Drug Testing and Analysis最新文献

Increasing oxygen transport through elevated hemoglobin concentration and red blood cell mass is a key objective of blood doping, commonly achieved via recombinant human erythropoietin (rHuEPO) administration or blood transfusions. While the Athlete Biological Passport (ABP) offers an effective indirect tool for detecting such manipulations, its sensitivity and specificity may be limited, particularly in cases involving microdoses or confounding physiological factors. To address these limitations, the identification of novel biomarkers that complement current ABP markers is essential.

This study presents a targeted metabolomics approach to discover candidate biomarkers of rHuEPO administration by analyzing polar metabolites in plasma and serum from two administration studies: one involving a single CERA injection, and the other using multiple doses of epoetin delta. Hydrophilic interaction chromatography hyphenated with tandem mass spectrometry enabled the selective and sensitive detection of a panel of polar endogenous metabolites.

Following data normalization and stringent quality control, generalized least squares models were applied to evidence temporal changes in metabolite signals. Among the most responsive and concordant markers across both studies were hypoxanthine and inosine, which showed significant and marked increases following rHuEPO administration. Notably, the relative increase of these metabolites coincided with the maximum in reticulocyte percentages, reflecting maximal erythropoietic activity. As intermediates in purine metabolism, their increases are likely tied to augmented purine turnover during red blood cell production. These findings suggest that hypoxanthine and inosine are promising candidate biomarkers to complement existing ABP parameters. However, further validation is required to confirm their reliability and applicability within the ABP framework.

Clenbuterol (Clb) is a β2-agonist drug included in the list of substances prohibited during and out of competition by the World Anti-Doping Agency (WADA-AMA). Several adverse analytical findings have been detected by accredited WADA laboratories, but athletes often claim that results are due to dietary contamination. In this contribution, bovine microsomal incubation and the excretion of bovine and human urinary metabolites of Clb were analyzed and compared using liquid chromatography electrospray Q-Exactive-Orbitrap mass spectrometry to determine differences in Clb metabolism. Urine samples were processed by solid-phase extraction prior to electrospray analysis in both the positive and negative ion modes. MS/MS experiments were obtained by parallel monitoring reaction (PRM) triggered by an inclusion ions list. The strategy for metabolite identification involved the search for typical biotransformation based on accurate mass shifts using diagnostic fragment ions from the parent drug. This approach successfully identified eight metabolites, including a novel N-methylated form of Clb, reported here for the first time. Additionally, four metabolites found exclusively in bovine urine offer significant potential for further research aimed at distinguishing unintentional doping.

Autologous blood transfusions (ABTs) are prohibited by the World Anti-Doping Agency (WADA), yet detecting autologous blood micro-transfusions (ABMTs) remains a challenge. Due to smaller transfused volumes, ABMTs cause attenuated biomarker changes, limiting detection sensitivity within the Athlete Biological Passport (ABP). This study assessed whether mRNA expression of 5-aminolevulinic acid synthase (ALAS2) and carbonic anhydrase 1 (CA1), measured from dried blood spots (DBS), could serve as sensitive biomarkers of ABMT. In a randomized, placebo-controlled design, 47 trained individuals (24 ♀; mean VO₂peak 56 ± 7 mL·min⁻¹·kg⁻¹) were allocated to an ABMT group (n = 23; ♀ = 12) or placebo group (n = 24; ♀ = 12). The ABMT group donated 450 mL of blood and received a 130 mL packed red blood cell reinfusion 4 weeks later. Blood sampling occurred regularly before and after both donation and reinfusion. ALAS2 and CA1 mRNA expression from DBS, and reticulocyte percentage (RET%) from venous blood, were analyzed. Following blood donation, ALAS2, CA1, and RET% increased by 270%, 200%, and 150%, respectively. However, no consistent group-level changes were observed after ABMT. Individualized analysis identified more outliers for ALAS2 than for CA1, and blinded interpretation of individual mRNA profiles achieved > 95% sensitivity and specificity for detecting ABMT. These findings suggest that ALAS2 mRNA expression, assessed via minimally invasive DBS sampling, is a promising biomarker for identifying ABMT. This approach may enhance current anti-doping strategies by improving sensitivity to small-volume autologous transfusions that evade detection through traditional ABP biomarkers.

Methamphetamine is relatively uncommon on the European drug market, but Swedish drug test laboratories repeatedly detect low methamphetamine concentrations in urine samples containing high amphetamine levels, warranting clinical evaluation for suspected polydrug use. Of 12,062 routine samples screened positive for amphetamines, 86% were confirmed positive (≥ 200 μg/L) for amphetamine, 2.1% for methamphetamine, and 4.0% for 3,4-methylenedioxymethamphetamine (ecstasy) by liquid chromatography–tandem mass spectrometry. Of the 259 methamphetamine-positive samples, 98% contained amphetamine concentrations above the reporting limit, consistent with the metabolic conversion of methamphetamine to amphetamine in the body. However, in most (69%) of these samples, the methamphetamine concentration was only < 2% of the amphetamine level, suggesting methamphetamine was not the primary drug taken. Chiral analysis of selected samples showed that after use of illicit street amphetamine with a racemic content of the l- and d-enantiomers, a similar l/d proportion was observed for methamphetamine. Similarly, in samples from patients receiving d-amphetamine-based ADHD medication, low d-methamphetamine levels were detected, even though the pharmaceutical products contained no methamphetamine. This observation, together with the parallel l/d-enantiomer distributions, supports amphetamine N-methylation as a trace, albeit quantitatively insignificant, metabolic pathway in humans. From both a clinical and forensic perspective, a low urinary methamphetamine concentration of less than a few percent of the amphetamine level therefore does not warrant further clinical evaluation for suspected polydrug use. The present findings further demonstrate that chiral analysis of both amphetamine and methamphetamine is an effective approach for distinguishing between illicit and therapeutic sources in positive screening drug tests for the amphetamines.