Drug Test Anal [JOURNAL]
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200 papers
Clostebol Metabolism by Different Routes of Administration: Selection of Diagnostic Urinary Markers.
de la Torre X, Colamonici C, Martínez Brito D … +2 more , de Oca Porto RM, Botrè F
Drug Test Anal · 2025 Dec · PMID 40955777 · Publisher ↗
The accidental contamination by the use of transdermal applications of clostebol acetate has been proven by the monitoring of its main urinary metabolite 4-chloro-3α-hydroxy-androst-4-en-17-one (M1). This work is aimed a...
The accidental contamination by the use of transdermal applications of clostebol acetate has been proven by the monitoring of its main urinary metabolite 4-chloro-3α-hydroxy-androst-4-en-17-one (M1). This work is aimed at describing clostebol metabolism in humans and at searching for specific metabolic markers or concentration thresholds allowing for distinguishing between an oral and a transdermal administration, helping to set up adequate criteria to be adopted by the antidoping community when incidental contamination is suspected. Urine samples were collected after the administration of a single dose of clostebol acetate orally (n = 3, males), a single transdermal dose (n = 1, male), and multiple transdermal administrations (n = 3, males, and n = 3, females). After enzymatic hydrolysis, liquid-liquid extraction, and the formation of trimethylsilyl derivatives, the samples were analyzed by gas chromatography coupled to tandem mass spectrometry and time-of-flight mass spectrometry. The metabolism of clostebol after oral and transdermal applications was described. Ten metabolites were detected after oral administration (M1-M10), but only five (M1-M4 and M9) could be detected after transdermal applications under the assay conditions applied. The use of concentrations of any metabolite might be difficult because of the interindividual variability in absorption, metabolism, and/or excretion. Instead, the ratios between M4 and M1 showed plausible results to discriminate between both administrations under the conditions described here. The intentionality of the use of one or other route of administration cannot be assessed.
Comparative Evaluation of Smart Sampling for hCG Determination: A New Potential Direction in Protein Biomarker Analysis From Dried Microsamples.
Mrsa A, Matijevic M, Dehnes Y … +2 more , Halvorsen TG, Reubsaet L
Drug Test Anal · 2025 Dec · PMID 40947129 · Publisher ↗
Since the early 20th century, sampling biological matrices like blood on paper (dried blood spots [DBS]) has been vital in clinical analysis. While DBS microsampling is convenient for small molecules, extensive sample pr...
Since the early 20th century, sampling biological matrices like blood on paper (dried blood spots [DBS]) has been vital in clinical analysis. While DBS microsampling is convenient for small molecules, extensive sample preparation can make LC-MS protein analysis impractical because of the time-consuming steps, especially for low-abundance proteins. Smart sampling, introduced in 2018, simplifies this by integrating sample preparation directly on the sampler. The work presented in this paper aims to compare a newly validated smart sampling method with two other methods: an in-house method based on immunocapture on magnetic beads and a commercial method that uses electrochemiluminescence immunoassay (ECLIA). The performance of the three hCG methods was compared using 21 single-blind serum samples. Linear regression analysis revealed strong correlations (all R > 0.91) between the actual sample concentrations and the results obtained from all three methods. Immunocapture with magnetic beads showed the strongest linear correlation (R = 0.974). To assess agreement between the methods, Bland-Altman analysis was conducted. The comparison between smart sampling and magnetic beads showed an average bias of -5.2, with no significant trend in variation across the sample concentration range of 0.5-75 ng/mL. The smart sampling and ECLIA comparison revealed a bias of 0.4 ± 4 ng/mL, indicating even better agreement and consistent results. This paper presents the first-ever comparison of a smart sampling method with existing methods. The results highlight smart sampling as a promising new approach for bioanalysis and boost the technique as a viable alternative in protein biomarker analysis from complex matrices using LC-MS.
From Kratom to Semi-Synthetic Opioids: The Rise and Risks of MGM-15.
