.

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Identification and quantification of predominant metabolites of synthetic cannabinoid MAB‐CHMINACA in an authentic human urine specimen  Voir?

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB‐CHMINACA, was investigated. Although unchanged MAB‐CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB‐CHMINACA metabolites from a commercial source. The MAB‐CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid‐phase extraction, and analyzed by liquid chromatography–tandem mass spectrometry with or without hydrolysis with ÎČ‐glucuronidase. Among the six MAB‐CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with ÎČ‐glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4‐monohydroxycyclohexylmethyl metabolite M1 (N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide) and a dihydroxyl (4‐hydroxycyclohexylmethyl and tert‐butylhydroxyl) metabolite M11 (N‐(1‐amino‐4‐hydroxy‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB‐CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB‐CHMINACA metabolites in an authentic human urine specimen. The MAB‐CHMINACA metabolites from the urine specimen of an abuser were extracted by a QuEChERS method, and analyzed by liquid chromatography‐tandem mass spectrometry. Among the six MAB‐CHMINACA metabolites tested, 2 predominant metabolites (monohydroxyl metabolite M1 and dihydroxyl metabolite M11) could be identified and quantified (2.17±0.15 and 10.2±0.3 ng/mL for M1 and M11, respectively). This is the first report dealing with in vivo identification and quantification of MAB‐CHMINACA metabolites in an authentic human urine specimen.

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Ultra‐fast LC–MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma  Voir?

A novel approach to high‐throughput, targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96‐well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC–MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow‐injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences. A novel approach to ultra‐rapid, high‐throughput LC–MS/MS analysis has been developed. The total analysis time was just 36 s per injection, or one 96‐well plate of prepared samples per hour, for the demonstrated application of clozapine and norclozapine analysis in human plasma samples. This approach is simple, and is likely to be ideally suited to a number of targeted assays.

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Replacing PAPS: In vitro phase II sulfation of steroids with the liver S9 fraction employing ATP and sodium sulfate  Voir?

In vitro technologies provide the capacity to study drug metabolism where in vivo studies are precluded due to ethical or financial constraints. The metabolites generated by in vitro studies can assist anti‐doping laboratories to develop protocols for the detection of novel substances that would otherwise evade routine screening efforts. In addition, professional bodies such as the Association of Official Racing Chemists (AORC) currently permit the use of in‐vitro‐derived reference materials for confirmation purposes providing additional impetus for the development of cost effective in vitro metabolism platforms. In this work, alternative conditions for in vitro phase II sulfation using human, equine or canine liver S9 fraction were developed, with adenosine triphosphate (ATP) and sodium sulfate in place of the expensive and unstable co‐factor 3â€Č‐phosphoadenosine‐5â€Č‐phosphosulfate (PAPS), and employed for the generation of six representative steroidal sulfates. Using these conditions, the equine in vitro phase II metabolism of the synthetic or so‐called designer steroid furazadrol ([1â€Č,2â€Č]isoxazolo[4â€Č,5â€Č:2,3]‐5α‐androstan‐17ÎČ‐ol) was investigated, with ATP and Na2SO4 providing comparable metabolism to reactions using PAPS. The major in vitro metabolites of furazadrol matched those observed in a previously reported equine in vivo study. Finally, the equine in vitro phase II metabolism of the synthetic steroid superdrol (methasterone, 17ÎČ‐hydroxy‐2α,17α‐dimethyl‐5α‐androstan‐3‐one) was performed as a prediction of the in vivo metabolic profile. Replacing the expensive and unstable PAPS cofactor with ATP and sodium sulfate provides a cost‐effective alternative to study in vitro phase II sulfation using the liver S9 fraction. The scope of this approach is demonstrated for the human, equine, and canine sulfation of six representative steroids. The in vitro metabolism of the designer steroids furazadrol and superdrol is also reported.

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The effect of antioxidants on the long‐term stability of THC and related cannabinoids in sampled whole blood  Voir?

The stability of cannabinoids is complex and crucial for the assessment of impaired driving caused by cannabis. Therefore, the effect of antioxidants on the long‐term stability of Δ9‐tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), 11‐hydroxy‐Δ9‐tetrahydrocannabinol (THC‐OH), and 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol (THC‐COOH) in whole blood samples preserved with fluoride citrate (FC) and fluoride oxalate (FX) mixtures was investigated at different temperatures. The measured concentrations of the cannabinoids in authentic whole blood preserved solely with FC or FX mixtures decreased significantly during prolonged storage at −20°C. On average, less than 5% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage interrupted by 5 thawing/freezing cycles. The rate of decrease was greatest in FC‐preserved blood. The repeated thawing/freezing of the samples accelerated the instability progression. At 5°C approximately 60% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage. No significant decrease was observed in samples stored at −80°C during the test period of 5 months. The instability at −20°C was to a great extend avoided by adding 30 mM ascorbic acid (ASC) to the samples before storage. Samples preserved with a combination of the FX mixture and ASC showed no significant decrease in the recovered concentrations during a 5‐month storage period interrupted by 6 thawing/freezing cycles. Samples preserved with a combination of the FC mixture and ASC showed almost similar improvements in cannabinoid stability. Other reducing agents such as sodium metabisulfite and glutathione also improved the stability in FX‐preserved blood stored at −20°C. THC and related cannabinoids were instable in whole blood stored at −20°C, especially when interrupted by several thawing/freezing cycles. The measured concentrations of THC and CBD in authentic field samples of whole blood preserved solely with fluoride citrate or fluoride oxalate mixtures decreased on average to less than 5% of the initial concentrations after 19 weeks of storage interrupted by 5 thawing/freezing cycles. The instability was avoided by adding 30 mM ascorbic acid (ASC) to the blood samples before storage.

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Drug residues in syringes and other injecting paraphernalia in Hungary  Voir?

The appearance and spread of new psychoactive substances (NPS) is a phenomenon seen throughout Europe since 2008. Synthetic cathinones, a group of NPS, have been self‐reported as the drug injected by the vast majority of people who inject drugs (PWID) in Hungary. This study aims at updating our knowledge of what exactly are the compounds injected by PWID. This multi‐site study analysed residues from used injecting drug paraphernalia collected from PWID via low‐threshold services and from public places in Budapest, Debrecen, Miskolc, Szeged, Békéscsaba and Pécs between March 2015 and February 2016. The paper describes the results of the chemical analysis of 2985 analytical samples created out of the 22 005 objects collected in this period. Active agents and their occurrences (compound cases) were identified using GC–MS. The study detected 161 different compounds, mostly synthetic cathinones (29%), non‐psychoactive compounds (14%), amines (12%), non‐psychoactive medications (12%) and other substances (32%). Of the 12 762 compound cases, 50% were cathinones, 18% substitution medications, 9% non‐controlled psychoactive substances and 24% other substances. Among compound cases, the most frequent cathinones were pentedrone (21%) and α‐PHP (12%). Among substitution medications, most were methadone (93%), and non‐controlled psychoactive substances were caffeine (74%) and nicotine (21%). Overall, the most prevalent substances were methadone (16%), pentedrone (10%) and caffeine (7%) with considerable variation detected among participating cities. Our results are consistent with previous self‐reported data showing a high prevalence of synthetic cathinone injection among PWID in Hungary. We also detected a large‐scale misuse of methadone by PWID. The injection of synthetic cathinones as the main pattern among Hungarian people who inject drugs and the large‐scale diversion and abuse of methadone are the main results of the study. The findings are based on the chemical analysis of used and discarded injecting paraphernalia collected throughout a 12‐month period involving seven study locations in Hungary.

