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news.gifArticles - - Drug Testing and Analysis

Drug Testing and Analysis


Wiley Online Library : Drug Testing and Analysis


Comparative pharmacokinetics of trandolapril, its active metabolite, and verapamil in human plasma of Egyptian population using HPLC–MS/MS  Voir?

Trandolapril and verapamil are commonly used antihypertensive drugs. However, there is a lack of available data on the change of their pharmacokinetics in patients with liver or kidney impairment and hence the need for dose adjustment. In the presented work, a high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for the monitoring of trandolapril, its active metabolite trandolaprilat, and verapamil in human plasma of patients with renal impairment and/or liver insufficiency. The chromatographic separation was achieved on a Gemini C18 reversed phase column using a gradient elution mode with a run time of 10 min. The mobile phase consisted of a mixture of methanol and 2% acetic acid. The electrospray ionization MS/MS analysis was performed in multiple reaction monitoring mode. The assay was validated as per FDA guidelines for bioanalytical method validation and proved to be suitable for the determination of therapeutic drug levels in plasma. The inter‐group changes in pharmacokinetic data were compared to that of healthy volunteers. The comparison showed a significant difference in the pharmacokinetic parameters between the studied groups. The presented results exhibit the benefits of the proposed assay as a validated analytical tool for the continuous drug monitoring.

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Keratinous matrices for the assessment of drugs of abuse consumption: a correlation study between hair and nails  Voir?

Keratinous matrices ‐ hair and nails ‐ accumulate substances over time and allow retrospective investigation of past consumption. Analysis of these matrices can provide information complementary to blood and urine analysis or can be used as standalone. So far, research has primarily focused on the detection of substances in hair, while studies in nails are scarce. In this study, we assessed concentrations of drugs of abuse and their metabolites in hair, finger‐ and toenails collected from the same individuals to evaluate differences and correlations between matrices. A total of 26 hair, 24 fingernail, and 18 toenail samples were collected. Samples were analyzed by a validated liquid chromatography‐tandem mass spectrometry method able to simultaneously detect the following compounds: amphetamine (AMP), methamphetamine, 3,4‐methylenedioxymethamphetamine (MDMA), 3,4‐methylenedioxyethylamphetamine, morphine (MOR), codeine (COD), 6‐monoacetylmorphine (6‐MAM), methadone (MTD), 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), cocaine (COC), benzoylecgonine (BE), and ecgonine methyl ester (EME). Strong positive correlations between hair, finger‐ and toenails were present for COC, BE, EME, AMP and MDMA. MOR, COD, 6‐MAM, MTD and EDDP showed positive trends. Concentrations were generally higher in nails compared to hair. Ratios between parent compounds and their metabolites were assessed for 6‐MAM/MOR, EDDP/MTD, BE/COC and EME/COC. Preliminary cut‐off concentrations for COC, BE, EME and AMP in finger‐ and toenails were proposed. In light of these results, nails can be considered as a useful alternative to hair for monitoring of long‐term drug consumption. However, care should be taken regarding the variability in the accumulation of compounds between the matrices.

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Different in vitro and in vivo tools for elucidating the human metabolism of alpha‐cathinone‐derived drugs of abuse  Voir?

In vitro and in vivo experiments are widely used for studying the metabolism of new psychoactive substances (NPS). The availability of such data is required for toxicological risk assessments and development of urine screening approaches. This study investigated the in vitro metabolism of the five pyrrolidinophenone‐derived NPS alpha‐pyrrolidinobutyrophenone (alpha‐PBP), alpha‐pyrrolidinopentiothiophenone (alpha‐PVT), alpha‐pyrrolidinohexanophenone (alpha‐PHP), alpha‐pyrrolidinoenanthophenone (alpha‐PEP, PV8), and alpha‐pyrrolidinooctanophenone (alpha‐POP, PV9). First, they were incubated with pooled human liver microsomes (pHLM) or pooled human liver S9 fraction (pS9) for identification of the main phase I and II metabolites. All substances formed hydroxy metabolites and lactams. Longer alkyl chains resulted in keto group and carboxylic acid formation. Comparing these results with published data obtained using pHLM, primary human hepatocytes (PHH), and authentic human urine samples, PHH provided the most extensive metabolism. Second, enzyme kinetic studies showed that the initial metabolic steps were formed by cytochrome P450 isoforms (CYP) CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 resulting in pyrrolidine, thiophene or alkyl hydroxy metabolites depending on the length of the alkyl chain. The kinetic parameters indicated an increasing affinity of the CYP enzymes with increase of the length of the alkyl chain. These parameters were then used to calculate the contribution of a single CYP enzyme to the in vivo hepatic clearance. CYP2C19 and CYP2D6 were mainly involved in the case of alpha‐PBP and CYP1A2, CYP2C9 and CYP2C19 in the case of alpha‐PVT, alpha‐PHP, alpha‐PEP, and alpha‐POP.

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Phase I metabolism of the recently emerged synthetic cannabinoid CUMYL‐PEGACLONE and detection in human urine samples  Voir?

Indole, indazole or azaindole based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl‐derivative, CUMYL‐PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ‐carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography tandem mass spectrometry and liquid chromatography high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL‐PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono‐hydroxylated at the γ‐carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL‐PEGACLONE uptake. Moreover, three immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross‐reactivity.

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Development of an immunochromatographic strip incorporating anti‐mitragynine monoclonal antibody conjugated to colloidal gold for kratom alkaloids detection  Voir?

A lateral flow‐based immunochromatographic strip was developed for the rapid detection of mitragynine (MG), a dominant alkaloid found in the leaves of kratom. Monoclonal antibody (mAb) against MG (anti‐MG mAb) was conjugated to colloidal gold and used as a recognition probe. MG‐ovalbumin conjugate (MG‐OVA) and goat anti‐mouse IgG were immobilized on the strip to produce a test zone and control zone, respectively. Based on the principle of a competitive assay, MG in a test sample competed with MG‐OVA resident in the test zone to bind with colloidal gold‐anti‐MG mAb, resulting in an inverse relation of color intensity at the test zone and MG amount. The limit of detection (LOD) of the immunochromatographic strip is determined at 1 mg/mL of MG by visual assessment and 0.60 mg/mL by Image J analysis. The developed immunochromatographic strip can determine MG in kratom cocktails and kratom leaf samples. It could serve as a rapid and simple diagnostic kit for the detection of MG in kratom samples.

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Effect of glucocorticoid administration on the steroid profile  Voir?

The steroid profile (SP) is a powerful tool to detect the misuse of endogenous anabolic androgenic steroids in sports, and it is included in the athlete biological passport (ABP). Glucocorticoids (GCs), which are widely prescribed in sports and are only prohibited in competition by systemic routes, inhibit the hypothalamic‐pituitary‐adrenal axis. Since the metabolites monitored in the SP have a partial adrenal origin, their excretion in urine might be altered by GCs consumption. The aim of the present work was to investigate if GCs administered by either systemic or local routes could influence the SP parameters. Three of the most frequently detected GCs in sports (prednisolone, betamethasone and triamcinolone acetonide) were administered to healthy male and female volunteers (n=40) using different administration routes (topical, oral and intramuscular administration at different doses). In total, 66 administrations of GCs were performed. Urine samples were collected before and after GCs administration. The SP was measured using gas chromatography‐mass spectrometry. The excretion rates of the SP metabolites decreased after systemic GCs administration. This excretion decrease showed to be associated with the dose and the administration route. However, the individual evaluation of the SP ratios (T/E, A/T, A/Etio, 5αAdiol/5ÎČAdiol and 5αAdiol/E) led to normal sequences for all the conditions tested. Therefore, GCs administration did not produce misinterpretations on the ABP evaluation. According to these results, GCs administration should not distort the establishment of normal ranges of the SP ratios, and does not need to be considered a confounding factor in the SP evaluation.