Gour A, Mukhopadhyay S, Henderson A … +7 more , Awad A, Seabra MA, Pullman M, León F, Cutler SJ, McCurdy CR, Sharma A
Drug Test Anal · 2025 Dec · PMID 40936282 · Publisher ↗
Kratom (Mitragyna speciosa), a plant native to Southeast Asia, has long been used for its stimulant and analgesic properties. 7-Hydroxymitragynine (7-HMG) is a potent and selective opioid agonist in vitro and demonstrate...
Kratom (Mitragyna speciosa), a plant native to Southeast Asia, has long been used for its stimulant and analgesic properties. 7-Hydroxymitragynine (7-HMG) is a potent and selective opioid agonist in vitro and demonstrates a potent opioid effect in living subjects, reversible by naloxone. It has been semi-synthesized into products that are readily available in retail and virtual shops. It is known that 7-HMG has earned the nickname "legal morphine," and has gained popularity among users seeking pain relief and/or a "high" comparable with prescription opioids. Medicinal chemistry efforts have led to synthetic 7-HMG derivatives such as MGM-15, where stereospecific saturation of the imine N(1)-C(2) double bond increases opioid receptor affinity and activity. Despite its higher in vitro opioid potency, MGM-15 is currently sold in the US for human consumption as a "research chemical" in tablet form, even though there is an absence of this being studied in humans and obviously no FDA approval. In this study, we analyzed commercially available MGM-labeled tablets using UPLC-MS/MS and subsequently evaluated the binding affinities of purified MGM-15 across multiple opioid receptors. Tablets contained an average of 10.9 ± 0.2 mg (10.7 to 11.2 mg) of MGM-15, with no naturally occurring kratom alkaloids or illicit substances detected. MGM-15 shows greater hMOR and hDOR binding affinities than 7-HMG, indicating the potential for higher opioid effects and risks, emphasizing the urgent need for more research to advise regulation and hopefully prevent misuse and harm.
Detection of Anabolic Androgenic Steroids and Steroid Esters-Comparing Dried Blood Spots Collection Devices and Urine Samples.
Suominen T, von Walden J, Harju L … +4 more , Pohanka A, Schulze J, Lehtihet M, Ekström L
Drug Test Anal · 2025 Dec · PMID 40925340 · Full text
Dried blood spots (DBS) have emerged as a promising complement, and in some settings, an alternative, to urine for anabolic androgenic steroid (AAS) testing, offering advantages such as minimal invasiveness, simplified s...
Dried blood spots (DBS) have emerged as a promising complement, and in some settings, an alternative, to urine for anabolic androgenic steroid (AAS) testing, offering advantages such as minimal invasiveness, simplified storage, and transportation. This study evaluated two DBS collection devices-cellulose-based Capitainer-B50 and polymer-based Tasso-M20-and compared results with traditional urine analysis. Ten self-reported AAS users were recruited and provided matched urine and DBS samples. High agreement between the two DBS devices was observed, although Capitainer-B50 showed a slightly greater detection rate, likely due to a higher sample volume (50 μL vs. 17.5 μL) improved analyte recovery, and lower background noise. Notably, DBS enabled detection of testosterone use in all 10 participants, while urine testing missed two cases with naturally low urinary testosterone/epitestosterone (T/E) ratios (most likely UGT2B17 del/del carriers). Moreover, the differentiation between prescribed and illicit use of testosterone esters was also possible in DBS, but not in urine testing, while nandrolone detection in DBS was limited at low concentrations. The findings support DBS as a sensitive and practical tool for AAS detection and provide critical advantages in detecting doping with testosterone esters in individuals with prescribed testosterone therapy and in UGT2B17 deletion carriers.
Impurity Profiling of ATS Synthesized From Ring-Substituted APAAN and MAPA Analogs.
Langone D, Painter B, Nash C … +4 more , Hulshof JW, Oldenhof S, Johnston MR, Kirkbride KP
Drug Test Anal · 2025 Dec · PMID 40921150 · Publisher ↗
Designer precursors for the synthesis of amphetamine-type stimulants pose a significant challenge to law enforcement. The precursors APAAN (α-phenylacetoacetonitrile) and MAPA (methyl α-acetylphenylacetate) became popula...
