Drug Testing and Analysis最新文献

The fast skeletal troponin activators (FSTAs) Reldesemtiv and Tirasemtiv were developed for patients suffering from neuro-degenerative diseases of the motor nervous system, e.g. amyotrophic lateral sclerosis (ALS). The drug candidates can increase the sensitivity of troponin C to calcium by selectively activating the troponin complex resulting in increased skeletal muscle contraction. Although the development of the drug candidates is currently discontinued because of missed end points in phase III clinical studies with patients with ALS, phase I clinical trials showed an increase in muscle contraction force in healthy humans. This effect could be abused by athletes to enhance performance in sports. As the substances are listed on the 2024 edition of the World Anti-Doping Agency's Prohibited List, the aim of this study was to identify and characterize metabolites of Reldesemtiv and Tirasemtiv to ensure their reliable identification in doping control analyses. The biotransformation of the drug candidates was studied in vitro using pooled human liver microsomes and 3D cultivated human hepatic cells of the cell line HepaRG, yielding a total of 11 metabolites of Reldesemtiv and eight of Tirasemtiv. In addition, a human elimination study was conducted to investigate the metabolism and elimination profile of Tirasemtiv and Reldesemtiv in vivo, suggesting the N-glucuronide of Tirasemtiv and hydroxylated 3-fluoro-2-(3-fluoro-1-methylcyclobutyl)pyridine as well as its glucuronide as suitable target analytes for routine doping controls. Applying a validating HPLC-MS/MS method, optimized to detect Reldesemtiv and Tirasemtiv in human urine, microdosing (50 μg) of each substance was traceable for 24-72 h.

The non-psychoactive cannabinoids cannabidiol (CBD) and cannabidiolic acid (CBDA) are available on the market in different forms, mostly for their anti-inflammatory and potential analgesic properties. These substances are prohibited during equine competitions. CBD and CBDA are naturally present in hemp straw, commonly used as a bedding substitute for wheat straw. Unfortunately, horses can eat it, which therefore could lead to a possible risk of positive findings for CBD/CBDA in biological samples after doping control tests. The goals of this study were, first, to provide recommendations on the use of hemp straw before competition and, second, to assess if discrimination between hemp bedding exposure and CBD oil administration is possible. Several CBD equine in vivo studies have been conducted, including one on hemp straw used as bedding and one after administration of CBD oil by topical and sublingual routes. In hemp straw, CBDA was detected in higher quantities than CBD, and other cannabinoids have been observed. After hemp straw exposure, CBDA was also detected in higher quantities than CBD in all urine samples. It appeared that hemp straw should not be used as bedding for equine competition except if a delay of at least 48 h is respected. Regarding the CBD oil product analysis, CBD was the main compound detected. After administration, 7-hydroxy CBD was identified in the urine. In conclusion, based on these data, we highlighted that it could be possible to discriminate the exposure of a horse to hemp straw from an administration of a CBD oil product thanks to the main presence of CBDA.

Despite the fact that drugs of abuse are illegal, a drug-free festival still remains an utopia in most settings. For law enforcement purposes, it is necessary to rapidly determine whether controlled substances are involved. On-site testing is a challenging task because drugs appear in different physical forms and concentrations. The aim of this study was to compare the performance of two spectroscopic techniques, Raman and Fourier transform-infrared (FT-IR), for the testing of drug seizures at a dance festival. First, samples were measured through packaging with Raman. Subsequently, homogenized samples were analysed with FT-IR. For MDMA tablets, a chemometric model was applied on the FT-IR spectra for the dose estimation. After the festival, results were confirmed in the forensic laboratory with gas chromatography coupled with mass spectrometry (GC-MS) and gas chromatography with flame ionization detection (GC-FID). In total, 166 samples of which 90 tablets, 53 powders, 16 crystals and 7 liquids were analysed. MDMA, cocaine and ketamine were the top three drugs seized. The Raman technique was suitable for powders and crystals (sensitivity of 100% and 81%, respectively). However, in comparison with FT-IR, Raman performance was lower for the analysis of liquids (sensitivity of 67%) and 'ecstasy'-like tablets (sensitivity of 41%). Overall, sensitivities above 95% were obtained with FT-IR. The MDMA doses of the tablets, determined on-site, ranged between 52 mg and 336 mg MDMA hydrochloride. For a quick identification of a variety of drugs on-site, the combination of Raman and FT-IR is recommended. It should be emphasized that optimized settings, in-house libraries and analysis by trained operators are essential to obtain correct results.