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Growth Hormone Isoform‐Differential Mass Spectrometry for Doping Control Purposes  Voir?

Mass spectrometry (MS) allows for monitoring growth‐hormone (GH) isoform compositions at high specificity. It is demonstrated, that this capability can be used to reliably detect alterations as elicited by (putative) doping with 22 kDa‐GH. Sample treatment consists in enzymatic protein cleavage, followed by two‐step liquid chromatography clean‐up, prior to analysis by MS. The protocol does not depend on antibodies for analyte extraction, at any stage. Therefore, MS opens an opportunity to independent confirmation, if combined with the antibody‐based isoform differential test presently used in practice. To probe for the fitness‐for‐purpose of this concept, GH‐free serum was spiked with pure 22 kDa‐ and 20 kDa‐GH covering a representative range of concentrations (0.5–9.4 ÎŒg/L), while the 22 kDa fraction was within a range of 80–85%, either, or at 100%, the latter simulating an administration of 22 kDa‐GH. Mean deviation of 22 kDa‐fractions found from expectation was within less than 3%. Beyond this, results by antibody‐free isoform‐differential MS, as described, were in line with those of the WADA‐approved antibody‐based test for 18 native sera and 3positive controls. In this context, relating 22 kDa‐GH to total‐GH rather than (22 kDa+20 kDa) was considered as an alternative strategy to earlier approaches. 20 kDa‐GH as additional measurand, next to 22 kDa‐ and total‐GH, however, provides useful extra‐information, as it directly indicates presence or absence of a non‐22 kDa‐GH form.

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Use of split‐free nLC‐MS/HRMS interface to improve the detection of α‐cobratoxin in equine plasma for doping control  Voir?

Cobra (Naja naja kaouthia) venom contains a toxin called α‐cobratoxin (α‐Cbtx) containing 71 amino acids (MW 7821 Da) with a reported analgesic power greater than morphine. In 2013, the first analytical method for the detection of α‐Cbtx in equine plasma was developed by Bailly‐Chouriberry et al. allowing the confirmation of the presence of α‐Cbtx at low concentrations (1‐5 ng/mL or 130‐640 fmol/mL) in plasma samples. In order to increase the method sensitivity and therefore to improve the detection of α‐Cbtx in post‐administration plasma samples, a nano‐liquid chromatography ‐ mass spectrometry/high resolution mass spectrometry (nLC‐MS/HRMS) method was developed. This new method allowed us to confirm the presence of α‐Cbtx in plasma samples spiked at 100 pg/mL (12.8 fmol/mL) and the detection of α‐Cbtx was obtained in plasma samples collected 72h post‐administration (50 pg/mL or 6.4 fmol/mL) which was defined as the limit of detection (LOD). The presented method is twenty‐fold more sensitive compared to the method previously described.

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GC‐MS/MS DETECTS POTENTIAL PREGABALIN ABUSE IN SUSCEPTIBLE SUBJECTS' HAIR  Voir?

Pregabalin, a GABA analogue, binds to the alpha 2 delta subunit of voltage‐dependent calcium channels. It is recognized as efficacious in pathologies such as epilepsy, neuropathic pain and anxiety disorders. Since pregabalin prescriptions have increased worldwide, reports of its abuse have been accumulating, mainly in patients with opioid abuse disorders. The present study investigated potential pregabalin abuse by means of hair analysis, a matrix that provides valuable retrospective information. Half of the pool of 280 susceptible patients had been occasional drug users and were being monitored for driving licence renewals. The other 140 patients had a history of opiate dependency and were monitored to assess compliance with methadone therapy. In view of determining pregabalin in hair samples, it was extracted in methanol, successfully derivatized to give the ethyl chloroformate derivative, and finally pregabalin was analyzed by gas chromatography/tandem mass spectrometry. Selectivity, linearity, limit of detection, limit of quantification, recovery, intra‐ and inter‐ day precision, and accuracy of the quantification procedure were appraised. Pregabalin limits of detection and quantification were 30 pg/mg and 50 pg/mg, respectively. We found 10.7% of hair samples from methadone patients and 4.29% from occasional drug users were positive to pregabalin without medical prescription. The mean pregabalin concentration in hair was higher than in consumers with medical indications (1.45 ng/mg vs 0.74 ng/mg). These results suggest that pregabalin possesses a significant abuse potential particularly among individuals attending opiate dependence services and that pregabalin abuse is a serious emerging issue, which should be carefully monitored.

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Effect of non‐prohibited drugs on the phase II metabolic profile of morphine. An in vitro investigation for doping control purposes  Voir?

The potential consequences of drug‐drug interaction on the strategies adopted by the anti‐doping laboratories to report an adverse analytical finding for morphine were investigated. We have evaluated in vitro the effects of fourteen drugs on the principal metabolic pathways of morphine. The selected drugs are among those most commonly used by the athletes, none of them presently included in the World Anti‐Doping Agency Prohibited List. The non‐prohibited drugs included four antifungals (fluconazole, itraconazole, ketoconazole and miconazole), six benzodiazepines (alprazolam, bromazepam, clonazepam, lorazepam, lormetazepam and triazolam), and four non‐steroidal anti‐inflammatory drugs (diclofenac, ibuprofen, ketoprofen and nimesulide). The in vitro assays were based on the use of either human liver microsomes or uridine 5'‐diphospho‐glucuronosyl‐transferases. Morphine and its glucuronides were determined by developed liquid chromatography‐mass‐spectrometry procedure after dilution with an aqueous solution containing their deuterated isotopologues as internal standards. Morphine is mainly excreted as phase II metabolites: about 70% of the parent compound is found to be biotransformed by the UGT2B7 to morphine‐3‐glucuronide (6065%) and morphine‐6‐glucuronide (5‐10%). A reduction of the enzymatic activity of the UGT2B7 was recorded in the presence of nine of the fourteen drugs under investigation (namely: ketoconazole, miconazole, itraconazole, diclofenac, ibuprofen, clonazepam, lorazepam, lormetazepam and triazolam), with a consequent significant reduction of the levels of the glucuronide metabolites. This phenomenon in vivo may affect the rate of the urinary excretion of morphine with the risk to report “false negative” results, especially in case of results close to the decision limit value set by the WADA.

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Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration  Voir?

In the fight against doping, the introduction of alternative markers to the steroid profile can be considered as an effective approach to improve the screening capabilities for the detection of testosterone (T) misuse. The aim of this study was to evaluate the potential of several T metabolites (cysteinyl conjugated and glucuronoconjugated resistant to enzymatic hydrolysis) to detect both the transdermal and the intramuscular administration of T. In Part I of the study, we studied the potential of these metabolites for the detection of T transdermal administration. Results revealed that resistant glucuronides can be a suitable complement to the current steroid profile. In this, Part II, dedicated to the intramuscular administration, we studied the potential of cysteinyl conjugated, resistant glucuronoconjugated and 1‐cyclopentenoylglycine (1‐CPG) for the detection of a single intramuscular injection of T cypionate. Possible differences in the excretion profile of all markers were explored between individuals with low basal (n=6) and medium basal (n=6) values of the testosterone/epitestosterone ratio (T/E). The results showed that all tested markers presented low intra‐individual stability in basal conditions. Despite this, all glucuronoconjugated markers and 1‐CPG, but not the cysteinyl conjugated markers, provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile. Based on the results obtained from the 2 parts of this study and from previously reported data, the potential applicability and the limitations of including these markers in the steroid profile are discussed. The use of alternative markers can improve the screening capabilities for the detection of testosterone misuse. In Part II of this study, we studied the potential of cysteinyl conjugated, resistant glucuronoconjugated and 1‐cyclopentenoylglycine (1‐CPG) for the detection of a single intramuscular injection of T cypionate. Glucuronoconjugated markers and 1‐CPG, but not cysteinyl conjugated markers, provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile.

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Sensitivity of doping biomarkers after administration of a single dose testosterone gel  Voir?