Designer precursors for the synthesis of amphetamine-type stimulants pose a significant challenge to law enforcement. The precursors APAAN (α-phenylacetoacetonitrile) and MAPA (methyl α-acetylphenylacetate) became popular in the previous decade and have since been restricted. Recently, a ring-substituted analog of MAPA used for the synthesis of MDMA (3,4-methylenedioxymethamphetamine) was detected, highlighting the potential for criminal misuse of substituted analogs of these designer precursors. Previous research has characterized the impurity profiles of methamphetamine synthesized from APAAN and MAPA. In this study, the impurity profiles of MDMA and PMMA (p-methoxymethamphetamine) synthesized from ring-substituted analogs of APAAN and MAPA were characterized. In addition, byproducts forming from the conversion of p-methyl and p-fluoro analogs of APAAN and MAPA into analogs of P2P were investigated. MDMA was synthesized from 3,4-methylenedioxy-substituted MAPA (methyl α-acetyl-[3,4-methylenedioxyphenyl]acetate, MAMDPA) via sodium cyanoborohydride reductive amination and hydrogenation with platinum oxide and methylamine. Four impurities originating from MAMDPA were detected in MDMA synthesized via reductive amination, all of which are 3,4-methylenedioxy analogs of the impurities detected in methamphetamine synthesized from MAPA. Synthesis of MDMA via the hydrogenation route only produced one characteristic impurity, which, due to its instability under acidic conditions, is not likely to be present in clandestine MDMA hydrochloride samples. PMMA was synthesized via sodium cyanoborohydride reductive amination of MeO-P2P produced from methoxyl-substituted APAAN (α-p-methoxyphenylacetoacetonitrile, APMPAAN). Five impurities were identified as originating from APMPAAN, two of which are proposed to be the most reliable markers for the use of APMPAAN in the synthesis of PMMA.
RETRACTION: Voltammetric Determination of Isoproterenol Using Multiwall Carbon Nanotubes-Ionic Liquid Paste Electrode.
Drug Test Anal · 2025 Oct · PMID 40904173 · Publisher ↗
Abstract loading — click title to view on PubMed.
Enantiomeric Determination of Medetomidine in Street Drug Samples (August 2024-February 2025) and Implications for Immunoassay Test Strip Analysis.
Sisco E, Ventura M, Shuda SA
Drug Test Anal · 2025 Dec · PMID 40903287 · Full text
Over the last several years, there has been an influx of α-agonists into the street drug supply, beginning with the proliferation of xylazine, a potent veterinary sedative. Since 2023, another sedative, medetomidine, has...
Over the last several years, there has been an influx of α-agonists into the street drug supply, beginning with the proliferation of xylazine, a potent veterinary sedative. Since 2023, another sedative, medetomidine, has been widely detected. Medetomidine, broadly, encompasses two enantiomers-dexmedetomidine and levomedetomidine-with the dex enantiomer being pharmacologically active and present in human-use pharmaceuticals. In this work, we investigate street drug samples containing medetomidine to better understand the enantiomeric makeup in the illicit supply. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze 100 drug product or paraphernalia residue samples. All samples were found to contain a racemic mixture of medetomidine. A subset of samples was analyzed by medetomidine immunoassay test strips, where racemic mixtures were not found to negatively affect results. All samples were also qualitatively analyzed using direct analysis in real time mass spectrometry (DART-MS) to identify commonly co-occurring compounds, which included fentanyl, xylazine, and local anesthetics. Parabens, preservatives found in licit injectable preparations of the drug, were not detected. This work provides a snapshot into the medetomidine makeup of the street drug supply, which needs to be continually monitored given the differences in pharmacology between the two enantiomers.
Identification of Tetrahydrocannabidiol Metabolites in Human Urine.
