Drug Testing and Analysis最新文献

Positive identification and reporting of therapeutic substances intended for human consumption in race-day equine and canine samples is a controversial topic. While inadvertent environmental exposure is a potential cause for the presence of these substances in race-day samples, intentional use cannot be ruled out given their therapeutic benefits. Pregabalin is widely prescribed in Australia to treat epilepsy, anxiety, and neuropathic pain in humans; however, it is also increasingly used as a recreational drug. Metformin is commonly used to treat type 2 diabetes in humans. Both pregabalin and metformin have no routine use on racing animals and should not be present in race-day samples taken from healthy animals. They are prohibited substances under the Rules of Racing with no established screening limits. Although therapeutic levels for these substances have been established in humans, such information is not available for animals. Pregabalin and metformin are analytically challenging molecules, more so when they are extracted from biological matrices routinely screened for hundreds of other compounds simultaneously. A simple extraction, followed by a targeted Ultra High-Pressure Liquid Chromatography Orbitrap™ Mass Spectrometry method utilising a reverse-phase C18 column, is presented. This method is effective in screening for pregabalin and metformin, in addition to more than 150 other compounds of interest in equine and canine urines. The prevalence of pregabalin and metformin in race-day equine and canine urine samples in Western Australia was monitored using this method over 12 months. More than 4000 urine samples were screened, and four samples were confirmed positive for these substances.

The determination of serum concentrations of testosterone (T) and 4-androstenedione (A4) was implemented into the steroidal module of the Athlete Biological Passport in 2023. Monitoring T, A4, and the ratio of T/A4 in a longitudinal manner enables the detection of the misuse of low-dose T administrations especially in female athletes, whereas urinary markers of the steroid profile may not be influenced significantly. In contrast to the urinary steroid profile, knowledge on confounding factors regarding serum concentrations of T and A4 is yet comparably scarce, and corroborating exogenous sources of the target analytes by isotope ratio mass spectrometry (IRMS) is desirable. In a recent study, it was demonstrated that carbon isotope ratios (CIRs) of serum steroids can be determined if analyte concentrations permit. The therein-employed method utilized 2D-GC/IRMS, and only a limited number of potential endogenous reference compounds were available. The here-presented approach uses complementary analyte purification strategies, addressing previous limitations. A high-performance liquid chromatography cleanup was developed and fully validated for serum steroids in order to enable all doping control laboratories to potentially implement this method alongside existing protocols for urinary steroids. Besides the already-investigated endogenous steroids cholesterol, dehydroepiandrosterone sulfate, androsterone sulfate, and epiandrosterone sulfate, two additional steroids were included in the test menu, namely, pregnenolone sulfate and 5-androstene-3β,17β-diol sulfate. Serum steroid concentrations down to 25 ng/mL were found to allow robust CIR determinations, and a reference population encompassing 124 male and female athlete samples was investigated to enable the calculation of population-based thresholds for all relevant steroid combinations.

Despite the International Federation of Horseracing Authorities (IFHA) regulation associated with heavy sanctions, the abuse of prohibited substances must be identified and deterred throughout horses' athletic careers, such as the administration of recombinant growth hormone (rGH). GH is naturally produced in mammal organisms to stimulate growth. Thus, rGH administration can enhance the performance of horses by expanding some physical abilities. As measuring endogenous GH levels is complex, an indirect strategy is to monitor GH-associated biomarkers in plasma as insulin-like growth factor 1 (IGF-1) levels. To prevent these misuses, the Equine Biological Passport (EBP) has been designed in France (GIE LCH) and Australia (ARFL-Racing NSW) to profile specific biological and chemical parameters in selected racehorses. In this study, we investigated individual limits as a complementary tool to a single limit to supervise the stability of IGF-1 profile over a racing season. The aim is to design custom limits based on the horse's history to detect any deviation below the single limit. The method was assessed using experimental data and then tested on EBP data from three thoroughbreds and three French trotters. Finally, individual limits have been added to the French EBP for IGF-1 monitoring.

Bisphosphonate drugs and myo-inositol trispyrophosphate are of concern to the racing industry and have been listed as prohibited substances in equine sports. The current bisphosphonate plasma screening analysis employed at the Australian Racing Forensic Laboratory involves the use of sequential solid-phase extraction procedures, passing the samples through a mixed mode cartridge, followed by a weak anion exchange cartridge. The eluates collected following the second extraction are then methylated and analysed by liquid chromatography-mass spectrometry. Under these extraction conditions, some bisphosphonates have shown poor recovery. To improve the extraction efficacy, the effects of cartridge chemistry were evaluated. In particular, the weak anion exchange cartridges used for screening were compared to an affinisep AttractSPE polymeric phase cartridge. The effectiveness of each extraction approach was assessed through both a visual comparison of signal to noise in extracted chromatograms and recovery measurements to determine the best approach for routine screening.

Bioanalysis, such as the quantification of drugs in different matrices, is of great importance in forensic toxicology. Nowadays, mainly so-called multianalyte approaches are used given their increased speed and effectiveness. However, such multianalyte procedures can be difficult to develop and maintain with sufficient robustness in the laboratory. One aspect of this is the tedious, manual preparation of spiking solutions containing such a great number of analytes. Therefore, the current study aimed to develop and validate a fast, simple, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 82 classic drugs and to evaluate an alternative autosampler-assisted automated approach for the preparation of spiking solutions. Simple protein precipitation of 200-μL whole blood was used followed by analysis by reversed-phase LC-MS/MS in advanced scheduled multiple reaction monitoring (MRM) mode. The method was fully validated according to international guidelines, including selectivity, recovery, matrix effects, linearity, bias/imprecision, processed-sample stability, and limits. Validation criteria were fulfilled for all analytes except for buprenorphine and five benzodiazepines. In the context of a multianalyte procedure, a (multipurpose) autosampler-assisted automatic preparation of calibrator spiking solutions proved comparable to manual preparation. Thus, automated preparation can overcome the frequently performed manual, time-consuming, and error-prone steps of multianalyte approaches and still allow for customized calibration ranges. Since its introduction, more than 8000 cases have been measured with the presented method, and 35 proficiency tests have been passed.

The applicability of urinary minimum reporting limits (MRLs) to determine in-competition use of prohibited substances is an evolving topic. Most stimulants are subject to a universal MRL, despite the wide range of commercially available dosages for commonly used stimulants. Further, it is unknown whether the urinary MRL is reflective of a pharmacological dose ingested after the start of the in-competition period. To evaluate whether urinary MRLs can distinguish between in-competition and out-of-competition use, a controlled administration study was performed with three commonly used stimulants-amphetamine, methylphenidate, and modafinil at relatively low but therapeutically relevant dosages. Four to six volunteers were administered a particular drug once per day for five consecutive days. Urine, serum, dried blood spots (DBS), and oral fluid (OF) were collected during the active administration period and for 48 h after cessation of use. For all participants, urinary concentrations for all target analytes exceeded the MRL even 48 h after cessation of use. In serum and DBS, most volunteers showed detectable amounts at 48 h post use. Peak concentrations were variable between target compounds even with similar administered dosages. Further, there was a reproducible difference between serum and DBS concentrations. Interpretation of results from OF measurements was challenging due to the inability to normalize for hydration status and OF viscosity. Analyte concentrations decreased steadily over the washout period but did not correlate across matrices for all target analytes. The study reiterates the challenges associated with determining in-competition use by relying on urinary concentrations.