Drug Testing and Analysis最新文献

Ethyl glucuronide (EtG) in hair is a reliable biomarker of alcohol consumption habits. Due to its small concentration incorporated into hair, analytical methods sensitive enough to reliably quantify EtG in this matrix are required. Sample preparation is critical in hair analysis, especially for EtG, for which extraction efficiency and matrix effect can strongly influence the results; furthermore, miniaturized methods are sought, to reduce solvent use and times of sample preparation. A micro extraction by packed sorbent (MEPS) procedure coupled to a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of EtG in human hair samples. Fifty milligrams of hair samples were cut into snippets and extracted in water. The cleanup of the extract was carried out by using a MEPS syringe packed with anion exchange sorbent (SAX); all parameters for conditioning, washing, loading and eluting steps were optimized and the eluted aqueous volume was directly injected in the LC-MS/MS system operating in the negative ionization mode. The method was fully validated assessing LOD, LOQ, calibration curve, repeatability, accuracy, matrix effect and carryover. The method was subsequently applied to QCs and authentic hair samples. The developed MEPS method is quick and effective, with low solvent purchase and discard costs, allowing the differentiation between social drinkers and chronic excessive alcohol consumers, according to the cut-offs established by the Society of Hair Testing (SoHT).

The amino-terminal propeptide of type III procollagen (P-III-NP) is used with IGF-I to detect the illicit use of growth hormone and to monitor growth hormone therapy. However, the only currently available assays for P-III-NP are immunoassays, which are not well harmonized. In addition, other fragments of type III procollagen may better evaluate collagen turnover. We aimed to develop a high-throughput assay using immunoaffinity enrichment coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify peptides belonging to three different regions of type III procollagen in human serum simultaneously. To facilitate higher throughput, we transferred the assay from microcentrifuge tubes to a 96-well plate format with partially automated pipetting. The method was linear (Pearson's R ≥ 0.994) over an estimated concentration range of 1.35-13.3 nM, 0.04-2.28 nM, and 0.26-5.1 nM for each surrogate peptide of P-III-NP, collagen degradation products, and the carboxyl-terminal propeptide, respectively. Intra-day and inter-day imprecision were both < 13.6%, and the results of robustness testing were also encouraging. The method was successfully applied to capillary blood samples obtained using Tasso+ microsampling devices. Modest correlation of P-III-NP concentration was observed between our new method and a WADA-approved immunoassay (N = 40, Pearson's R = 0.789) with a significant bias of -87.8%. Our method simultaneously quantifies four peptides belonging to three regions of type III procollagen in human serum. High bias between assays highlights the need for common higher-order calibrators or reference materials to help improve the comparability of results across laboratories.

Blood collection is an important facet of anti-doping testing, forming the basis of the hematological module of the athlete biological passport (ABP). Presently, whole blood is collected via venepucture under standardized conditions, in accordance with the World Anti-doping Agency's International Standard for Testing and Investigations (ISTI). Advances in capillary whole blood collection technology now afford the ability to collect microvolumetric capillary whole blood from the upper arm (or other suitable vascular location such as the abdomen) that is "needle-free" and virtually painless. Previous work has demonstrated the suitability and feasibility of capillary whole blood compared with venous using the Tasso+ device. Recently, other microcapillary devices have received FDA approval increasing the options available to the anti-doping community. The present study extends previous work, by collecting microliter capillary whole blood samples via two different FDA approved devices (RedDrop and the Tasso+ device) to compare with venous blood collected simultaneously. Ten participants provided three matched blood samples (2× capillary and 1× venous) in accordance with WADA ABP guidelines for blood sample collection, for a total of 30 samples. Capillary samples from both devices showed excellent laboratory agreement with venous blood for all CBC parameters, with the exception of platelets. Excellent laboratory agreement was also observed between the two microcapillary collection devices. Irrespective of the device, microcapillary blood collection provides a valid alternative to venous blood collection for ABP purposes.