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Analytical characterization of three cathinone derivatives, 4‐MPD, 4F–PHP and bk‐EPDP, purchased as bulk powder from online vendors  Voir?

Novel emerging drugs of abuse, also referred as new psychoactive substances, constitute an ever‐changing mixture of chemical compounds designed to circumvent legislative controls by means of chemical modifications of previously banned recreational drugs. One such class, synthetic cathinones, namely ÎČ‐keto derivatives of amphetamines, has been largely abused over the past decade. A number of new synthetic cathinones are detected each year, either in bulk powders/crystals or in biological matrices. It is therefore important to continuously monitor the supply of new synthetic derivatives and promptly report them. By using complementary analytical techniques (i.e. one‐ and two‐dimensional NMR, FT‐IR, GC–MS, HRMS and HPLC‐UV), this study investigates the detection, identification and full characterization of 1‐(4‐methylphenyl)‐2‐(methylamino)pentanone (4‐methylpentedrone, 4‐MPD), 1‐(4‐fluorophenyl)‐2‐(pyrrolidin‐1‐yl)hexanone (4F–PHP) and 1‐(1,3‐benzodioxol‐5‐yl)‐2‐(ethylamino)‐1‐pentanone (bk‐EPDP), three emerging cathinone derivatives. This paper describes the detection, identification and full structural elucidation of three emerging cathinone derivatives, 4‐MPD, 4F–PHP and bk‐EPDP, purchased as bulk powders online from ‘legal high' websites.

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Chiral analysis of amphetamines in hair by liquid chromatography–tandem mass spectrometry: compliance‐monitoring of attention deficit hyperactivity disorder (ADHD) patients under Elvanseź therapy and identification after controlled low‐dose application  Voir?

Amphetamine (AMP) is used as an illicit drug and also for the treatment of attention deficit hyperactivity disorder (ADHD). Respective drugs most often contain the enantiomer (S)‐AMP as active compound or (S)‐AMP is formed from the prodrug lisdexamfetamine (Elvanseź) whereas the illicit drug is usually traded as racemate ((R/S)‐AMP). A differentiation between the use of the medically prescribed drug and the abuse of illicit street amphetamine is of great importance, for example in retrospective consumption monitoring by hair analysis. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the chiral separation and quantitation of (S)‐ and (R)‐AMP in hair was developed. For this purpose, 20 mg hair was extracted and derivatized with N‐(2,4‐dinitro‐5‐fluorophenyl)‐L(S)‐valinamide L(S)‐(DNPV) to yield amphetamine diastereomers. Baseline separation of the resulting diastereomers was achieved on a high‐pressure liquid‐chromatography system (HPLC) coupled to a Sciex QTRAPź 5500 linear ion trap quadrupole mass spectrometer. The method was successfully validated. Analysis of hair samples from nine Elvanseź patients revealed only (S)‐AMP in eight cases; one subject showed both enantiomers indicating a (side‐) consumption of street amphetamine. The analysis of the 16 amphetamine users' samples showed only racemic amphetamine. Furthermore, it could be shown in a controlled study that (S)‐AMP can be detected after administration of even very low doses of lisdexamfetamine and dexamphetamine, which can be of interest in forensic toxicology and especially in drug‐facilitated crime (DFC). The method now enables the retrospective compliance‐monitoring of ADHD patients and the differentiation between medically prescribed intake of (S)‐amphetamine and abuse of illicit street amphetamine. This study describes the successful development and validation of an LC–MS/MS method for the qualitative and quantitative analysis of (S)‐amphetamine and (R)‐amphetamine in hair. With this method it is now possible to differentiate between the intake of medically prescribed (S)‐amphetamine and illegal street amphetamine by the use of hair analysis. Furthermore, it could be shown that after a low oral dose of lisdexamfetamine, (S)‐amphetamine can still be detected in hair which can be of great importance in forensic toxicology.

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Ephedra alkaloid contents of Chinese herbal formulae sold in Taiwan  Voir?

Consumption of Ephedra alkaloids is prohibited in‐competition by the World Anti‐Doping Agency (WADA). In Taiwan, colds are often treated with Chinese herbal formulae containing Herba Ephedrae. We screened products sold in Taiwan and preliminarily assessed their relationships with WADA threshold violations. Fifty‐six concentrated powder products, including 19 Chinese herbal formulae that contained Herba Ephedrae, were collected. The content of Ephedra alkaloids, namely ephedrine (E), methylephedrine (ME), norpseudoephedrine (NPE; cathine), pseudoephedrine (PE), and norephedrine (NE; phenylpropanolamine), was determined using a validated high‐performance liquid chromatography method. The results revealed that the phenotypic indicators of the collected products, E/PE and E/total ratios, were 1.52–4.70 and 0.49–0.72, respectively, indicating that the Herba Ephedrae species in these products was probably E. sinica or E. equisetina, but not E. intermedia. The contents of E, ME, NPE, PE, and NE and the total alkaloid contents in the daily doses of the products were 0.45–34.97, 0.05–4.87, 0.04–3.61, 0.15–12.09, and 0.01–2.00 mg and 0.68–53.64 mg, respectively. The alkaloid contents followed a relatively consistent order (E > PE > ME ≈ NPE > NE), even for products from different manufacturers. We calculated that single doses of 50.0% and 3.6% of the products would result in the WADA thresholds of E and NPE being exceeded, respectively. Our data provide critical information for athletes and medical personnel, who should be wary of using complex Chinese herbal formulae in addition to over‐the‐counter products. This is the first study to systematically determine the Ephedra alkaloid contents of the Chinese herbal formula products sold in Taiwan. Using their E/PE and E/total ratios, we identified that the Herba Ephedrae species used in the products was probably E. sinica or E. equisetina, but not E. intermedia. Among the 56 tested samples, we discovered that a single dose of half of them would probably result in a threshold violation.

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Influence of bleaching and coloring on ethyl glucuronide content in human hair  Voir?

Ethyl glucuronide (EtG) is increasingly used in forensic toxicology as a marker for alcohol use in analyses of hair samples, especially in abstinence control. Some cosmetic treatments are considered to markedly reduce the EtG content. In view of especially many women with coloured hair the present study was performed to further investigate the effect of a variety of colouring procedures (bleaching, tinting, permanent and semi‐permanent dyeing, henna) on the EtG content. Untreated hair samples (n = 12, EtG 13.9–64.7 pg/mg) were re‐analyzed (gas chromatography‐ negative chemical ionization mass spectrometry, 0.8 pg/mg quantification limit) after different treatment procedures. A decrease of the EtG content of at least 10% occurred in every case. The reduction in comparison to the untreated hair was expectedly high for permanent dyeing and bleaching with 18.1% of the initial content (median, range 0.0–50.9%) and 18.4% (0.0–46.7%), respectively. For henna this was 38.3% (0.0–83.0%), for tinting 70.4% (29.0–90.8%), for semi‐permanent dyeing 41.9% (0.0–77.4%). With permanent hair dye the EtG content was decreased to below 7 pg/mg in 10 of 12 cases, in 3 cases even below the LOD (0.2 pg/mg). Surprisingly henna treatment without oxidative component had a marked influence, EtG was below 2 pg/mg in 2 of 12 samples. The study showed that all tested coloration procedures markedly affected the deposited EtG content. Even temporary or henna coloration may have a marked effect. The present data support the recommendation to exclude hair samples with colour manipulations for analysis on the EtG content as a precaution in alcohol abstinence programs. Copyright © 2017 John Wiley & Sons, Ltd. Different hair colouring procedures were performed with 12 EtG positive hair samples, including treatment with henna. The expected marked reductions in EtG contents even below the LOD were found. Unexpectedly, treatment with henna also affected EtG content massively.