Schirmer W, Mösch I, Schürch S … +1 more , Weinmann W
Drug Test Anal · 2025 Dec · PMID 40897389 · Full text
Tetrahydrocannabidiol (H4CBD) is an emerging semisynthetic cannabinoid, which has been known since 1940. Like hexahydrocannabinol (HHC), it is easily obtained by hydrogenation of available phytocannabinoids, in the case...
Tetrahydrocannabidiol (H4CBD) is an emerging semisynthetic cannabinoid, which has been known since 1940. Like hexahydrocannabinol (HHC), it is easily obtained by hydrogenation of available phytocannabinoids, in the case of H4CBD by hydrogenation of cannabidiol (CBD). H4CBD shows a weak affinity for the CB receptor, but it is unclear if H4CBD shows psychoactive properties, as reports from users are divided. Only a few countries have placed H4CBD under their narcotic substance law, for example, France and Switzerland. The aim of this study was to identify human Phase I and II metabolites in urine as potential forensic targets. The H4CBD used for this study was bought from an online store and analyzed beforehand using GC-MS. The Phase I and II metabolites were identified using LC-HR-MS/MS and GC-MS after trimethylsilylation. The found H4CBD metabolites were carboxylated, hydroxylated, and bishydroxylated species and their glucuronides with hydroxylation and carboxylation positions on the alicyclic moiety and on the side chain. The tentatively identified metabolites were the carboxylic acids 5″-COOH-H4CBD and 7-COOH-H4CBD, the hydroxylated metabolites (1R,6R)-OH-H4CBD, (1R,6S)-OH-H4CBD, two epimers of 2″-OH-H4CBD, and both epimers of 7-OH-H4CBD. The identified bishydroxylated metabolites were side-chain hydroxylated derivatives of 7-OH-H4CBD. Various other hydroxylated metabolites were found, but their exact hydroxylation positions could not be determined. Some ESI+ spectra of the metabolites showed very unusual fragmentation patterns, like the loss of both oxygens from the resorcinol moiety with subsequent ring contraction and the appearance of radical cations for Phase II metabolites. These unusual patterns were noticed for H4CBD and its side-chain-altered metabolites.
Identification of Candidate Blood Biomarkers of Recombinant Human Erythropoietin Administration Using Targeted Polar Metabolomics by HILIC-MS/MS.
Salamin O, Ramic L, Nicoli R … +3 more , Rudaz S, Guillarme D, Kuuranne T
Drug Test Anal · 2025 Dec · PMID 40887900 · Full text
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 tr...
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.
Identification of Novel THC Analogs: Chloro Derivatives of Hexahydrocannabinol and Tetrahydrocannabibutol Butanoate in an Oil Product Obtained in Japan.
Tanaka R, Kawamura M, Ito M … +1 more , Kikura-Hanajiri R
Drug Test Anal · 2025 Nov · PMID 40880172 · Publisher ↗
Since around 2021, products such as e-cigarette liquid cartridges, herbal products, and gummy products, claiming to contain tetrahydrocannabinol (THC) analogues, have been seen for sale on the internet. Recently, product...
Since around 2021, products such as e-cigarette liquid cartridges, herbal products, and gummy products, claiming to contain tetrahydrocannabinol (THC) analogues, have been seen for sale on the internet. Recently, products claiming to contain other THC derivatives have appeared. In this study, we identified the ingredients in products distributed on the internet that claim to contain THC derivatives. The e-cigarette cartridge product analyzed in this study was obtained from Japan in September 2024. One milligram of the oil product was treated with 1 mL of acetonitrile under ultrasonication. The resulting solutions were used for gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) measurements. After isolating and purifying unknown components from the product, structural analysis was performed by measuring H, C nuclear magnetic resonance (NMR) and various two-dimensional NMR (COSY, HMQC, HMBC, and NOESY) and LC with hybrid quadrupole time-of-flight MS. The analysis revealed that chlorinated HHCs (i.e., (9R)-2-chloro-HHC, (9S)-2-chloro-HHC, (9R)-4-chloro-HHC, (9S)-4-chloro-HHC, (9R)-2,4-dichloro-HHC, and (9S)-2,4-dichloro-HHC) were the major components, and chlorinated dihydro-iso-THCs (i.e., 10-chloro-dihydro-iso-THC, 8-chloro-dihydro-iso-THC, and 8,10-dichloro-iso-THC) were the minor components isolated and identified from the product. Furthermore, Δ-THCB-O-butanoate, a compound in which the hydroxyl group at the C1 position of ΔTHCB was butanoylated, was detected.