Detecting the use of bisphosphonates (BPs) in equine athletes is of interest to regulators and laboratories due to the threat to welfare issues for the potential to provide analgesic effects and manipulating bone structure. The detection of BPs in biological matrices is challenging due to erratic biological elimination and inconsistent analytical recoveries. Therefore, complementary approaches are needed to provide evidence of their misuse in racehorses. BPs have two sub-classes: nitrogenous and non-nitrogenous. This study investigated plasma elimination following administration of one example from each sub-class, together with changes in endogenous eicosanoid and corticosteroids. Zoledronic acid (ZA) and tiludronic acid (TA) were administered by IV infusion to 8 thoroughbred horses with an 11-month washout period between each administration. Sample preparation for quantification of BPs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilised a two-step solid phase extraction (SPE) consisting of polymeric reversed-phase followed by weak anion exchange prior to derivatisation using trimethyl orthoacetate. Endogenous biomarkers were analysed after protein precipitation and SPE with polymeric reversed-phase prior to liquid chromatography-high resolution mass spectrometry (LC-HRMS) using data independent acquisition. The LC-MS/MS analysis showed ZA was undetectable after 8 h post-administration while TA was detected up to the final collection point of 28 days post-administration. The LC-HRMS analysis utilised targeted (i.e., prior inclusion list of compounds) approaches to monitor level changes of eicosanoid and corticosteroid biomarkers. Putative biomarkers were identified and now subject to validation for translation into routine sample analysis for improved retrospectivity to detecting BP misuse in equine plasma.

After the Scottish Prison Service (SPS) introduced mail photocopying procedures in December 2021, a shift in smuggling methods was observed for synthetic cannabinoid receptor agonists (SCRAs) and other new psychoactive substances (NPS) from drug-infused papers back to traditional sample matrices (e.g., tablets and powders), although new matrices also emerged. This study reports on waxy- or putty-like materials as a novel drug preparation for SCRAs and other drugs seized from UK prisons. In 2023, 22 of these new preparations were seized from Scottish prisons, with eight found in sealed vape pods. The materials were positive for SCRAs, phytocannabinoids, novel benzodiazepines, and/or gabapentinoids. Additionally, 11 preparations were seized from an English prison, all containing the SCRAs MDMB-4en-PINACA and MDMB-INACA. MDMB-INACA was pharmacologically characterized using in vitro CB₁ and CB₂ bioassays, revealing moderate efficacy but low potency at CB₁. Furthermore, the in vitro CB₁ bioassay was also used to evaluate the CB₁ activating potential of extracts from eight seized samples. Six of these showed high CB₁ activity, whereas the samples lacking SCRAs or containing only MDMB-INACA showed no or only weak CB₁ activity, respectively. Lastly, applying the bioassay as an activity-based "untargeted" screening method effectively identified the presence of SCRAs in one waxy preparation, which was initially not detected by gas chromatography-mass spectrometry (GC-MS). This underscores the effectiveness of the bioassay for evaluating these new waxy- or putty-like materials for the presence of SCRAs.

Counterfeit pharmaceuticals are a subclass of falsified and substandard medicines. They are illicit products, purporting to be genuine medicines, that are made and sold by criminal organisations. They represent a significant risk to patient safety, as well as a financial and reputational threat to the companies who make the genuine medicines. It is essential to have analytical methods to determine if suspect samples seized by law enforcement agencies are counterfeit, with mass spectrometry (MS) being a commonly used technique in forensic cases. Speed-to-answer is vital to enable law enforcement agencies to progress investigations, as well as for pharmaceutical companies so that they can notify health authorities of the circulation of counterfeit medicines. In this work, an atmospheric solids analysis probe (ASAP)-MS was assessed as a fast and simple-to-use approach to analyse tablets on a commercially available instrument. Complementing the analytics with real-time sample recognition software demonstrated that the classification of tablets as authentic or counterfeit could be achieved quickly (< 2 min) and without the need for MS interpretation skills. Authentication of five tablets (two authentic pharmaceuticals, one placebo and two counterfeits containing the correct active pharmaceutical ingredient [API] but at lower quantities than in the genuine medicine and with different excipient contents) of unknown origin was achieved with 100% success. This creates the opportunity to deploy the end-to-end workflow as a tool for non-scientists, such as law enforcement officers and border control staff, for use in-territory to obtain fast answers and make data-led decisions to control the illegal trading of medicines.