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Identification, isolation, and synthesis of seven novel impurities of anti‐diabetic drug Repaglinide  Voir?

Seven unknown impurities in Repaglinide bulk drug batches at below 0.1% (ranging from 0.05 to 0.10%) were detected by an ultra‐performance liquid chromatographic (UPLC) method. These impurities were isolated from the crude sample of Repaglinide using preparative high performance liquid chromatography (prep‐HPLC). Based on liquid chromatography‐electrospray ionization‐mass spectrometry (LC‐ESI/MS) study, the chemical structures of seven new impurities (8, 9, 10, 11, 13, 14, and 16) were presumed and characterized as 4‐(cyanomethyl)‐2‐ethoxybenzoic acid (8), 4‐(cyanomethyl)‐2‐ethoxy‐N‐(3‐methyl‐1‐(2‐(piperidin‐1‐yl)phenyl)butyl)benzamide (9), 4‐(2‐amino‐2‐oxoethyl)‐2‐ethoxy‐N‐(3‐methyl‐1‐(2‐(piperidin‐1‐yl)phenyl)butyl) benzamide (10) and 2‐(3‐ethoxy‐4‐((3‐methyl‐1‐(2‐(piperidin‐1‐yl)phenyl)butyl) carbamoyl) phenyl) acetic acid (11) and 4‐(cyanomethyl)‐N‐cyclohexyl‐2‐ethoxybenzamide (13), 2‐(4‐(cyclohexylcarbamoyl)‐3‐ethoxyphenyl) acetic acid (14) and N‐cyclohexyl‐4‐(2‐(cyclohexylamino)‐2‐oxoethyl)‐2‐ethoxybenzamide (16). The complete spectral analysis, proton nuclear magnetic resonance (1H NMR), 13C NMR, MS, and infrared (IR) confirmed the proposed chemical structures of impurities. Identification, structural characterization, formation, and their synthesis was first reported in this study. The impurity 11 was crystallized and structure was solved by single crystal X‐ray diffraction. Seven unknown impurities in repaglinide 1 bulk drug batches at below 0.1 level were detected by an ultra performance liquid chromatographic (UPLC) method. The spectral analysis e.g. 1H NMR, 13C NMR, MS and IR confirmed the proposed chemical structures of impurities. Identification, complete characterization data and their synthesis were reported for the first time.

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Analysis of RBC‐microparticles in stored whole blood bags – a promising marker to detect blood doping in sports?  Voir?

Blood doping in sports is prohibited by the World Anti‐Doping Agency (WADA). To find a possible biomarker for the detection of blood doping, we investigated the changes in blood stored in CPDA‐1 blood bags of eight healthy subjects who donated one unit of blood. Aliquots were taken on days 0, 14, and 35. Platelet‐free plasma was prepared and stored at −80°C until analysis on a flow cytometer dedicated for the analysis of microparticles (MPs). Changes in the number of red blood cell (RBC) ‐MPs were highly significant (p < 0.0001) with a mean of 219 (10^3/ÎŒL) on day 0 changing to 23 120 (10^3/ÎŒL) on day 14 and 29 310 (10^3/ÎŒL) on day 35. We conclude that RBC‐MPs seem to be a promising biomarker for doping control but confirmation by a transfusion study is necessary. The goal of this study was to investigate red blood cell microparticles (RBC‐MPs) during storage in CPDA‐1 blood bags. Blood was analyzed on day 0 and after 14 and 35 days of storage using the new Apogee Flow Systems A‐60 Micro‐Plus flow cytometer. The number of RBC‐MPs increased significantly during the study (approx. 100‐fold). RBC‐MPs have the potential as possible a biomarker for the detection of autologous blood transfusion.

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Identification of pyrolysis products of the new psychoactive substance 2‐amino‐1‐(4‐bromo‐2,5‐dimethoxyphenyl)ethanone hydrochloride (bk‐2C‐B) and its iodo analogue bk‐2C‐I  Voir?

2‐Amino‐1‐(4‐bromo‐2,5‐dimethoxyphenyl)ethanone hydrochloride (bk‐2C‐B) has recently emerged as a new psychoactive substance (NPS). It is most commonly consumed orally, although there are indications that it might also be ingested by inhalation or ‘smoking'. Information about the stability of bk‐2C‐B when exposed to heat is unavailable and the potential for pyrolytic degradation and formation of unknown substances available for inhalation prompted an investigation using a simulated ‘meth pipe' scenario. Twelve products following pyrolysis of bk‐2C‐B were detected and verified by organic synthesis of the corresponding standards. In addition, 2‐amino‐1‐(4‐iodo‐2,5‐dimethoxyphenyl)ethanone hydrochloride (bk‐2C‐I) was characterized for the first time and subjected to pyrolysis as well. Similar products were formed, which indicated that the replacement of the bromo with the iodo substituent did not affect the pyrolysis pattern under the conditions used. Two additional products were detected in the bk‐2C‐I pyrolates, namely 1‐(2,5‐dimethoxyphenyl)‐ethanone and 1‐iodo‐4‐ethenyl‐5‐methoxyphenol. The potential ingestion of pyrolysis products with unknown toxicity adds an element of concern. Copyright © 2017 John Wiley & Sons, Ltd. A study of the pyrolysis of two new beta keto analog of 2‐(4‐bromo‐2,5‐dimethoxyphenyl) ethan‐1‐amine (2C–B), bk‐2C‐1. Products were formed by deamination, loss of the bk oxygen and halogenation.

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lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG‐4592) and its main metabolites into routine doping controls  Voir?

The utility of hypoxia‐inducible factor (HIF) prolyl hydroxylase inhibitors as a therapeutic means of treating patients suffering from anaemia has been demonstrated for various clinical settings. However, besides this intended use, HIF stabilizers can be the subject of misuse in amateur and elite sports due to their erythropoietic properties, as recently proven by several cases of adverse analytical findings in doping control testing. Consequently, to allow for adequate and comprehensive test methods, knowledge of the drug candidates' metabolism and analytical options enabling appropriate detection windows in sports drug testing samples (i.e., blood and urine) is essential to doping control laboratories. In the present study, a novel HIF prolyl hydroxylase inhibitor referred to as Roxadustat (FG‐4592) and main plasma‐ and urine‐derived metabolites were investigated in the context of routine doping control analytical approaches. Liquid chromatography‐mass spectrometry‐based test methods were used to study the target analytes' dissociation pathways following electrospray ionization and collision‐induced dissociation. Diagnostic precursor‐product ion pairs were selected to enable the implementation of the intact drug Roxadustat and selected metabolites into multi‐analyte initial testing procedures for plasma and urine specimens. The assays were validated in accordance to guidelines of the World Anti‐Doping Agency (WADA) and results demonstrated the suitability (fitness‐for‐purpose) of the employed analytical methods with detection limits ranging from 0.05 to 1 ng/mL and 1 to 5 ng/mL for urine and plasma, respectively. Subsequently, elimination study plasma and urine samples collected up to 167 h post‐administration were analyzed using the validated methods, which suggested the use of different target analytes for blood and urine analyses with FG‐4592 and its glucuronide, respectively, for optimal detection windows. Additionally, a light‐induced rearrangement product (photoisomer) of Roxadustat resulted in the formation of an additional compound of identical mass. Copyright © 2017 John Wiley & Sons, Ltd. Updating and expanding routine doping control assays is vital for efficient sports drug testing programs. HIF stabilizers have been identified as a new class of substances being misused in sports, and the comprehensive analysis of FG‐4592 (Roxadustat) inclusive of its main metabolites and a light‐induced rearrangement artifact is presented.