Differentiating Positional Isomers of the 2-(Dimethoxyphenyl)-N-(2-(trifluoromethoxy)benzyl)ethanamine Series: A Chromatography-Mass Spectrometry Approach.
Kupriyanova OV, Shevyrin VA, Sadykova RG … +1 more , Shafran YM
Drug Test Anal · 2025 Nov · PMID 40879633 · Publisher ↗
Synthetic derivatives of phenylethanamine, specifically 2-(2,5-dimethoxyphenyl)-N-((2-substituted)benzyl)ethanamines, regularly appear on the global market of new designer drugs and have been reported to be associated wi...
Synthetic derivatives of phenylethanamine, specifically 2-(2,5-dimethoxyphenyl)-N-((2-substituted)benzyl)ethanamines, regularly appear on the global market of new designer drugs and have been reported to be associated with adverse effects in humans who consume them. At the same time, their positional isomers may lack psychoactive effects. These compounds are not currently regulated by legislation and could potentially be used for medical purposes in the future. Continuing the study of the properties of 2-(2,5-dimethoxyphenyl)-N-((2-substituted)benzyl)ethanamines with different substituents, a poorly studied series of 2-(dimethoxyphenyl)-N-(2-(trifluoromethoxy)benzyl)ethanamines (NBOMe(F)) with a trifluoromethoxy group (OCF) in the ortho-position of the N-benzyl fragment is of interest. Gas chromatographic analysis revealed the existence of a critical pair of isomers when using an HP-5 type column, the differentiation of which is difficult under varying temperature conditions of the column thermostat. Their separation was achieved on a DB-17MS column under isothermal conditions at a temperature of 210°C. A method using thin-layer chromatography for the differentiation of this critical pair of isomers is also proposed. Retention indices of the isomeric compounds of the NBOMe(F) series and their N-trifluoroacetyl derivatives have been determined, which can serve as an additional identification criterion for gas chromatographic analysis. It has been shown that differentiation of NBOMe(F) isomers using electron ionization mass spectra is only possible if the spectra of both the isomers themselves and their N-trifluoroacetyl derivatives, which exhibit significant differences, are recorded. As an alternative method for differentiating the isomeric compounds, an approach using high-performance liquid chromatography-tandem mass spectrometry, which does not require derivatization, is proposed.
Post-Mortem Identification and Toxicological Findings of Fluetonitazepyne and Isotonitazepyne.
Kriikku P, Pelander A, Jylhä A … +1 more , Ojanperä I
Drug Test Anal · 2025 Nov · PMID 40879310 · Full text
Nitazepyne-type substances, such as fluetonitazepyne and isotonitazepyne, are among the latest additions to the group of nitazenes-highly potent and dangerous opioids that have emerged on the illicit drug market in recen...
Nitazepyne-type substances, such as fluetonitazepyne and isotonitazepyne, are among the latest additions to the group of nitazenes-highly potent and dangerous opioids that have emerged on the illicit drug market in recent years. In early 2025, five death cases in Finland tested positive for fluetonitazepyne and one for isotonitazepyne in urine drug screening. The median (range) concentration of fluetonitazepyne in post-mortem femoral blood was 1.7 (0.4-9.5) μg/L, and the concentration of isotonitazepyne was 1.4 μg/L. Other psychoactive substances were detected in all cases. A peak corresponding to O-dealkylated fluetonitazepyne, the 4-OH-nitazepyne metabolite, was present in all fluetonitazepyne-positive urine samples and was later used in the identification of isotonitazepyne in one fatal case. This metabolite proved useful as a marker compound for nitazepyne-type benzimidazole opioids in a urine screening.