Micro‐doping with testosterone (T) is challenging to detect with the current doping tests. Today, the methods available to detect T are longitudinally monitoring of urine biomarkers in the Athlete Biological Passport (ABP) and measuring the isotopic composition of excreted biomarkers to distinguish the origin of the molecule. In this study, we investigated the detectability of a single dose of 100 mg T gel in 8 healthy male subjects. We also studied which biomarkers were most sensitive to T gel administration, including blood biomarkers. The ABP successfully detected T gel administration in all 8 subjects. The most sensitive ratio was 5αAdiol/E, however, all ratios showed atypical findings. Isotope ratio mass spectrometry (IRMS) was performed on 5 subjects and only 2 met all the criteria for a positive test according to the rules set by the World Anti‐Doping Agency (WADA). The other 3 showed inconclusive results. Other markers that were affected by T gel administration, not used for this detection today, were serum dihydrotestosterone (DHT) and T as well as reticulocyte count and percentage in whole blood. miRNA‐122 was not significantly affected by the single T dose. A single dose of 100 mg T gel is possible to detect with today's doping tests. Since a single dose of T gel has an impact on some hematological biomarkers, access to both modules of the ABP when evaluating the athletes' profiles will increase the possibility to detect micro‐doses of T. In addition, serum DHT and T may be a useful addition to the future endocrine module of the ABP. ABP could detect a single dose of T gel in all of the subjects when three baseline values were present. On the other hand, IRMS only showed positive in 40% of the samples investigated. In addition, serum hormones, such as DHT, should be further investigated as possible complementary biomarkers in longitudinal testing.

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Detection of Sotatercept (ACE‐011) in human serum by SAR‐PAGE and western single blotting  Voir?

A method for the detection of Sotatercept (ACE‐011, ACVR2A‐Fc) in human serum is presented. The method is a modification of a recently published protocol for Luspatercept (ACE‐536, ACVR2B‐Fc), another erythropoiesis stimulating fusion protein. Out of 27 tested antibodies against either the extracellular domain of ACVR2A or the full‐length protein, only 4 antibodies bound strongly enough to Sotatercept for usage with immunoprecipitation followed by SAR‐PAGE and Western single blotting. The adapted protocol allows the detection of 0.1 ng/mL Sotatercept in just 50 ÎŒL human serum. None of the 3 commercial ACVR2‐ELISAs was able to detect Sotatercept and the 2 tested surrogate proteins, even in the ÎŒg/mL range. As for Luspatercept, only IPG‐IEF/2D‐PAGE generated discrete isoforms. Due to the long serum half‐life, the SAR‐PAGE method will be able to detect Sotatercept for several weeks and will be very useful in doping testing. Our recently published SAR‐PAGE method for the detection of Luspatercept (ACE‐536, ACVR2B‐Fc) in human serum was adapted accordingly in order to detect Sotatercept (ACE‐011, ACVR2A‐Fc). The final protocol allows the detection of Sotatercept down to 0.1 ng/mL in just 50 ÎŒL of serum. Due to its high sensitivity, the method will be very useful for anti‐doping control.

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Synthesis of a novel polymeric magnetic solid phase extraction adsorbent for selective extraction of amphetamine from urine samples coupled with high performance liquid chromatography  Voir?

A novel pH‐responsive block copolymer (Poly ethylene glycol‐b‐poly (N,N‐dimethylaminoethylmethacrylate‐co‐maleic acid) was designed for the decoration and stabilization of magnetic nanoparticles (MNPs) as an efficient magnetic nano adsorbent for extraction of amphetamine (AM) from biological urine samples to be determined by high performance liquid chromatography–ultra violet detector (HPLC–UV). Full characterization of the synthesized polymeric magnetic nanoparticles (PMNPs) were followed by various techniques like Fourier transform infrared (FT–IR) spectroscopy, powder x‐ray diffraction (XRD), scanning electron microscopy (SEM), and vibrating sample magnetometer (VSM). Important extraction parameters including pH, amount of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, agitation rate (rpm), and ionic strength of the extraction medium were studied and optimized. Under optimized extraction conditions, good linearity was observed in the concentration range of 30–2000 ng/mL for AM. The amount of the qe was calculated as 0.18 (mg/g). The method was applied in determination of AM from positive urine samples with the recovery of 99.84%. Results indicated that the proposed method could be applied in clinical and forensic laboratories for simple, selective, and fast determination of AM from urine samples. A novel pH‐responsive block copolymer (Poly ethylene glycol‐b‐poly (N,N‐ dimethylaminoethylmethacrylate‐co‐maleic acid) was designed for the decoration and stabilization of magnetic nanoparticles (MNPs) to apply as an efficient magnetic nano adsorbent for extraction of amphetamine (AM) from biological urine samples to be determined by high performance liquid chromatography ultra violet detector (HPLC‐UV).

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Surveillance of drug abuse in Hong Kong by hair analysis using LC‐MS/MS  Voir?

The aim of this study is to reveal the habits of drug abusers in hair samples from drug rehabilitation units in Hong Kong. With the application of liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology, a total of 1771 hair samples were analyzed during the period of hair testing service (January 2012 to March 2016) provided to 14 drug rehabilitation units including non‐governmental organizations (NGOs), rehabilitation centers, and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously, including ketamine, norketamine, cocaine, benzoylecgonine, cocaethylene, norcocaine, codeine, MDMA, MDA, MDEA, amphetamine, methamphetamine, morphine, 6‐acetylmorphine, phencyclidine, and methadone. The results showed that ketamine (77.2%), cocaine (21.3%), and methamphetamine (16.5%) were the frequently detected drugs among those drug abusers, which is consistent with the reported data. In addition, the usage of multiple drugs was also observed in the hair samples. About 29% of drug‐positive samples were detected with multiple drug use. Our studies prove that our locally developed hair drug‐testing method and service can be a valid tool to monitor the use of abused drugs, and which could facilitate rehabilitation program management. With the application of LC‐MS/MS technology, a total of 1,771 hair samples were analyzed from drug rehabilitation units including non‐governmental organizations (NGOs), rehabilitation centers and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously. The usage of multiple drugs was observed in the hair samples. Our studies prove that our locally developed hair drug testing method and service can be a valid tool to monitor the use of abused drugs.

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Annual banned‐substance review: Analytical approaches in human sports drug testing  Voir?

Several high‐profile revelations concerning anti‐doping rule violations over the past 12 months have outlined the importance of tackling prevailing challenges and reducing the limitations of the current anti‐doping system. At this time, the necessity to enhance, expand, and improve analytical test methods in response to the substances outlined in the World Anti‐Doping Agency's (WADA) Prohibited List represents an increasingly crucial task for modern sports drug‐testing programs. The ability to improve analytical testing methods often relies on the expedient application of novel information regarding superior target analytes for sports drug‐testing assays, drug elimination profiles, alternative test matrices, together with recent advances in instrumental developments. This annual banned‐substance review evaluates literature published between October 2016 and September 2017 offering an in‐depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2017 Prohibited List. Detection assays for drugs and methods of sports doping published between 2016 and 2017 are critically reviewed and evaluated in context with the Prohibited List 2017 as established by the World Anti‐Doping Agency.

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A simple validated multi‐analyte method for detecting drugs in oral fluid by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS)  Voir?

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. 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, and Δ9‐ tetrahydrocannabinol. Thirty‐seven analytes were validated using the developed UPLC–MS/MS multi‐analyte 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%. 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. A UPLC‐MS/MS method was developed to identify and quantitate 37 commonly abused drugs in oral fluid. Drugs of interest included amphetamines, benzodiazepines, cocaine, opiates, opioids, phencyclidine and tetrahydrocannabinol. Sample preparation and extraction are simple, and analysis times short. Validation showed satisfactory performance at relevant concentrations. The possibility of contaminated samples as well as the interpretation in relation to well‐knows matrices, such as urine, will demand further study.