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Detection of the recently emerged synthetic cannabinoid 5F–MDMB‐PICA in ‘legal high' products and human urine samples  Voir?

Indole or indazole‐based synthetic cannabinoids (SCs) bearing substituents derived from valine or tert‐leucine are frequently abused new psychoactive substances (NPS). The emergence of 5F–MDMB‐PICA (methyl N‐{[1‐(5‐fluoropentyl)‐1H–indol‐3‐yl]carbonyl}‐3‐methylvalinate) on the German drug market is a further example of a substance synthesized in the context of scientific research being misused by clandestine laboratories by adding it to ‘legal high' products. In this work, we present the detection of 5F–MDMB‐PICA in several legal high products by gas chromatography–mass spectrometry (GC–MS) analysis. To detect characteristic metabolites suitable for a proof of 5F–MDMB‐PICA consumption by urine analysis, pooled human liver microsome (pHLM) assays were performed and evaluated using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QToF‐MS) techniques to generate reference spectra of the in vitro phase I metabolites. The in vivo phase I metabolism was investigated by the analysis of more than 20 authentic human urine specimens and compared to the data received from the pHLM assay. Biotransformation of the 5‐fluoropentyl side chain and hydrolysis of the terminal methyl ester bond are main phase I biotransformation steps. Two of the identified main metabolites formed by methyl ester hydrolysis or mono‐hydroxylation at the indole ring system were evaluated as suitable urinary biomarkers and discussed regarding the interpretation of analytical findings. Exemplary analysis of one urine sample for 5F–MDMB‐PICA phase II metabolites showed that two of the main phase I metabolites are subject to extensive glucuronidation prior to renal excretion. Therefore, conjugate cleavage is reasonable for enhancing sensitivity. Commercially available immunochemical pre‐tests for urine proved to be unsuitable for the detection of 5F–MDMB‐PICA consumption. Copyright © 2017 John Wiley & Sons, Ltd. Signals for metabolites of the SC 5F‐PB‐22 during routine LC‐MS/MS screening of urine specimens for SC metabolites were identified as metabolites of the novel synthetic cannabinoid SC 5F‐MDMB‐PICA by LC‐QToF‐MS analysis. In this study the detection 5F‐MDMB‐PICA in herbal blends adn the identification of phase I metabolites suitable for proving 5F‐MDMB‐PICA consumption by LC‐MS/MS and LC‐Q‐ToF‐MS analyses of authentic urine samples is reported. Commercial available immunochemical pre‐tests were tested for the detection of 5F‐MDMB‐PICA consumption.

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Report on an anti‐doping operation in Guadeloupe: High number of positive cases and inferences about doping habits  Voir?

Anti‐doping controls in non‐major events are relatively infrequent and athletes that compete only in these events are less likely to be controlled. The French Anti‐Doping Agency carried out an anti‐doping operation during a regional cycling competition in Guadeloupe. The urine and serum samples were analysed by the French anti‐doping laboratory. Out of 42 athletes, 7 were positive for one or more substances prohibited by the World Anti‐Doping Agency. Four serum samples contained continuous erythropoietin receptor activator (CERA) and one a recombinant erythropoietin. However, no traces were found in the corresponding urines. One of the athletes positive for CERA was also positive for growth hormone (GH), identified using the GH isoform test. The same serum was negative with the GH biomarkers test, probably because of the brief interval between injection and control (less than a day). The stimulants heptaminol and dimethylbutylamine, as well as the glucocorticoid prednisone and its metabolite prednisolone, were also found. Strikingly, 16.6% of the controlled athletes were using one or more prohibited drugs. These findings indicate that doping is widespread in athletes competing regionally and that CERA is still a popular drug for endurance sports. They underline the need for more controls, particularly blood sampling during non‐major competitions. Copyright © 2017 John Wiley & Sons, Ltd. The results of an anti‐doping operation performed during a regional cycling race in Guadeloupe in March 2016 are presented. Serum and urine samples from 42 athletes were analysed. Four CERA, one rEPO and one GH were identified in serum samples. Stimulants heptaminol and DMBA and glucocorticoids prednisone/prednisolone were also found in some urines. Altogether, 7 of 42 athletes tested (16.6%) were reported with adverse analytical findings. Interestingly, CERA and rEPO were not retrieved in urine, and would have been missed without serum analyses.

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Flubromazolam – Basic pharmacokinetic evaluation of a highly potent designer benzodiazepine  Voir?

Since their first appearance on the Internet in 2012, designer benzodiazepines established as an additional, quickly growing compound class among new psychoactive substances. Data regarding pharmacokinetic parameters, metabolism, and detectability for new compounds are limited or often not available. One of these compounds, flubromazolam (8‐bromo‐6‐(2‐fluorophenyl)‐1‐methyl‐4H‐[1,2,4]triazolo[4,3‐a][1,4]benzodiazepine), the triazolo‐analogue of flubromazepam, has been offered on the Internet from 2014 on. The purpose of the present study was to assess the period of detectability in biological samples along with preliminary basic pharmacokinetic parameters of the designer benzodiazepine flubromazolam. To investigate these, one of the authors ingested a capsule containing 0.5 mg of the drug. Metabolism studies and suitability tests for the detection with immunochemical assays were performed with the samples obtained from the self‐experiment and five authentic case samples. Flubromazolam and its mono‐hydroxylated metabolite were detectable by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in urine for up to 6.5 and 8 days, respectively (lower limit of quantification (LLOQ) flubromazolam: 0.1 ng/mL). Peak serum concentrations were as low as 8 ng/mL (8 h post ingestion). Glucuronides were also detected. The terminal elimination half‐life could be estimated in the range of 10–20 h. Immunochemical assays yielded negative results for serum samples and positive results for urine samples for up to five days post ingestion. The presented data demonstrate the detectability of a single uptake of 0.5 mg of flubromazolam in hair samples collected two weeks after drug uptake by LC–MS3 (cmax 0.6 pg/mg; LOD 0.01 pg/mg). The detected metabolites were in good agreement with those described in other studies. Copyright © 2017 John Wiley & Sons, Ltd. Designer benzodiazepines are a quickly growing class of compounds among new psychoactive substances. We report on the period of detectability in biological samples and present basic pharmacokinetic data of the highly potent, rather long‐acting designer benzodiazepine flubromazolam, a compound which might well be used in drug facilitated crimes.

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Oral absorption, distribution, metabolism, and excretion of icaritin in rats by Q‐TOF and UHPLC–MS/MS  Voir?