Testing for Meldonium, a Doping Agent, in Human Hair.
Kintz P, Alvarez JC, Gheddar L
Drug Test Anal · 2025 Dec · PMID 40878895 · Publisher ↗
Meldonium has been developed in the 70s in Latvia and is currently used in a limited number of countries for heart-related diseases, such as heart attack, failure, or angina pectoris. Due to its metabolic properties (dec...
Meldonium has been developed in the 70s in Latvia and is currently used in a limited number of countries for heart-related diseases, such as heart attack, failure, or angina pectoris. Due to its metabolic properties (decrease of lactate production, increase of glycogen use, and protective action again oxidative stress), meldonium has been abused by numerous athletes to enhance their performance. The drug has been prohibited by the World Anti-Doping Agency since 2016 and is on the S4.4.3 list (metabolic modulators) of the prohibited substances at all times. As athletes can challenge their anti-doping violation involving meldonium, there is an interest in testing for it in hair in order to document their pattern of exposure. Such hair application can be complicated to develop, as meldonium has a chemical formula close to an amino acid and presents an ionized fraction, which are limiting factors for drug incorporation into hair. Liquid chromatography coupled to tandem mass spectrometry was used. The drug was extracted from hair after methanol incubation in an ultrasound bath and separation on a BEH HILIC column. Linearity was verified from 0.5 to 100 pg/mg (R = 0.9943). The limit of detection was 0.1 pg/mg. Although their meldonium regimen was unknown, the drug was identified in the proximal hair segment (0 to 1 cm) of three consumers at 0.7, 6.1, and 17 pg/mg, highlighting for the first time the incorporation in hair of this molecule.
Biosynthesis and Identification of Clenbuterol Metabolites in Urine and In Vitro Microsome Incubation Samples Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry: A Comparison Between Human and Bovine Metabolism.
Gómez-Tagle A, Bressan C, Ventura R … +3 more , Álvarez-Sanchez A, Cardenas-Yong E, Velasco-Bejarano B
Drug Test Anal · 2025 Dec · PMID 40859598 · Full text
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 acc...
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.
mRNA Biomarkers in Dried Blood Spots May Improve Detection of Autologous Blood Micro-Transfusions Using an Individualized Approach.
Andersen AB, Oliveira JA, Loria F … +5 more , Bejder J, Salamin O, Kuuranne T, Nordsborg NB, Leuenberger N
Drug Test Anal · 2025 Nov · PMID 40781904 · Full text
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 at...
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 VOpeak 56 ± 7 mL·min·kg) 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.
Estimation of Plasma Volume by Machine Learning to Improve the Interpretation of the Athlete Biological Passport.
Krumm B, Lewis L, Mørkeberg J … +4 more , Schumacher YO, d'Onofrio G, Moreillon B, Faiss R
Drug Test Anal · 2025 Nov · PMID 40781891 · Full text
The identification of confounding factors related to plasma volume (PV) fluctuations is crucial for appropriate qualitative interpretations of Athlete Biological Passport (ABP) profiles. As part of ongoing efforts to rem...
The identification of confounding factors related to plasma volume (PV) fluctuations is crucial for appropriate qualitative interpretations of Athlete Biological Passport (ABP) profiles. As part of ongoing efforts to remove PV variance from the concentration-based biomarkers such as hemoglobin concentration ([Hb]), a new machine learning model for blood volume (BV) estimation using a single complete blood count analysis was applied within the ABP framework. Forty existing ABP profiles from elite athletes and healthy control subjects were used. PV was estimated using a machine learning model trained on a previous dataset. First, a visual display of the estimated PV shift was added in overlay of individual profiles. Alternatively, individual [Hb] thresholds were adjusted in a new graphical profile to account for PV variations. Finally, a set of ABP profiles with PV estimations was presented to ABP experts to assess the model's relevance in interpreting hematological data. A moderate correlation was found between measured and estimated PV in both men (r = 0.40, p < 0.0001) and women (r = 0.39, p < 0.0001), supporting the validity of the estimation model. In addition, ABP experts favorably assessed the available PV information, particularly the visual representation of PV. This novel estimation model offers distinct advantages (e.g., same biomarkers currently analyzed from routine ABP analyses) and could therefore be of particular interest. Further application of this model in the presence of specific and transient confounding factors may allow to confirm these results.