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Semi‐quantitative analysis of tramadol, dextromethorphan, and metabolites in decomposed skeletal tissues by ultra performance liquid chromatography quadrupole time of flight mass spectrometry  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 minutes post‐dose. Rats (n=4) received 75 mg/kg dextromethorphan and were euthanized by CO2 asphyxiation approximately 45 minutes post‐dose. Remains decomposed to skeleton outdoors and vertebral bones were collected. Bones were cleaned, dried, and pulverized to a fine powder. Bones 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 minutes 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. A rapid and sensitive method for analysis of dextromethorphan (DXM), tramadol (TRAM) and selected metabolites by dynamic methanolic extraction, filtration/pass‐through extraction (FPTE) and UPLC‐qTOF‐MS is described. Analysis time, sensitivity and analyte compatibility are superior to those in previous methods based on SPE and GC‐MS. The method was applied to analysis of bone of rats acutely exposed to either DXM or TRAM by intraperitoneal injection. All analytes included in method validation were detected in all samples analyzed by this method.

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Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration  Voir?

Although the introduction by the World Anti‐Doping Agency (WADA) of the steroid module of the athlete biological passport (ABP) marked an important step forward in the screening of testosterone (T) misuse, it still remains one of the most difficult challenges in doping control analysis. The urinary determination of alternative markers has been recently reported as a promising tool for improving the screening of T oral administration. However, their evaluation for other, commonly used, administration routes is still required. The main goal of this study is the evaluation of the potential of 2 groups of metabolites (cysteinyl conjugated and glucuronoconjugated) after transdermal and intramuscular administration of T. Their suitability was evaluated in individuals with both low basal (L‐T/E) and medium basal (M‐T/E) values of T/E. In this Part I, we evaluated the urinary excretion profile of these 2 groups of T metabolites after the administration of 3 doses of T gel to 12 volunteers (6 L‐T/E and 6 M‐T/E) for 3 consecutive days. For this purpose, 9 different concentration ratios (5 cysteinyl conjugated and 4 glucuronoconjugated markers) were studied. Both, the intra‐individual variability and the detection windows (DW) obtained by each ratio were evaluated. Cysteinyl conjugates showed a general low intra‐individual variability and DWs that were shorter than any other tested marker. Despite the relatively large intra‐individual variability, the DWs reached by glucuronoconjugates (2–3 days) were similar to those obtained by markers currently included in the ABP. Overall; this evaluation advises for the introduction of additional glucuronoconjugated markers in the screening of transdermal T administration. The screening of testosterone misuse still remains one of the most difficult challenges in doping control analysis. In this study, we evaluated the potential of 2 groups of metabolites (cysteinyl conjugates and resistant glucuronoconjugates) for the detection of transdermal administration of T. Detection windows reached by glucuronoconjugates were similar to those currently obtained, whereas poorer results were obtained with cysteinyl conjugates.

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Development and validation of an ultrahigh performance liquid chromatography‐high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy  Voir?

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA‐carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography–high resolution tandem mass spectrometry (UHPLC–HRMS/MS) with full‐scan/data‐dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (HILIC) column (100 x 2.1 mm, 1.7 ÎŒm). Serum samples (250 ÎŒL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 to 2000 ng/mL for HGA and from 10 to 1000 ng/mL for MCPA‐carnitine. HGA and MCPA‐carnitine showed acceptable accuracy and precision (bias ‐3.0% to 1.1%; RSD 9.2% to 12.4%). The limit of quantification (LOQ) was defined as the lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from ‐79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples, respectively. Applicability was tested in 10 authentic AM cases. In all specimens, relevant amounts of HGA and MCPA‐carnitine were found (570–2000 ng/mL; ~8.5–150 ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA‐carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM. Hypoglycine A (HGA), an amino acid present in sycamore maple seeds, is suspected to provoke equine atypical myopathy (AM). The aim of this study was the development of a fast and easy ultra high performance liquid chromatography‐high resolution tandem mass spectrometry method for reliable identification and sensitive quantification of HGA and its metabolites especially methylene cyclopropyl acetic acid carnitine (MCPAC) in horse serum. The fully validated method could be successfully applied to real cases of suspected AM.

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Elimination profile of triamcinolone in urine following oral administration  Voir?

Triamcinolone (T) is a glucocorticoid commonly used to relieve inflammation and treat arthritis, severe allergies, and asthma; however, it is banned by the World Anti‐Doping Agency in competition for athletes when administered orally, intravenously, intramuscularly, or rectally. The minimum required performance limit (MRPL) for urinary T is 30 ng/mL. However, the data about the urinary excretion of T after oral administration is limited. We investigate the elimination profile and determine whether single‐dose administration of T would cause a positive doping result. Twelve healthy volunteers received a single‐dose of 4‐mg T rally, and urine samples were collected for 24 hours. A validated liquid chromatography–tandem mass spectrometry method was used to determine urinary T levels. Non‐compartmental modeling was used to estimate the pharmacokinetic parameters. All the urinary T concentrations were much higher than the MRPL. The peak urinary T concentration was 3211.4 ± 860.3 ng/mL (mean ± SD), time to peak concentration was 1.7 ± 0.9 hours, and the estimated elimination half‐life was 4.4 ± 2.8 hours. About 27.76% of the consumed dose was eliminated via urine within 24 hours of intake. After a single‐dose oral administration, urinary T concentrations still exceeded the MRPL after 24 hours. This information could be useful for limiting the misuse of T. Athletes should be aware when using T in competition and acquire approval for a therapeutic use exemption prior to use. Moreover, the elimination profile of orally administered T may be crucial information for distinguishing different dosage routes. After a single‐dose oral administration of triamcinolone, urinary concentrations of triamcinolone still exceeded the MRPL after 24 h. The urinary triamcinolone level was much higher 24 h after receiving triamcinolone orally than after a local injection. The elimination profile of orally administered triamcinolone may be crucial information for distinguishing different dosage routes.

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Functional characterization of CYP2C19 variants in nebivolol 4‐hydroxlation in vitro  Voir?

Cytochrome P450 2C19 (CYP2C19) allelic variants are thought to play an important part in inter‐individual variability in drug metabolism. We evaluated the in vitro hydroxylation of nebivolol by 31 CYP2C19 alleles identified in a Chinese Han population recently. Wild‐type CYP2C19*1B and 30 isoforms were highly expressed in insect cells, and the enzymatic activities of CYP2C19 variants towards nebivolol hydroxylation were characterized. Among the 30 CYP2C19 alleles, most of the recombinant CYP2C19 variants exhibited no or significantly low activity compared with CYP2C19*1B. Three variants, CYP2C19*29 (K28I), L16F, and CYP2C19*23 (G91R), showed increased intrinsic clearance of >140% CYP2C19*1B. Combined with a previous study on the effects of CYP2D6 variants on nebivolol metabolism, our comprehensive analyses on the enzymatic activities of CYP2C19 variants towards nebivolol in the present study may contribute to determination of the optimal doses of nebivolol for the treatment of hypertension and understanding of “individualized” medication. The enzymatic activities of the CYP2C19 variants expressed in insect cells towards nebivolol hydroxylation were characterized. Most of the recombinant variants exhibited no or significantly low activities compared with CYP2C19*1B. Three variants showed increased intrinsic clearance of >140% CYP2C19*1B. Our study may contribute to determination of the optimal doses of nebivolol for the treatment of hypertension.

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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 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 from the 1940s to the 1960s. Germans first tested mescaline as a “truth drug” in a military context. In the 1940s, the United States military started testing hallucinogenic substances as truth drugs for 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 compounds 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 1953/1954 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. In 1955, the military changed focus to lysergic acid diethylamide (LSD), but some interest in mescaline‐like compounds remained for their ability to change mood and habit without interfering with cognition and sensory perception. Based on the known documents, it remains unclear (but probable) whether any of the mescaline derivatives tested were being used operationally. This article describes the context in which MDA, MDMA, and other mescaline‐like compounds were synthesized and explored for their possible “speech‐inducing” effects for military and intelligence purposes between the 1940s the 1960s. Because mescaline was found to produce too much mental irritation and hallucinations, compounds with more specific effects were synthesized. MDA, MDE and MDMA (a.k.a. Ecstasy) were among the substances tested further in human and animal experiments.