Icaritin (ICT) displays numerous pharmacological activities for the treatment of various cancers, osteoporosis, inflammation, and angiocardiopathy. The absorption, distribution, metabolism, and excretion of ICT still remain unknown. ICT was administered to rats at 2 mg/kg for intravenous injection or 40 mg/kg for oral route. Major metabolite of ICT was identified using quadrupole time‐of‐flight (Q‐TOF), and ICT and its major metabolite were quantified in plasma, tissues, urine, faeces, and bile by ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). A total of 24 metabolites of ICT in plasma were identified and mono C‐7 glucuronide glucuronidated icaritin (GICT) was the major metabolite of ICT after oral administration. The absolute bioavailability of ICT was 4.33% although ICT was rapidly absorbed into the blood. For oral administration, concentrations of GICT at various time points was 6.38–8.81‐fold higher than those of ICT, and the area under the curve (AUC) of GICT was about 8‐fold higher than that of ICT, while AUC values of ICT and GICT were almost equal for intravenous injection. Approximately 65.7% ICT and 42.7% GICT were distributed in liver and kidney, respectively. Unabsorbed ICT was mainly excreted as the parent form in faeces with at least 60% of administered dose during 24 h, whereas absorbed ICT was predominantly excreted as GICT from urine with 2.74% of administered dose accounting for 63.28% of absorbed drug. ICT was rapidly absorbed into the blood although a large amount of ICT remained unabsorbed, and then rapidly and mainly metabolized to GICT. ICT mainly distributed in liver, while GICT predominantly distributed in kidney. Absorbed ICT and GICT were predominantly excreted via urine, and unabsorbed ICT was mainly excreted as the parent form in faeces. Copyright © 2017 John Wiley & Sons, Ltd. Absorption, distribution, metabolism, and excretion of ICT were first investigated. Metabolites of ICT were first identified by Q‐TOF.

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Issue Information  Voir?

No abstract is available for this article.



Racing chemistry: A century of challenges and progress  Voir?

Horseracing has been called ‘one of the first quintessentially modern sports'. Its urge towards standardization, its mathematically set odds, its concern with weights, and its pioneering embrace of drug‐testing reflect an empirical temperament crucial to its transformation from a gentleman's pastime to a global industry funded by wagering. Ironically, in the late nineteenth century, it was modern science itself, and in particular the purification and synthesis of the drugs of nature, that turned the doping of racing animals – a practice recorded in antiquity – into an organized criminal enterprise. This paper presents original research into the history of racing chemistry in Australia in the context of developments in the field worldwide. Using a case‐study approach based on extensive archival materials, it reveals unpublished diaries kept by an analyst working at Sydney Racing Laboratory in the 1950s that document conflicts between scientists over identification of performance drugs in racing animals. The author presents evidence that augments and revises earlier narratives concerning the history of the establishment of laboratory control at Australian racetracks and the removal of the country's first official analyst for racing, Miss Jean Kimble. The Kimble case illustrates the inevitable political, professional, and personal pressures that bear upon drug‐testing in sports, and also conflicts between scientists over standards and priorities. Copyright © 2016 John Wiley & Sons, Ltd. Jean Kimble peering into a microscope with camera attachment and the official group portrait taken at the AORC's second annual conference, held on May 18, 1949 in New York City. Kimble is standing in the middle row fifth from the left. The woman next to her, who is dressed in darker clothing is another female scientist of that era, Elsie Bellows. The man sitting in the front row, third from the right is Charles Morgan, the AORC President at the time.

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Challenges in detecting substances for equine anti‐doping  Voir?

The artificial increase of the physical capability of horses using drugs is well known in racing and other equine sports. Both illicit and therapeutic substances are regarded as prohibited substances in competition in most countries. Some countries make distinctions for a few, specific drugs which are, however, allowed for use in other countries. The primary objective in the case of doping control is the detection of any trace of drug exposure, either parent drug or any of its metabolites, using the most powerful analytical methods which are generally based on chromatographic/mass spectrometric techniques. Of major concern in horseracing is the absence of a single organization regulating the anti‐doping framework; instead of this, individual racing authorities provide rules and regulations often resulting in variations in the applied doping control programmes of different countries. The aim of this paper is to review the recent literature (approximately from 2012 to mid‐2016) to highlight the numerous and diverse challenges faced in doping control of racing and equestrian sports, including the detection of designer drugs (anabolic steroids or stimulants) and of other emerging prohibited substances, such as peptides and noble gases in horse urine and plasma. Moreover, the application of ‘omics' techniques (especially of metabolomics) deserves attention for establishing possible fingerprints of drug abuse as well as the evolution of instrumental analysis resulting a powerful ally in the fight against doping in equine sports. Copyright © 2017 John Wiley & Sons, Ltd. In equine anti‐doping, the detection of an enormous number of prohibited substances (or methods) must be controlled compared to those in human sports, as the health of two individuals must be protected: the human's and the horse's. Diverse drug classes must be analyzed in multi‐targeted methods with a minimal drop in sensitivity. The use of innovative techniques, such as omics, and the evolution of instrumental techniques offer a powerful contribution to the fight for clean equine sports.

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A review of designer anabolic steroids in equine sports  Voir?

In recent years, the potential for anabolic steroid abuse in equine sports has increased due to the growing availability of designer steroids. These compounds are readily accessible online in ‘dietary' or ‘nutritional' supplements and contain steroidal compounds which have never been tested or approved as veterinary agents. They typically have unusual structures or substitution and as a result may pass undetected through current anti‐doping screening protocols, making them a significant concern for the integrity of the industry. Despite considerable focus in human sports, until recently there has been limited investigation into these compounds in equine systems. To effectively respond to the threat of designer steroids, a detailed understanding of their metabolism is needed to identify markers and metabolites arising from their misuse. A summary of the literature detailing the metabolism of these compounds in equine systems is presented with an aim to identify metabolites suitable for incorporation into screening protocols by anti‐doping laboratories. The future of equine anti‐doping research is likely to be guided by the incorporation of alternate testing matrices into routine screening, the improvement of in vitro technologies that can mimic in vivo equine metabolism, and the improvement of instrumentation or analytical methods that allow for the development of untargeted screening, and metabolomics approaches for use in anti‐doping screening protocols. Copyright © 2016 John Wiley & Sons, Ltd. Designer steroids pose a growing threat to the integrity of equine sports. Studies of the in vivo and in vitro metabolism of designer steroids in equine systems are critically reviewed, highlighting recommended target analytes, analytical approaches, and future directions.

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Doping control analysis of anabolic steroids in equine urine by gas chromatography‐tandem mass spectrometry  Voir?

Anabolic steroids are banned substances in equine sports. Gas chromatography‐mass spectrometry (GC‐MS) has been the traditional technique for doping control analysis of anabolic steroids in biological samples. Although liquid chromatography‐mass spectrometry (LC/MS) has become an important technique in doping control, the detection of saturated hydroxysteroids by LC‐MS remains a problem due to their low ionization efficiency under electrospray. The recent development in fast‐scanning gas‐chromatography‐triple‐quadrupole mass spectrometry (GC‐MS/MS) has provided a better alternative with a significant reduction in chemical noise by means of selective reaction monitoring. Herein, we present a sensitive and selective method for the screening of over 50 anabolic steroids in equine urine using gas chromatography‐tandem mass spectrometry (GC‐MS/MS). Copyright © 2016 John Wiley & Sons, Ltd. A sensitive method was developed for the simultaneous detection of 52 steroids in equine urine using GC‐MS/MS. The developed method has enhanced sensitivity and selectivity for detection of free and conjugated steroids. It has acceptable precision and recovery to be used as a qualitative screening method on a regular basis.

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Application of testosterone to epitestosterone ratio to horse urine – a complementary approach to detect the administrations of testosterone and its pro‐drugs in Thoroughbred geldings  Voir?

Detection of testosterone and/or its pro‐drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra‐high performance liquid chromatography tandem mass spectrometry (UHPLC‐MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post‐race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi‐Bolicź) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd. The use of steroid ratios may provide a supplementary approach to aid in differentiating the administration of testosterone and its pro‐drugs and unusual conditions resulting in elevated testosterone concentrations in geldings. In the current study, an upper limit of 5 was proposed for the ratio of urinary testosterone: epitestosterone (T:E). Analysis of atypical urine samples showed that the T:E ratio remained below the proposed limit, whilst it was exceeded following the administration of a range of separate testosterone‐related steroids.