Origin and Interpretation of Low Methamphetamine Levels Found in Amphetamine-Positive Urine Samples: Support for Methylation of Amphetamine as a Minor Metabolic Pathway.
Helander A, Andersson A, Villén T
Drug Test Anal · 2025 Nov · PMID 40776552 · Full text
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...
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.
Response to Letter to Editor From Brown et al.
Handelsman DJ, Bermon S
Drug Test Anal · 2025 Nov · PMID 40774829 · Publisher ↗
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Comment on "Biology and Management of Male-Bodied Athletes in Elite Female Sports" by Handelsman and Bermon.
Brown GA, Shaw B, Shaw I … +8 more , Joyner MJ, Hunter SK, Senefeld JW, Tucker R, Hilton E, Lundberg TR, Carlson C, Kirk C
Drug Test Anal · 2025 Nov · PMID 40774828 · Publisher ↗
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Ethylated Phosphorylcholine as a New Marker for Alcohol Consumption: A Proof of Concept.
Dumitrascu C, Iturrospe E, Scheir E … +16 more , Timmermans P, Fransen E, Van Puymbroeck M, Van Nieuwenhove G, Busschots M, D'Hondt D, Van Goethem A, Claeys W, Van Rafelghem B, Baetens E, Jorens PG, Gys C, Jacobs W, Neels H, Covaci A, van Nuijs ALN
Drug Test Anal · 2025 Nov · PMID 40760722 · Publisher ↗
Alcohol consumption is widespread worldwide and a leading cause of injuries, morbidity, and mortality. Accurately detecting alcohol use with reliable biomarkers is crucial in clinical and forensic settings. Direct alcoho...
Alcohol consumption is widespread worldwide and a leading cause of injuries, morbidity, and mortality. Accurately detecting alcohol use with reliable biomarkers is crucial in clinical and forensic settings. Direct alcohol biomarkers, i.e., ethanol (EtOH), ethyl glucuronide (EtG), ethyl sulphate (EtS), phosphatidylethanol 16:0/18:1 (PEth) reflect short- and long-term consumption. Nevertheless, complementary biomarkers with improved specificity and sensitivity are needed to better assess alcohol use, including generating a detailed timeline of consumption. In vitro exposure of HepaRG liver cells to EtOH resulted in the generation of ethylated phosphorylcholine (EtOChoP). This is the first study to investigate the in vivo presence of EtOChoP and its occurrence in medico-legal samples. Proof-of-concept and observational studies assessed EtOChoP, PEth, EtG, EtS, and EtOH in whole blood, and, when available, other matrices were analyzed for EtG, EtS (plasma, serum, urine, hair), EtOH (urine), and EtOChoP (plasma, serum). A single alcohol exposure event (0.5 g/kg EtOH, with blood EtOH concentration peaking at 0.76 g/L at 100 min) led to EtOChoP presence, and, similar to short-term biomarkers (e.g., EtOH, EtG, and EtS in whole blood), EtOChoP was not detected in the following day(s). However, in the observational study, EtOChoP remained detectable even when short-term biomarkers were absent, resembling long-term biomarkers (PEth and hair EtG). Notably, 14% of samples were positive only for EtOChoP, highlighting the need for additional biomarkers. These findings identify EtOChoP as a promising alcohol (ab)use biomarker formed after EtOH consumption and possibly accumulating during chronic drinking. EtOChoP could potentially differentiate between recent drinking and chronic problematic drinking in individuals with high PEth levels.
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