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A real‐time in vitro assay to evaluate the release of macromolecules from liposomes  Voir?

The availability of a real‐time assay to experimentally investigate the release of encapsulated proteins would be beneficial given the interest in the use of liposomes as a drug delivery vehicle. Although simple assays for small molecular weight substances exist, assays to evaluate macromolecules do not. Here we describe a method that detects the release of model macromolecules from liposomes in real time. The assay employs the intermolecular distance‐dependent phenomenon of fluorescence resonance energy transfer (FRET) between the fluorophore donor, fluorescein (FITC), and fluorescent quencher, QSYź9. The macromolecular species were conjugated to the markers fluorescein (44kDa dextran) and QSYź9 (67 kDa bovine serum albumin, BSA). Following confirmation of quenching between FITC‐Dex and QSYź9‐BSA, liposomes were loaded with the macromolecular markers and subjected to various treatments (high‐pressure extrusion and Triton X solubilisation) to cause release from liposomes. An increase in FITC fluorescence was observed when liposomes were subjected to extrusion cycles. Surprisingly, the addition of Triton X did not cause an increase in fluorescence probably because the FRET pair became associated with mixed micelles. This assay method should be useful in studies to investigate the mechanisms by which macromolecules are released from liposomes, particularly when liposomes are exposed to release‐triggers (eg, temperature change, pH change, ultrasound). Such understanding will underpin the formulation of triggered liposomal delivery systems for macromolecules. The assay described employs fluorescence resonance energy transfer (FRET) between fluorophore donor, fluorescein (FITC), and fluorescent quencher, QSYź9. The macromolecular species were conjugated to the markers fluorescein (44kDa dextran) and QSYź9 (67 kDa bovine serum albumin). Upon dilution, an increase in FITC fluorescence was observed; thus, confirming FRET. Following this confirmation, liposomes were loaded with the macromolecular markers and subjected to various treatments to cause release from liposomes. An increase in FITC fluorescence was used to confirm release.

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Analysis for higenamine in urine by means of ultra‐high‐performance liquid chromatography–tandem mass spectrometry: Interpretation of results  Voir?

Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine and is present in many plants. The substance may be also found in supplements or nutrients, consumption of which may result in violation of anti‐doping rules. Higenamine is prohibited in sport at all times and included in Class S3 (ÎČ‐2‐agonists) of the World Anti‐Doping Agency (WADA) 2017 Prohibited List. The presence of higenamine in urine samples at concentrations greater than or equal to 10 ng/mL constitutes an adverse analytical finding (AAF). This work presents a new metabolite of higenamine in urine sample which was identified by means of ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Samples were prepared according to 2 protocols – a Dilute and Shoot (DaS) approach and a method involving acid hydrolysis and double liquid‐liquid extraction (LLE). To meet the requirements typical for a confirmatory analysis, the screening procedure was further developed. In samples prepared by the DaS method, 2 peaks were observed; the earlier one was specific for higenamine and the later one unknown. MS scan analysis showed mass about 80 Da higher than that of higenamine. In turn, in samples prepared in accordance with the protocol involving hydrolysis, an increase in the area under peak for higenamine was observed, while the second peak was absent. It seems that the described strategy of detection of higenamine in urine avoids false negative results. Higenamine is excreted in the unchanged form and as a sulfate conjugate, thus it can be monitored in the dilute‐and‐shoot methods. Application of a method involving acid hydrolysis and double liquid‐liquid extraction (LLE) caused an increase in the peak area of higenamine, compared to the Dilute and Shoot method. Thus, this strategy of detection of higenamine in urine avoids false negative results.

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Sample preparation method for the combined extraction of ethyl glucuronide and drugs of abuse in hair  Voir?

Often in hair analysis, a small hair sample is available while the analysis of a multitude of structurally diverse substances with different concentration ranges is demanded. The analysis of the different substances often requires different sample preparation methods, increasing the amount of required hair sample. When segmental hair analysis is necessary, the amount of hair sample needed is further increased. Therefore, the required sample amount for a full analysis can quickly exceed what is available. To combat this problem, a method for the combined hair sample preparation using a single extraction procedure for analysis of ethyl glucuronide with liquid chromatography‐multistage fragmentation mass spectrometry/multiple reaction monitoring (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 clean‐up. 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 (MDMA); 3,4‐methylenedioxyamphetamine (MDA); 3,4‐methylenedioxy‐N‐ethylamphetamine (MDE); morphine; 6‐monoacetylmorphine; codeine; acetylcodeine; cocaine; benzoylecgonine; norcocaine; cocaethylene; methadone; 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) and methylphenidate. In conclusion, as only 1 sample preparation is needed with 1 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. The development and validation of a hair sample preparation with a combined extraction of ethyl glucuronide and drugs of abuse is described. The combined sample preparation allows a reduction of the amount of needed hair sample for an optimal analysis. Additionally, the cost and time of sample preparation when analysis of both substance groups is required can be slightly reduced.

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Analysis of new psychoactive substances in oral fluids by means of microextraction by packed sorbent followed by ultra‐high‐performance liquid chromatography–tandem mass spectrometry  Voir?

In recent years, new drugs, commonly known as new psychoactive substances (NPS), appeared on the market, which include, among others, synthetic cannabinoids, cathinones, and tryptamine analogs of psilocin. The aim of this work was to develop and validate a new method for simultaneous screening and quantification of 31 NPS in oral fluid by ultra‐high‐performance liquid chromatography‐tandem mass spectrometry (UHPLC–MS/MS). The chosen target analytes represented different chemical and toxicological NPS classes, such as synthetic cathinones, piperazines, phenethylamines, synthetic cannabinoids, and their metabolites. The procedure involved a rapid sample preparation based on protein precipitation followed by clean‐up utilizing microextraction by packed sorbent (MEPS); the quantitative analysis was performed by UHPLC–MS/MS. The MEPS clean‐up, regardless of non‐quantitative recoveries for some analytes, provided an effective removal of interfering compounds, as demonstrated by reduced matrix effects found at different concentrations for all the analytes. The validation protocol, based on SWGTOX guidelines, demonstrated the suitability of the proposed method for quantitative analysis: linearity range ranged over 3 or 4 orders of magnitude; precision and accuracy tests gave RSD% values below 25%, and accuracy ranged from 85.9% to 107%, accomplishing SWGTOX requirements. Limits of detection (LODs) ranged between 0.005 ng/mL and 0.850 ng/mL and limits of quantification (LOQs) from 0.015 to 2.600 ng/mL. A simple, fast, and reliable procedure based on ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/ MS) was developed for the simultaneous determination of 31 new psychoactive substances belonging to different chemical classes such as synthetic cathinones, piperazines, phenethylamines, synthetic cannabinoids, and their metabolites in OF samples. The procedure involves a rapid sample preparation method based on a protein precipitation followed by clean‐up based on MEPS. The method has been validated according to the SWGTOX guidelines.

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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 liquid chromatography–quadrupole time of flight–mass spectrometry (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 liquid chromatography–tandem mass spectrometry (LC–MS/MS) which was applied to 576 hair samples. 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. o‐/m‐/p‐hydroxy cocaine as well as m‐/p‐hydroxy benzoylecgonine and m‐/p‐hydroxy norcocaine were detected in hair samples of cocaine users.

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Drug residues in used syringes in Switzerland: A comparative study  Voir?