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Evidence of boldenone, nandrolone, 5(10)‐estrene‐3ÎČ‐17α‐diol and 4‐estrene‐3,17‐dione as minor metabolites of testosterone in equine  Voir?

The detection of boldenone, nandrolone, 5(10)‐estrene‐3ÎČ,17α‐diol, and 4‐estrene‐3,17‐dione in a urine sample collected from a gelding having been treated with testosterone (500 mg ‘Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these ‘testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)‐estrene‐3ÎČ,17α‐diol, and 4‐estrene‐3,17‐dione could indeed be detected and confirmed in the early post‐administration urine samples from all six geldings. Although boldenone has been reported to be present in urine after testosterone administration, there has been no direct evidence reported that boldenone, nandrolone, 5(10)‐estrene‐3ÎČ,17α‐diol, and 4‐estrene‐3,17‐dione are metabolites of testosterone in geldings. Subsequent in vitro experiments involving the incubation of testosterone with horse liver microsomes, liver, adipose and muscle tissues, and adrenal cortex homogenates failed to provide evidence that these four substances are minor metabolites of testosterone. An administration trial using ‘Testosterone Suspension 100' supplemented with 13C–labelled testosterone (500 mg, 1:1 ratio, injected intramuscularly) was performed. The similarities of the excretion curves of 12C–testosterone and 13C–testosterone in urine suggest that there was minimal kinetic isotope effect. 13C–Labelled boldenone, nandrolone and 4‐estrene‐3,17‐dione were detected but not 5(10)‐estrene‐3ÎČ,17α‐diol and its 13C–counterpart. This is the first unequivocal evidence of boldenone, nandrolone and 4‐estrene‐3,17‐dione being metabolites of testosterone in geldings. In view of these results, caution should be exercised when interpreting findings of boldenone, nandrolone and/or 4‐estrene‐3,17‐dione together with a relatively high level of testosterone in gelding urine. Copyright © 2017 John Wiley & Sons, Ltd. Low levels of boldenone, nandrolone, 5(10)‐estrene‐3ÎČ,17α‐diol, and 4‐estrene‐3,17‐dione could detected and confirmed in the early post‐administration urine samples in six gelding horses administered with ‘Testosterone Suspension 100'. A tracer experiment was carried out by administering a 1:1 mixture of 13C–testosterone and Testosterone Suspension 100. Indeed, 13C–labelled boldenone, 4‐estrene‐3‐17‐dione and nandrolone were detected in post‐administration urine and provide the first unequivocal evidence that these steroids are metabolites of testosterone in geldings.

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In vitro phase I metabolism of selective estrogen receptor modulators in horse using ultra‐high performance liquid chromatography‐high resolution mass spectrometry  Voir?

Selective estrogen receptor modulators (SERMs) are chemicals that possess the anti‐oestrogenic activities that are banned ‘in' and ‘out' of competition by the World Anti‐Doping Agency (WADA) in human sports, and by the International Federation of Horseracing Authorities (IFHA) in horseracing. SERMs can be used as performance‐enhancing drugs to boost the level of androgens or to compensate for the adverse effects as a result of extensive use of androgenic anabolic steroids (AASs). SERMs have indeed been abused in human sports; hence, a similar threat can be envisaged in horseracing. Numerous analytical findings attributed to the use of SERMs have been reported by WADA‐accredited laboratories, including 42 cases of tamoxifen and 2 cases of toremifene in 2014. This paper describes the identification of the in vitro phase I metabolites of tamoxifen and toremifene using ultra‐high performance liquid chromatography‐high resolution mass spectrometry (UHPLC‐HRMS), with an aim to identify potential screening targets for doping control in equine sports. A total of 13 and 11 in vitro metabolites have been identified for tamoxifen and toremifene, respectively, after incubation with homogenized horse liver. The more prominent in vitro biotransformation pathways include N‐desmethylation, hydroxylation, and carboxylation. In addition, this is the first report of some novel metabolites for both tamoxifen and toremifene with hydroxylation occurring at the N‐methyl moiety. To our knowledge, this is the first study of the phase I metabolism of tamoxifen and toremifene in horses using homogenized horse liver. Copyright © 2017 John Wiley & Sons, Ltd. Identification of in vitro phase I metabolites of tamoxifen and toremifene as potential screening targets for the detection of their misuses in racehorces. N‐Hydroxymethylated metabolites were first reported in equine.

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Doping control study of AICAR in post‐race urine and plasma samples from horses  Voir?

Acadesine, 5‐aminoimidazole‐4‐carboxamide‐1‐ÎČ‐D‐ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate‐activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term ‘exercise pill'. Its use has been classified as gene doping by the World Anti‐Doping Agency (WADA), and since it is endogenous, it may only be possible to control deliberate administration of AICAR to racehorses after establishment of an appropriate threshold. Herein we report our studies of AICAR in post‐race equine urine and plasma samples including statistical studies of AICAR concentrations determined from 1,470 urine samples collected from thoroughbreds and standardbreds and analyzed in Australia, France, and Hong Kong. Quantification methods in equine urine and plasma using liquid chromatography‐mass spectrometry were developed by the laboratories in each country. An exchange of spiked urine and plasma samples between the three countries was conducted, confirming no significant differences in the methods. However, the concentration of AICAR in plasma was found to increase upon haemolysis of whole blood samples, impeding the establishment of a suitable threshold in equine plasma. A possible urine screening cut‐off at 600 ng/mL for the control of AICAR in racehorses could be considered for adoption. Application of the proposed screening cut‐off to urine samples collected after intravenous administration of a small dose (2 g) of AICAR to a mare yielded a short detection time of approximately 4.5 h. Copyright © 2017 John Wiley & Sons, Ltd. AICAR is an endogenous AMPK activator in many mammals, including horses. It has attracted considerable attention in the field of doping control because of its potential endurance enhancing effect. This paper presents the statistical evaluation of AICAR in horse urine using population data acquired in three countries. A urinary screening cut‐off at 600 ng/mL could be considered for adoption for the control of excessive AICAR in equine urine.

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Control of methylxanthines in the competition horse: pharmacokinetic/pharmacodynamic studies on caffeine, theobromine and theophylline for the assessment of irrelevant concentrations  Voir?

Methylxanthines positives in competition samples have challenged doping control laboratories and racing jurisdictions since methylxanthines are naturally occurring prohibited substances and often constituents of feed. For theobromine, an international threshold (renamed in International Residue Limit, IRL) of 2 ”g/mL in urine has been established. On the basis of the data presented herein, a threshold or rather an IRL for theobromine in plasma of 0.3 ”g/mL was proposed and was thereupon approved by the International Federation of Horseracing Authorities (IFHA). Official recommendations for reporting caffeine and theophylline are still lacking. The aim of the study was to investigate IRLs for theobromine in blood and for caffeine and theophylline in blood and urine. Therefore, a set of six administrations were carried out including both single i.v. and single oral administrations of caffeine, theobromine and theophylline. Plasma and urine concentrations were determined using a validated liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Applying the Toutain model approach an effective plasma concentration (EPC) of caffeine was estimated at 3.05 ”g/mL, irrelevant concentrations in blood (IPC) and urine (IUC) approached 6 and 12 ng/mL, respectively. EPC of theobromine was calculated with 3.80 ”g/mL, and irrelevant concentrations of theobromine were determined at 8 ng/mL in plasma and at 142 ng/mL in urine. Toutain modelling of the theophylline data produced an EPC, IPC, and IUC of 3.20 ”g/mL, 6 ng/mL, and 75 ng/mL, respectively. The obtained irrelevant concentrations were used to postulate IRLs for theobromine in plasma and for caffeine and theophylline in plasma and urine. Copyright © 2016 John Wiley & Sons, Ltd. Methylxanthines are naturally occuring prohibited substances necessitating an adequate consideration in doping control by racing and equestrian authorities. However, international rules for the control of methylxanthines are in place for theobromine only. The present paper attempts to assist racing laboratories and authorities in reporting and interpreting methylxanthines findings in a transparent, consistent and defensible manner. Therefore PK/PD studies were carried out including i.v. and oral administrations of caffeine, theobromine and theophylline. Plasma and urine concentrations were determined by LC‐MS/MS. Toutain modelling on the obtained data was applied to investigate irrelevant concentrations of methylxanthines in the horse. These irrelevant concentrations were used to propose international residue limits for methylxanthines in the competition horse.