An improved screening workflow and a robust capillary flow LC-MS confirmatory method for the detection of recombinant human erythropoietin (rHuEPO) has been implemented to increase the sensitivity of rHuEPO detection and to reduce the number of suspect samples committed to confirmatory testing. The influence of repeated dosing of epoetin-β on the detection window of rHuEPO in equine plasma was assessed using the optimised method. Samples were initially assessed using an economical R&D Human EPO Duo-Set ELISA Development System. Samples indicating a result greater than the batch baseline were analysed using the complementary R&D Human EPO Quantikine IVD ELISA kit. All samples recording an abnormal screening result were subjected to confirmatory analysis. Confirmation of rHuEPO in plasma (≥2.5 ml) in the range of 4-13 mIU/ml (n = 6) was achieved using immunoaffinity enrichment, tryptic digestion, and capillary flow LC-MS/MS. Four horses were administered a single dose of epoetin-β (10,000 IU) via the subcutaneous and intravenous routes, on two occasions, seven days apart. The excretion profile was rapid with epoetin-β detection times of 48 to 72 h following each administration, with no appreciable difference observed between the two routes of administration. This workflow has been shown as an effective anti-doping strategy related to rHuEPO misuse and supports the use of out-of-competition testing of horses in the 2 to 3-day period prior to race-day.

The identification of trimetazidine, a medicine used for treating stable angina pectoris and for preventing angina attacks, has been recently observed in doping cases involving high profile athletes from various countries over the world. In all the files where the authors have been involved, the urine concentration of trimetazidine was low (<2 ng/mL), and the athletes argued that contamination was the source of their adverse analytical finding. It is possible to challenge imposed sanctions in relation to an adverse analytical finding, but it is the responsibility of the athlete to demonstrate he/she is innocent and can qualify for no fault or negligence. When the delay between the urine collection and the notification of the violation was not too long (less than 6 months), these athletes requested a head hair test. Trimetazidine was analyzed by an original LC-MS/MS method involving pH 9.5 borate buffer overnight incubation of 20 mg and subsequent solvents extraction in presence of trimetazidine-D8 used as internal standard. Linearity was verified from 1 to 200 pg/mg (R² = 0.9987). Limit of detection of the method was 0.1 pg/mg. The hair specimen of a male subject, collected 4 weeks after single oral ingestion of 20 mg trimetazidine, tested positive at 146 pg/mg in the corresponding segment. Concentrations of trimetazidine measured in several hair specimens (n = 5) collected from athletes challenging their anti-doping rule violation were below 1 pg/mg, which is consistent with incidental exposure due to contamination. This is the first evidence that trimetazidine is incorporated in human hair after a single therapeutic dose administration.

Several protocols for the analysis of amphetamine-type stimulants (ATS) in hair have been developed over the years, with microextraction by packed sorbent (MEPS) being used for drugs like opiates, cocaine and ketamine. However, concerning ATS determination in hair samples, this approach has only been applied so far to amphetamine (AMP) and methamphetamine (MAMP). This study aimed at developing and validating a MEPS-based procedure for the determination in hair of not only AMP and MAMP but also of 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 1-(1,3-benzodioxol-5-yl)propan-2-yl (ethyl)amine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-aminobutane (MBDB) as well. Hair, 50 mg, was incubated with 1 M sodium hydroxide (NaOH) at 45°C overnight, neutralization with 10 M hydrochloric acid (HCl) and centrifugation followed. The design of experiments approach was used for MEPS optimization, with the final optimized conditions including conditioning (250 μL methanol and deionized water), loading (18 × 100 μL) and elution (7 × 100 μL 2% NH₄OH in acetonitrile). The eluted extract was evaporated to dryness and underwent microwave-assisted derivatization with N-methyl-bis(trifluoroacetamide) (MBTFA), and it was afterwards injected onto the gas chromatography-mass spectrometer (GC-MS). The obtained recoveries ranged between 8% and 14% for AMP, 14% and 20% for MAMP, 10% and 15% for MDA, 18% and 28% for MDMA, 25% and 43% for MDE and 34% and 52% for MBDB, and the method was linear from 0.2 to 5.0 ng/mg. Precision and accuracy were in accordance with international method validation guidelines. This novel method involving MEPS coupled to GC-MS offers a swift, eco-friendly and cost-effective alternative to traditional procedures for detecting these AMPs in hair samples.