Harm reduction services, including needle‐exchange programmes, have been implemented in Switzerland for over 20 years. Their main aim is to lessen the negative social and/or physical consequences associated with illicit drug consumption and, therefore, improve prevention messages. To this end, knowledge of illicit drug consumption practices is necessary. Periodic self‐report surveys are the primary source of data for monitoring drug users' behaviour. Analysis of residual content of used syringes can bring further and objective knowledge about consumed products through analytically confirmed data. Used syringes were sampled in 2 syringe‐exchange facilities in Lausanne. These structures are a bus where the users bring back their syringes (ABS) and an automatic injecting kit dispenser (AIKD). Once syringes were collected, a validated gas chromatography–mass spectrometry (GC–MS) method was implemented in order to detect drugs (licit or illicit) contained in the residual content of used syringes. Cocaine was the most common drug detected alone (39% in ABS and 31% in AIKD), followed by the simultaneous detection of heroin and cocaine (12% and 17%) and heroin and midazolam (12% and 17%). The differences between the illicit drugs distribution of used syringes collected in AIKD and ABS were not statistically significant. Analysis of residual content of used syringes as a monitoring tool is an original approach that has already led to a better understanding of the habits of drug‐injection users. Over the long term, this approach is a powerful tool to track and detect new consumption practices in a quasi‐real‐time. Analysis of the content of used syringes provides information about the actual composition of illicit drugs consumed by injecting drugs users. The present study allows data confrontation between 2 sampling campaigns conducted in 2015 and 2016. During this campaign, cocaine was the most common drug detected alone (39% in 2015 and 31% in 2016), followed by the simultaneous detection of heroin and cocaine (12% and 17%) and heroin and midazolam (12% and 17%).

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Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction–tandem mass spectrometry to detect hCG use  Voir?

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti‐Doping Agency (WADA) list of prohibited substances. The majority of WADA‐accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography–tandem mass spectrometry (LC–MS/MS). In this study we measured the concentration of intact hCG, free ÎČ‐subunit (hCGÎČ) and ÎČ‐subunit core fragment (hCGÎČcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC–MS/MS method. Mean concentrations of intact hCG, hCGÎČ and hCGÎČcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGÎČ and hCGÎČcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG. Concentrations of intact hCG, free ÎČ‐subunit (hCGÎČ) and ÎČ‐subunit core fragment (hCGÎČcf) were measured in 570, 275, and 256 male urine samples, respectively, by an immunoextraction‐LC‐MS/MS method. Mean concentrations of intact hCG, hCGÎČ and hCGÎČcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper 97.5th percentile for intact hCG, hCGÎČ and hCGÎČcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. A threshold of 1.0 IU/L for intact hCG is recommended to detect doping with hCG.

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Fatality involving ocfentanil documented by identification of metabolites  Voir?

The use of new psychoactive substances (NPS) has rapidly increased over the last decade. 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 the 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. This work reports a fatality involving ocfentanil (OcF), a fentanyl derivative, documented by the identification of OcF metabolites. CYP‐ and UGT‐ dependent metabolites of OcF were in vitro investigated using human liver microsome incubation and liquid chromatography with high‐resolution mass spectrometry detection, and subsequently confirmed in post‐mortem samples. Four OcF metabolites were in vitro produced (including O‐desmethylOcF as suggested most abundant metabolite of OcF) and all except one were observed in blood and bile post‐mortem samples.

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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?

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. Camel hair samples were collected from 30 non‐racing dromedary camels along with 3 racing camels in Al Ain, UAE and were decontaminated, pulverised, sonicated, and extracted prior to analysis. A liquid chromatographic–mass spectrometric method was employed to determine the levels of glucocorticoids in the hair samples. The 4 drugs of interest, namely hydrocortisone, dexamethasone, flumethasone and methylprednisolone, and an internal standard were quantified in camel hair samples. All 4 of the glucocorticoids were detected in camel hair samples with concentrations ranging between 31 and 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 (1130 pg/mg), flumethasone (2576 pg/mg), methylprednisone (1156 pg/mg) and dexamethasone (29 pg/mg). The authors believe this is the first report of a test for corticosteroids in camel hair. The new test has been validated according to Food and Drug Administration (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. We are confident that we are reporting a novel, very robust, reliable, and inexpensive method for the estimation of glucocorticoids in camel hair. This novel hair test is a ground‐breaking innovation to add to the repertoire of camel blood and urine tests already in place for camel health and disease. This test will act as a deterrent and tackle glucocorticoids abuse in camel racing, which will be a big milestone in streamlining camel racing across the globe.

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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 has been used in combat since WWI, there is 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 toward 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. We developed an in vitro testing procedure to investigate the scavenging potential of N‐acetylcysteine (NAC) against the chemical blister agent sulfur mustard (SM). We found that even though reaction between NAC and SM occurred, NAC did not diminish SM concentration by chemical scavenging to a relevant extent. The developed in vitro testing procedure can easily be modified and is therefore suitable to analyze the scavenging potential of other therapeutics against SM.

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

Each year, synthetic cannabinoids occur in high numbers on the illicit drug market, but data on their detectability are rarely available. 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. Six subjects inhaled smoke derived from 2 and 3 mg JWH‐018. The drug and 10 of its metabolites were analyzed in oral fluid samples collected during the following 12 hours using the Quantisal collection device by liquid chromatography–tandem mass spectrometry (LC–MS/MS). 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‐life 1.69 hours), detection of the drug over 6–12 hours (median 8 hours) 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). The detection of JWH‐018 in oral fluid requires high analytical sensitivity even 1 hour after inhalation. The pharmacokinetic properties of inhaled JWH‐018 are similar to those of THC. Times for detection are typically less than 12 hours. High variability of the oral fluid/serum ratio precludes extrapolation of oral fluid concentrations to blood. Pharmacokinetics of JWH‐018 in oral fluid was similar to that of THC after controlled inhalation. Contamination subsided within 1 hour and concentrations below 1 ng/mL were detectable after 6–12 hours. Concentrations in oral fluid and serum were not related.

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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 their extensive metabolism, parent compounds are hardly detectable in urine. Therefore, knowledge of 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, and CUMYL‐4CN‐B7AICA with a 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 hour 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 5 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. The aim was the elucidation of the metabolism of the new synthetic cannabinoids CUMYL‐PINACA, 5F‐CUMYL‐PINACA, CUMYL‐4CN‐BINACA, 5F‐CUMYL‐P7AICA, and CUMYL‐4CN‐B7AICA with a focus on the analytical and interpretational differentiation of the compounds. Some of the metabolic transformations lead to structurally identical metabolites, and should not be used as markers for the intake of a particular parent compound. Furthermore, constitutional isomers containing either an indazole or an azaindole core structure should also be differentiated by retention time rather than by their mass spectra alone.

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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?

To evaluate adherence to treatment, we developed and validated a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for baclofen quantification in hair.Twenty mg was washed twice with dichloromethane, incubated in phosphate buffer (pH 5) for 10 minutes at 95°C, then extracted by liquid‐liquid extraction in alkaline condition. Baclofen‐d4 was used as the internal standard. This method was applied to assess compliance in4 treated alcohol‐dependent patients (3 dead and one living). Blood quantification of baclofen and ethanol were performed in the 4 cases. Hair ethylglucuronide (ethanol metabolite, EtG) measurement (2x3 cm) was associated in 1 patient. Baclofen quantification in hair was validated over the range 10–5000 pg/mg. The accuracy was within 96.0%–110.9% and the precision was less than 9.3%. Baclofen segmental (3x2cm) 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 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 3 deceased patients, the non‐segmental analysis of hair showed baclofen concentrations of 15, 545, and 2475 pg/mg. The low concentrations in the 2 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. The measurement of baclofen in hair could allow to determine the compliance of an alcohol dependent patient to its treatment. Coupled to hair ethylglucuronide determination, these biomarkers could be used both to assess patient's compliance and therapeutic failure in compliant patients.