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Pharmacokinetics of betamethasone in plasma, urine, and synovial fluid following intra‐articular administration to exercised thoroughbred horses  Voir?

The use of corticosteroids, such as betamethasone, in performance horses is tightly regulated. The objective of the current study was to describe the plasma pharmacokinetics of betamethasone as well as time‐related urine and synovial fluid concentrations following intra‐articular administration to horses. Twelve racing‐fit adult Thoroughbred horses received a single intra‐articular administration (9 mg) of a betamethasone sodium phosphate and betamethasone acetate injectable suspension into the right antebrachiocarpal joint. Blood, urine, and synovial fluid samples were collected prior to and at various times up to 21 days post drug administration. All samples were analyzed using tandem liquid chromatography‐mass spectrometry. Plasma data were analyzed using compartmental pharmacokinetic modeling. Maximum measured plasma betamethasone concentrations were 3.97 ± 0.23 ng/mL at 1.45 ± 0.20 h. The plasma elimination half‐life was 7.48 ± 0.39 h. Betamethasone concentrations were below the limit of detection in all horses by 96 h and 7 days in plasma and urine, respectively. Betamethasone fell below the limit of detection in the right antebrachiocarpal joint between 14 and 21 days. Results of this study provide information that can be used to regulate the use of intra‐articular betamethasone in the horse. Copyright © 2017 John Wiley & Sons, Ltd. Following intra‐articular administration to exercised Thoroughbred horses, betamethasone was detected for up to 96 h and 7 days in plasma and urine, respectively. Betamethasone fell below detectable levels in synovial fluid between 14 and 21 days post administration and therefore urine and blood concentrations do not appear to be good indicators of drug concentrations at the effector site and by extrapolation may not be useful in determining the duration of pharmacologic effect.

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Pharmacokinetics and pharmacodynamics of meldonium in exercised thoroughbred horses  Voir?

Although developed as a therapeutic medication, meldonium has found widespread use in human sports and was recently added to the World Anti‐Doping Agency's list of prohibited substances. Its reported abuse potential in human sports has led to concern by regulatory authorities about the possible misuse of meldonium in equine athletics. The potential abuse in equine athletes along with the limited data available regarding the pharmacokinetics and pharmacodynamics of meldonium in horses necessitates further study. Eight exercised adult thoroughbred horses received a single oral dose of 3.5, 7.1, 14.3 or 21.4 mg/kg of meldonium. Blood and urine samples were collected and analyzed using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were determined using non‐compartmental analysis. Maximum serum concentrations ranged from 440.2 to 1147 ng/mL and the elimination half‐life from 422 to 647.8 h. Serum concentrations were below the limit of quantitation by days 4, 7, 12 and 12 for doses of 3.5, 7.1, 14.3 and 21.4 mg/kg, respectively. Urine concentrations were below the limit of detection by day 44 following administration of 3.5 mg/kg and day 51 for all other dose groups. No adverse effects were observed following meldonium administration. While the group numbers were small, changes in heart rate were observed in the 3.5 mg/kg dose group (n = 1). Glucose concentrations changed significantly in all dose groups studied (n = 2 per dose group). Similar to that reported for humans, the detection time of meldonium in biological samples collected from horses is prolonged, which should allow for satisfactory regulation in performance horses. Copyright © 2017 John Wiley & Sons, Ltd. Following oral administration of 3.5, 7.1, 14.3 or 21.4 mg/kg meldonium to exercised thoroughbred horses, serum concentrations were below the limit of quantitation LOQ by days 4, 7, 12 and 12 for doses of 3.5, 7.1, 14.3 and 21.4 mg/kg, respectively. Urine concentrations were below the limit of detection by day 44 following administration of 3.5 mg/kg and day 51 for all other dose groups. Heart rate was increased in the 3.5 mg/kg dose group and glucose concentrations increased in all dose groups post‐administration.

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Interlaboratory trial for the measurement of total cobalt in equine urine and plasma by ICP‐MS  Voir?

Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma study, has been undertaken. Quantification of cobalt in the biological samples was performed by inductively coupled plasma‐mass spectrometry (ICP‐MS). Ring tests were based on the analysis of 5 urine samples supplemented at concentrations ranging from 5 up to 500 ng/mL and 5 plasma samples spiked at concentrations ranging from 0.5 up to 25 ng/mL. The results obtained from the different laboratories were collected, compiled, and compared to assess the reproducibility and robustness of cobalt quantification measurements. The statistical approach for the ring test for total cobalt in urine was based on the determination of percentage deviations from the calculated means, while robust statistics based on the calculated median were applied to the ring test for total cobalt in plasma. The inter‐laboratory comparisons in urine and in plasma were successful so that 97.6% of the urine samples and 97.5% of the plasma samples gave satisfactory results. Threshold values for cobalt in plasma and urine were established from data only obtained by laboratories involved in the ring test. Copyright © 2017 John Wiley & Sons, Ltd. In order to control the misuse of cobalt, It was necessary to establish an international threshold for cobalt in plasma and/or in urine. An international collaboration, consisting of an interlaboratory comparison, between 5 laboratories for the urine study and 8 laboratories for plasma study has been successfully undertaken.

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Doping control analysis of lithium in horse urine and plasma by inductively coupled plasma mass spectrometry  Voir?

Lithium salts are commonly prescribed to treat bipolar disorder in humans. They are effective for the treatment of acute mania and the prophylaxis of manic relapses through long‐term use. Although there is no reported legitimate therapeutic use of lithium in horses, its potential mood‐stabilizing effect, low cost, and ready availability make lithium salt a potential agent of abuse in equine sports, especially for equestrian competition horses. Lithium can be found in soil, plants, and water, as such it is naturally present in the equine body, thus a threshold is necessary to control its misuse in horses. This paper describes the validation of quantification methods for lithium in equine urine and plasma using inductively coupled plasma mass spectrometry (ICP‐MS). Based on a population study of lithium in horse urine and an administration study using a single oral dose of lithium chloride (100 mg) to mimic the daily lithium intake from a diet rich in lithium, a urinary threshold of 5 ÎŒg/mL was proposed. Applying this urinary threshold to two other administration studies (a single oral dose of 65 g of lithium chloride, and a single intravenous dose of 2.54 g of lithium chloride), excessive lithium in urine could be detected for 8 days and 2.5 days respectively. The concentrations of lithium in plasma following these three lithium chloride administration trials were also studied. Copyright © 2017 John Wiley & Sons, Ltd. Lithium salts are commonly prescribed to treat bipolar disorder in humans. As they have mood‐stabilizing effect and are orally active and inexpensive, lithium salts are attractive doping agents in equine sports. This paper describes the validation of quantification methods for lithium in equine urine and plasma using inductively coupled plasma mass spectrometry (ICP‐MS). Population studies of lithium in horse urine and horse administration studies of lithium chloride were carried out. A urinary threshold of lithium was proposed based on these studies.