The use of differential mobility spectrometry at low pressure coupled to liquid chromatography-mass spectrometry (LC-vDMS-MS) was investigated for the analysis of 13 drugs of abuse (DoA) including the following: cocaine, ecgonine methyl ester, cocaethylene, benzoylecgonine, norcocaine, tramadol, isomeric pairs of metabolites; O-desmethyl-cis-tramadol and N-desmethyl-cis-tramadol, and cannabinoids: Δ⁹-tetrahydrocannabinol, Δ⁹-tetrahydrocannabidiol, 11-hydroxy-Δ⁹-tetrahydrocannabinol, 11-nor-9carboxy-Δ⁹-tetrahydrocannabinol, and 11-nor-9carboxy-Δ⁹-tetrahydrocannabinol glucuronide. Different parameters were optimized for isomeric separation, such as LC mobile phase composition (20%-100% methanol acetonitrile and isopropanol, flow rate: 8-100 μL/min) and DMS separation voltage. Methanol and acetonitrile significantly affected the compensation voltage of the analytes and improved DMS separation. A short trap/elute LC-vDMS-SIM/MS screening method of 1 min was developed to quantify 11 drugs of abuse (except THC/CBD), in addition to a 4-min LC-vDMS-SIM/MS method to identify and quantify five cannabinoids including the isomers THC/CBD and three THC metabolites. THC is the principal psychoactive constituent of cannabis and is a controlled substance in comparison to its isomeric counterpart CBD; this highlights the importance and challenges to resolve these isomeric pairs by analytical techniques. The signal responses were linear over a concentration range of 0.005-10 μg/mL for the DoA and 1-1000 ng/mL for cannabinoids. The intraday and interday precision were better than 12.2% and accuracy better than 115%. Urine samples from subjects who tested positive for THC and/or cocaine during roadside drug testing were evaluated to assess the performance of the methods LC-vDMS-SIM/MS and LC-MRM/MS. Results show that the developed LC-vDMS-SIM/MS method presents similar performance to LC-MRM/MS with improved sample throughput.

The metabolism of 3-chloromethcathinone (3-CMC) was studied after controlled administration in a murine model using the dried blood spot (DBS) technique for the sampling, storage and purification of blood samples. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was used for the identification of metabolites and investigation of their fragmentation pattern. Subsequently, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for their identification and 3-CMC quantification in routine workload. The main metabolites identified were two stereoisomers of dihydro-CMC, N-demethyl-CMC, and dihydro-N-demethyl-CMC. The stability of 3-CMC and of its metabolites deposited on DBS was evaluated by replicate analyses after 30, 50, and 90 days, demonstrating a decrease in concentration. It was more pronounced for 3-CMC, with -67% and -82% percentage deviation from the initial concentrations, and for N-demethyl 3-CMC (decrease comprised between -48% and -88%) than for the di-hydro metabolites, ranging from -5% to -37%. Regardless, all of them were detectable till 90 days after deposition as DBS. The possibility of identifying 3-CMC and its metabolites with high sensitivity is an invaluable tool for the diagnosis of exposure to the substance, also in low doses or after some hours, and for various applications in clinical and forensic toxicology, such as driving under the influence, drug-facilitated crimes, and addiction to intoxications. DBS demonstrated to be a reliable technique for the sampling, storage, and purification of the blood specimen for 3-CMC and metabolite detection.

The screening of drugs in plasma and urine often requires initial extraction (such as liquid-liquid extraction and solid-phase extraction) before the samples are submitted to instrumental analyses. These extraction procedures are often laborious and time-consuming. In this manuscript, a high-throughput automated assay based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 600-μL plasma aliquot, which is subjected to solid phase extraction (SPE) using OASIS HLB 96-well SPE with Biotage Extrahera system, evaporation, and reconstitution in a 96-well collection plate. LC-HRMS analyses were carried out on a Thermo Q-Exactive Mass spectrometer coupled with Thermo UHPLC system equipped with Thermo Accela ALS 2.4.0 autosampler linked to ACE Excel column. Drug targets were detected by retention time and accurate mass, with a mass tolerance window of 5 ppm in positive and negative ionization mode. The screening method was validated for over 300 drug targets in a 13-min run. Validation data including sensitivity, specificity, extraction recovery, and precision are presented. As the method employs full-scan mass spectrometry, unlimited number of drug targets can theoretically be incorporated into this method.

Malodorants comprise notoriously smelling mercaptans and might be applied for crowd control. Because exposure to malodorants may lead to irritation of the respiratory system, choking, and coma, bioanalytical verification of poisoning might be required in a medical and forensic context. We herein present the detection and identification of novel biomarkers of exposure to ethyl mercaptan, n-butyl mercaptan, tert-butyl mercaptan, and iso-amyl mercaptan. These alkyl thiol compounds were found to form disulfide adducts in human serum albumin (HSA) in plasma in vitro with the only non-disulfide-bridged Cys³⁴ residue and with other residues being part of the disulfide-bridged pattern in HSA. After proteinase K-catalyzed proteolysis, adducts of all mercaptans were detected simultaneously as the tripeptide Cys³⁴*ProPhe and the dipeptides Cys³⁶⁹*Tyr, ValCys³¹⁶*, and Cys^x*Ala (x denominates either Positions 91, 200, 253, 361, and/or 448) by a sensitive micro-liquid chromatography-electrospray ionization tandem mass spectrometry (μLC-ESI MS/MS) method working in the scheduled multiple reaction monitoring (sMRM) mode. Time- and concentration-dependent adduct formations while exposure and proteolysis were investigated and the suitability of adducts as biomarkers of exposure was elaborated. Adducts at Cys³⁴ showed the lowest limits of identification (LOIs, 6 nM to 1.2 μM mercaptan in plasma) and superior stability in plasma at 37°C. Therefore, Cys³⁴*ProPhe appears as the most promising target to prove exposure to mercaptans at least in vitro.