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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 has become a routine procedure in forensic toxicology. Hair coloration leading to loss of incorporated xenobiotics and to false negative results has turned out to be a major problem. Currently only colored extracts provide hints of manipulations but not bleaching. A liquid chromatographic–tandem mass spectrometric (LC–MS/MS) 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 minutes with concentrations up to 12% for 40 minutes. 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 (light‐blond 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. Hair samples of 21 people were treated with different concentrations of hydrogen peroxide. Results were compared with PTCA concentrations of 12 hair samples treated with different hair colorations. Results showed a marked increase of PTCA depending on hydrogen peroxide concentration and treatment time. Therefore PTCA can be used as a direct marker for oxidative hair treatment and may be useful to identify bleached hair samples in laboratory routine.

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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 the athlete biological passport (ABP) is underpinned by understanding normal fluctuations of its biomarkers to environmental or medical conditions, for example, 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, 3000 m). Athletes received either oral, intravenous (IV) or placebo iron supplementation, commencing 2 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 3 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 10 athletes returning abnormal values at 99% specificity, 2 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. The integrity of the athlete biological passport (ABP) is underpinned by understanding the normal fluctuations of its biomarkers to environmental or medical conditions. The combined impact of altitude and iron supplementation on the ABP was evaluated in endurance‐trained athletes undertaking 3 weeks of simulated altitude training whilst receiving oral, intravenous, or placebo iron supplementation, Individual ABP thresholds for haemoglobin concentration, reticulocyte percentage, and OFF score were calculated using the adaptive model and assessed at 99% and 99.9% specificity.

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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 3, 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 3 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. Three days' intake of codeine decreases serum levels of total testosterone. In co‐use of codeine and testosterone, they do not interact with each other. Codeine use may be a confounder when interpreting the Athlete Biological Passport.

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Detection of in utero cannabis exposure by umbilical cord analysis  Voir?

According to the 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 the 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 the umbilical cord by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with dual ionization source. Umbilical cord samples (0.5 g) were homogenized in methanol and extracted by solid‐phase extraction. Reversed‐phase chromatographic separation was performed in 14 minutes, and 2 transitions per analyte were monitored in multiple reaction monitoring mode. Method validation included linearity (1–10 to 20–200 ng/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 (1–10 ng/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 (1.6–19.1 ng/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. 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 umbilical cord's usefulness to detect cannabis in utero exposure.

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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 hour after intake) and 7.9 ng/mL (10.5 hours 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 hour after ingestion as well as in 90% of the samples collected 10.5 hours after administration. Serum concentrations of the Phase I metabolite prothipendyl sulfoxide averaged 4.3 ng/mL (1 hour after intake) and 3.6 ng/mL (10.5 hours 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. Prothipendyl and prothipendyl sulfoxide concentrations were determined in serum samples of 50 psychiatric patients. 40 mg doses caused average prothipendyl serum concentrations of 18.0 ng/mL (1 hour after intake) and 7.9 ng/mL (10.5 hours after intake), while 80 mg doses caused averages of 42.6 ng/mL and 15.2 ng/mL at the mentioned times of sampling. The utility of clinical serum concentrations is demonstrated by discussing two death cases involving prothipendyl findings.

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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 (GC–C–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 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 the creation and characterization of 3 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 ∆Δή13CVPDB values from 8 randomly selected ampoules of all 3 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. Uniform steroid carbon isotopic standards were created for use by and harmonize results among the World Anti‐Doping Agency accredited laboratories in synthetic steroid detection. Batches of 3 standards that contain ~20 mg of a single steroid in each ampoule were characterized for their carbon isotope composition and are suitable for use as daily working standards.

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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 summarizes current research and suggests the future of presumptive color testing. In this review, we describe the development of chemical color tests used by a large number of agencies worldwide as the first step in illicit drug identification. Further, we review the chemistry behind the color changes and classify common color test using this knowledge. Finally, we describe the challenges faced by color tests today and propose the future of color testing.

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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. Cocaine analogue impregnated into a variety of textiles can be extracted with dilute acidified water (0.5 M sulfuric acid) and quantitatively measured by using Raman spectroscopy.The use of internal standard potassium thiocyanate (KSCN) in the method removes the need for textile matrix matched calibration.

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Roadside 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 (DREs) 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 they pertain 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 the driver should still be considered the most important aspect for arrest and further action. The Alere DDSź2 mobile roadside drug testing system was evaluated as a presumptive screening device for drugs in oral fluid. All control subjects tested negatively and the corresponding oral fluid and blood samples analyzed in the laboratories were confirmed as negative; 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.

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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 solid‐phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) method. EtG positive hair samples were obtained from 3 regular drinkers and incubated for 0, 2, 4, 6, 8, and 10 hours at room temperature. EtG concentrations in hair were reduced after 2 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 2 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 (eg. 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). The influence of chlorinated swimming pool water on EtG concentrations in hair in comparison to deionized water (Milli‐Q) containing no chlorine was investigated. 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. These results clearly demonstrate that washout effects are caused by water and have a significant impact on EtG concentrations in hair.

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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 amphetamine production in recent years. This assumption is based on seizure data but 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 gas chromatography–mass spectrometry (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 nuclear magnetic resonance (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 analysis of 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 proof 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. New marker compounds were identified and characterised which indicate if amphetamine was clandestinely produced from the pre‐precursor α‐phenylacetoacetonitrile (APAAN). The APAAN markers were searched in over 580 amphetamine cases from Dutch and German police seizures going back as far as 2009 to determine the significance of these markers. The prevalence of the markers illustrates an increasing trend of APAAN use from 2010 on which is in agreement with seizure data.

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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 ultra‐high 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% to 81% and the process efficiency from 81% to 98%. Limit of detection was within 0.0005–0.001 mg/kg and limit of identification ranged from 0.001 to 0.005 mg/kg. In the retrospective analysis of 2339 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. The number of fentanyl and fentanyl analogues‐related overdose deaths and acute intoxications has grown dramatically worldwide during the last years. We utilized a screening method using high resolution mass spectrometry in MSE mode to identify 50 fentanyl analogues. We observed that along all fentanyl analogues with a 4‐anilidopiperidine core there is a class‐specific collision‐induced cleavage useful for their tentative identification without using reference standards. Furthermore, the presented method creates the possibility of screening for new synthetic 4‐anilidopiperidine fentanyl analogues.

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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. These guidelines for Legally Defensible Workplace Drug Testing by use of oral fluid 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.

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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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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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Issue Information  Voir?

No abstract is available for this article.



Doping control analysis at the Rio 2016 Olympic and Paralympic Games  Voir?

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3–21, 2016) and the XV Paralympic Games (September 7–18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti‐Doping Agency (WADA)‐accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state‐of‐the‐art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America. Copyright © 2017 John Wiley & Sons, Ltd. Preparation, logistics, and analyses performed in the Brazilian Doping Control Laboratory for the Olympic and Paralympic Games 2016, Rio de Janeiro.

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Identification and characterization of novel long‐term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC‐CI‐MS/MS  Voir?