Roxadustat (FG-4592), an orally active hypoxia-inducible factor prolyl hydroxylase stabilizer, has been shown to enhance erythropoiesis by increasing endogenous erythropoietin. It is indicated for the treatment of anemia and chronic kidney disease and is approved for clinical use in several countries, including the European Union, Japan and others. Due to its reasonably anticipated performance-enhancing effect in athletes, roxadustat is prohibited for use in sports at all times. A few cases of adverse analytical findings in routine doping controls have been reported worldwide, some of which were claimed to be the result of contaminated dietary supplements. The present study offers new data demonstrating the long-term excretion pattern of roxadustat. Even after a single-dose administration, roxadustat can remain detectable in urine for 8 months, albeit at very low concentrations (<10 pg/mL). Following three times a week treatment with 70 to 100 mg of roxadustat, the drug was still detectable in the urine of anemic patients for between 9 and 18 months after treatment was discontinued. Lastly, an athlete who admitted use of roxadustat for almost a year (50 mg 3 to 5 times a week) has now tested positive multiple times over the course of 15 months (the first test being 12 months after the drug was discontinued), with an estimated concentration of roxadustat between 3 and 8 pg/mL. Altogether, these findings indicate the unusually prolonged terminal excretion kinetics of roxadustat, a property that testing authorities should consider in their results management process.

Detection and monitoring of biomarkers related to doping is particularly suitable for the development of analytical strategies dedicated to indirect detection of banned substances. Previous studies in horses have already allowed the investigation of transcriptomic biomarkers in equine blood associated with reGH and rHuEPO administrations. Our most recent developments continue to focus on the discovery and monitoring of transcriptomic biomarkers for the control of ESAs, and a collaborative study with WADA-accredited doping control laboratories has recently been initiated to conduct a pilot study. In humans, three mRNAs (ALAS2, CA1, and SLC4A1) were previously observed to be differentially expressed after blood doping and were associated with immature red blood cells, the so-called circulating reticulocytes. In horses, circulating reticulocytes are rarely observed even after rHuEPO administration. With the improved primers that detect the equine orthologues of the human mRNAs from the ALAS2, CA1, and SLC4A1 genes, we can now report the first evidence of the detection of the three biomarkers in equine blood. In addition, an upregulation of the mRNA levels of the three genes was observed after analysis of blood samples collected from MirCERA-treated animals, with kinetics similar to those previously documented in humans. Our data suggest that ALAS2 and CA1 are promising indirect biomarkers for the detection of recombinant EPO abuse in horses, as observed in humans.

Thymosin $\beta$ 4 (TB4) is a ubiquitous, highly conserved and abundant peptide among mammals with a critical role in cytoskeleton organization. In spite of its yet non-authorized use as a medicine and being forbidden by the IFHA, the FEI, and the WADA, intelligence and doping control laboratories reported numerous products available online claiming to contain a synthetic acetylated fragment of TB4 or TB4 itself, promoted as a growth factor with regenerative properties. In this article, the first estimation of the endogenous TB4 concentration in racing horses' blood samples was performed through a population study. We reveal that this concentration does not significantly depend on gender, age, nor horse breed. We highlight that the TB4 concentration increases significantly and rapidly in plasma stored at 4°C when not separated from blood cells due to cell lysis. Finally, we also demonstrate that the detection of a non-natural synthesis impurity is possible in equine plasma after a single dose administration of a TB4 containing product to a horse.