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Identification of porcine relaxin in plasma by liquid chromatography‐high resolution mass spectrometry  Voir?

Relaxin (RLX) has demonstrated diverse pharmacological effects in various scientific and clinical studies. The emergence of porcine relaxin (pRLX) has raised concerns on the doping potential of pRLX. There have also been speculations in the horseracing industry on its covert use. To control the abuse of pRLX in equine sports, a method to detect pRLX effectively and to provide its unequivocal identification in equine biological samples is required. This paper reports on the detection and confirmation of pRLX in equine plasma by liquid chromatography‐high resolution mass spectrometry. pRLX was isolated from equine plasma by immunoaffinity purification using anti‐pRLX antibody‐coated magnetic beads. Anti‐pRLX antibody was generated in‐house by purifying antisera from rabbits immunized with pRLX. The isolated pRLX was subjected to reduction of their disulfide bonds to obtain their respective A‐ and B‐chains. The extracts were then further purified and concentrated prior to reversed‐phase LC separation and high resolution accurate mass measurement. As detection of the A‐chains was far more sensitive than that of the B‐chains, the A‐chain of pRLX was set as the targets for detection and confirmation. The limit of detection for pRLX was around 0.005 ng/mL (~ 0.86 fM) and the limit of confirmation was around 0.02 ng/mL (~ 3.4 fM). It was observed that method sensitivity was improved at least 5‐fold by using an EASY‐spray column and emitter in place of the conventional ESI source. The applicability of this method was demonstrated by the identification of pRLX and its metabolites in equine plasma obtained after subcutaneous administration. To our knowledge, this is the first report of a validated mass spectrometry method for the unequivocal confirmation of pRLX in any biological fluid. Copyright © 2016 John Wiley & Sons, Ltd. Relaxin is an insulin‐like protein with multiple and diverse pharmacological functions. Intelligence has shown that RLX has already been abused to improve racehorse performance. A sensitive and selective method for the identification of pRLX in equine plasma is presented in this paper.

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Confirmatory analysis of etanercept in equine plasma by LC‐MS for doping control  Voir?

Etanercept is a protein‐based medication for the treatment of human patients with rheumatoid arthritis and other autoimmune‐based diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography‐mass spectrometry (LC‐MS) method for confirmation of etanercept in equine plasma. Etanercept was extracted from plasma by anti‐human IgG antibody linked to magnetic beads. The analyte was reduced and alkylated, and then digested by trypsin. Tryptic peptides (T1 from human tumour necrosis factor receptor 2 of etanercept, T15 and T27 from human IgG1 of the protein) were employed for detection and confirmation of the analyte. The limit of detection was 5 ng/mL, and the limit of confirmation 10 ng/mL. This method is specific for confirmation of etanercept, as assessed using the results from BLAST and SEQUEST searches. The results from SEQUEST searches also revealed an unexpected unique specificity of product ion spectrum of IgG1 T27 with only a single product ion for identification of etanercept. It is the first report for such a finding, to the authors' knowledge. The method was successful in analyses of the plasma samples collected post administration of etanercept to horses. Etanercept was detected up to 11 days post administration. This method will be helpful for confirmation of etanercept or other protein‐based drugs consisting of human IgG1, in equine drug testing. Copyright © 2016 John Wiley & Sons, Ltd. The present study has established an LC‐MS method for confirmation of etanercept in equine plasma. This is the first reported method for confirmation of etanercept in plasma by LC‐MS.

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Two complementary methods to control gonadotropin‐releasing hormone vaccination (Improvacź) misuse in horseracing: Enzyme‐linked immunosorbent assay test in plasma and steroidomics in urine  Voir?

Since the availability on the European market of the vaccine Improvacź dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone‐releasing hormone or gonadotropin‐releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvacź vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti‐GnRH antibodies was detected up to 200 days after the first injection. Anti‐GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme‐linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John Wiley & Sons, Ltd. Two new analytical approaches are proposed to the horseracing industry to prevent Improvacź misuse. These are an enzyme‐linked immunosorbent assay test to detect anti‐gonadotropin‐releasing hormone antibodies in plasma and a complementary study with urine by steroidomics. The two methods allow the control of immunological castration.

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Intelligence‐based anti‐doping from an equine biological passport  Voir?

The move towards personalized medicine derived from individually focused clinical chemistry measurements has been translated by the human anti‐doping movement over the past decade into developing the athlete biological passport. There is considerable potential for animal sports to adapt this model to facilitate an intelligence‐based anti‐doping system. Copyright © 2017 John Wiley & Sons, Ltd. The move towards personalized medicine derived from individually‐focused clinical chemistry measurements has been translated by the human anti‐doping movement over the past decade into developing the athlete biological passport (ABP). There is considerable potential for animal sports to adapt this model to facilitate an intelligence‐based anti‐doping system.

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RNA sample preparation applied to gene expression profiling for the horse biological passport  Voir?

The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis‐stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal species have suggested that modification of gene expression offers promising new methods of improving the window of detection of banned substances by targeting their effects on blood cell gene expression. In this context, the present study describes the possibility of using a modified version of the dedicated Human IVD (in vitro Diagnostics) PAXgeneź Blood RNA Kit for horse gene expression analysis in blood collected on PAXgeneź tubes applied to the horse biological passport. The commercial kit was only approved for human blood samples and has required an optimization of specific technical requirements for equine blood samples. Improvements and recommendations were achieved for sample collection, storage and RNA extraction procedure. Following these developments, RNA yield and quality were demonstrated to be suitable for downstream gene expression analysis by qPCR techniques. Copyright © 2017 John Wiley & Sons, Ltd. The present study described the possibility to use a modified version of the dedicated human IVD PAXgeneź Blood RNA Kit for gene expression analysis from horse blood collected on PAXgeneź tubes applied to the horse biological passport.

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Equine performance genes and the future of doping in horseracing  Voir?

A horse's success on the racetrack is determined by genetics, training and nutrition, and their translation into physical traits such as speed, endurance and muscle strength. Advances in genetic technologies are slowly explaining the roles of specific genes in equine performance, and offering new insights into the development of novel therapies for diseases and musculoskeletal injuries that cause early retirement of many racehorses. Gene therapy approaches may also soon provide new means to artificially enhance the physical performance of racehorses. Gene doping, the misuse of gene therapies for performance enhancement, is predicted to be the next phase of doping faced by horseracing. The risk of gene doping to human sports has been recognised for almost 15 years, and the introduction of the first gene doping detection tests for doping control in human athletes is imminent. Gene doping is also a threat to horseracing, but there are currently no methods to detect it. Efficient and accurate detection methods need to be developed to deter those looking to use gene doping in horses and to maintain the integrity of the sport. Methods developed for human athletes could offer an avenue for detection in racehorses. Development of an equine equivalent test will first require identification of equine genes that will likely be targeted by gene doping attempts. This review focuses on genes that have been linked to athletic performance in horses and, therefore, could be targeted for genetic manipulation. The risks associated with gene doping and approaches to detect gene doping are also discussed. Copyright © 2017 John Wiley & Sons, Ltd. Gene doping is predicted to be the next phase of doping faced by horseracing. Development of an equine gene doping test will first require identification of equine genes that will likely be targeted by gene doping attempts. This review focuses on genes that have been linked to athletic performance in horses and, therefore, could be targeted for genetic manipulation.

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Dernière mise à jour : 27/11/2013 @ 22:54

 
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