The search for metabolites with longer detection times remains an important task in, for example, toxicology and doping control. The impact of these long‐term metabolites is highlighted by the high number of positive cases after reanalysis of samples that were stored for several years, e.g. samples of previous Olympic Games. A substantial number of previously alleged negative samples have now been declared positive due to the detection of various long‐term steroid metabolites the existence of which was unknown during the Olympic Games of 2008 and 2012. In this work, the metabolism of oxymesterone and mesterolone, two anabolic androgenic steroids (AAS), was investigated by application of a selected reaction monitoring gas chromatography–chemical ionization–triple quadrupole mass spectrometry (GC‐CI‐MS/MS) protocol for metabolite detection and identification. Correlations between AAS structure and GC‐CI‐MS/MS fragmentation behaviour enabled the search for previously unknown but expected AAS metabolites by selection of theoretical transitions for expected metabolites. Use of different hydrolysis protocols allowed for evaluation of the detection window of both phase I and phase II metabolites. For oxymesterone, a new metabolite, 18‐nor‐17ÎČ‐hydroxymethyl‐17α‐methyl‐4‐hydroxy‐androst‐4,13‐diene‐3‐one, was identified. It was detectable up to 46 days by using GC‐CI‐MS/MS, whereas with a traditional screening (detection of metabolite 17‐epioxymesterone with electron ionization GC‐MS/MS) oxymesterone administration was only detectable for 3.5 days. A new metabolite was also found for mesterolone. It was identified as 1α‐methyl‐5α‐androstan‐3,6,16‐triol‐17‐one and its sulfate form after hydrolysis with Helix pomatia resulted in a prolonged detection time (up to 15 days) for mesterolone abuse. Copyright © 2017 John Wiley & Sons, Ltd. By using GC‐CI‐MS/MS, novel long‐term metabolites for oxymesterone and mesterolone were detected and characterized. The new oxymesterone metabolite was detectable up to 46 days by using GC‐CI‐MS/MS, whereas with a traditional screening (detection of metabolite 17‐epioxymesterone with GC‐EI‐MS/MS) oxymesterone administration was only detectable for 3.5 days.

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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 to detect 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 a well‐known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 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 could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, 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. Copyright © 2017 John Wiley & Sons, Ltd. The ability of adult zebrafish to produce metabolities of sibutramine and stanozolol was evaluated in this work. 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). The fishes were able to produce several metabolites including demethylated, hydroxylated, dihydroxylated and reduced derivatives, all of which have already been detected in human urine.

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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. Copyright © 2017 John Wiley & Sons, Ltd. All investigated administration studies resulted in a significant and prolonged detectability of the exogenous steroid using carbon isotope ratios of epiandrosterone sulfate. These findings, together with the presented simplified sample preparation and measurement method, allow for using epiandrosterone as a promising complement to currently applied target analytes excreted glucuronylated. The method was fully validated and is fit‐for‐purpose.

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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 World Anti‐Doping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography‐combustion‐isotope ratio mass spectrometry (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 high performance liquid chromatography (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 endogenous reference compound (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. Copyright © 2017 John Wiley & Sons, Ltd. The GC‐C‐IRMS analysis of AICAR, a naturally occurring AMKP activator prohibited analogue, was implemented by adjustment with regard to a published protocol. The derivatization step as well as an additional purification step were optimized. The performances of this new protocol were assessed thanks to the repeated analysis of reference material and spiked urine samples. Variability of the 13C values comparable to the published protocol were obtained with a good reproducibility of the TMS‐derivatives formation.

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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. A method was developed to count blood cells, immature reticulocytes and red blood cells, in dried blood spots. The method was tested for detection of autologous blood transfusions and compared to the current athlete biological passport method.

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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; limit of detection (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. Copyright © 2017 John Wiley & Sons, Ltd. Several immunological strategies were tested in order to develop a detection method for Luspatercept and other ACVR2B‐Fc fusion proteins in serum samples (ELISA, IEF‐PAGE, SDS/SAR‐PAGE plus Western blotting). While Luspatercept can be determined in human serum with a commercial ACVR2B‐ELISA (LOD ca 15.6 ng/mL; 100 ÎŒL serum), the developed SAR‐PAGE method is more sensitive and allows the detection in only 50 ÎŒL serum down to ca 1.0 ng/mL. Considering the long serum half‐life (15–16 days) and high doses used for stimulating erythropoiesis, both the ELISA‐ and SAR‐PAGE methods will be able to detect Luspatercept misuse over a period of several weeks.

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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. Copyright © 2017 John Wiley & Sons, Ltd. 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. Therefore, this technique has the potential to be further developed for the detection of foreign genetic material in elite athletes.

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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, and amphetamines. Clenbuterol is present in a large number of doping samples from Olympic and non‐Olympic athletes that have adverse analytical findings (AAFs). In some cases, the presence of these substances 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 min with good separation. The method developed can also be used for the analysis of other biological matrices such as urine, serum, and 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 has not been reported previously. The information shown could be used by national anti‐doping organizations and the athletes with AAFs attributed to clenbuterol. Copyright © 2017 John Wiley & Sons, Ltd. In this study, 26 products with racemic clenbuterol were evaluated using a new analytical methodology for the resolution of R‐(−) and S‐(+)‐enantiomers of clenbuterol by LC–MS/MS. Both clenbuterol enantiomers were present in all products analyzed and the ratio was nearly 1. All products displayed a 50:50 ratio of clenbuterol enantiomers. No sample contained only one clenbuterol enantiomer.

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

According to the 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 (ie, 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. Copyright © 2017 John Wiley & Sons, Ltd. Immunopurification procedure developed for the isolation of EPO on immunomagnetic beads that is suitable for SAR‐PAGE and IEF analysis, removing the necessity of carrying the double‐blotting for either urine or serum/plasma samples. The generated IEF and SAR‐PAGE EPO profiles by single‐blotting analysis meet all the acceptance criteria. The method was shown to be rapid, robust and low‐cost.

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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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Application of HBOCs electrophoretic method to detect a new blood substitute derived from the giant extracellular haemoglobin of lugworm  Voir?

Manipulation of blood and blood components is prohibited in sports by the World Anti‐Doping Agency (WADA). This includes the use of blood substitutes to increase oxygen transport, like haemoglobin‐based oxygen carriers (HBOCs), which are compounds derived from haemoglobin. Despite their medical interest, the first generation of HBOCs had serious adverse effects and was abandoned. However, new studies are now exploiting the properties of marine worm haemoglobins, which circulate as giant extracellular complexes with high oxygen‐binding capacities. HEMOXYCarrierź (HC), developed by Hemarina, is one of the most advanced and promising HBOCs, and HC may become a tempting doping tool for athletes in the future. Here, HC detection in plasma/serum was evaluated with the method used to detect the first HBOCs, based on electrophoresis and heme peroxidase properties. An HC‐derived product was identified in human plasma up to 72 h after in vitro incubation at 37 °C. HC degradation also induced methemalbumin formation. After injecting HC at the effective dose of 200 mg/kg into mice, the HC‐derived product was detected only for a few hours and no accumulation of methemalbumin was observed. Due to this limited detection window in vivo, measuring specific worm globin degradation products by mass spectrometry might be an alternative for future anti‐doping analyses. Copyright © 2016 John Wiley & Sons, Ltd. The aim of this work was to test the capacity of detection of a new kind of haemoglobin‐based oxygen carrier (HBOC) issued from a marine worm. The electrophoretic method used for detection of the first‐generation HBOC was applied. The highlights of this work are: HEMOXYCarrierź produced by HEMARINA is a future blood substitute with doping potential. Detection of this product in plasma/serum using the electrophoretic HBOC method is applicable but limited in vivo to a few hours. Research of peptides specific of worm globins in blood or urine could increase the window of detection.

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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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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 (AAFs) in the 2011 FIFA Women's World Cup. Our preliminary study has suggested that musk ingestion did not lead to AAFs 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 ∆Δή13C‐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. Copyright © 2017 John Wiley & Sons, Ltd. In this study, musk samples were collected, and an administration study of musk was carried out. After musk administration, fluctuations in steroid profile could be observed, and adverse analytical finding were abstained in the IRMS test.

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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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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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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. Copyright © 2017 John Wiley & Sons, Ltd. In vitro metabolism of small prohibited peptides yielded several new metabolites which were identified by liquid chromatography coupled to high resolution mass spectrometry. Incubation experiments were performed with different blood specimens and rec. amidase.

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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 Anti‐Doping Agency (WADA), athlete biological passport (ABP), implemented through the global database, 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 and 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. Copyright © 2017 John Wiley & Sons, Ltd. The longitudinal evaluation of the IRMS data can 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. We describe an IRMS screening method allowing to increase the throughput of samples with adequate selectivity and producing results that overlap with the already validated confirmation method. The applicability has been demonstrated during a major event without compromising the detection capacity.

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

 
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