Drug Test Anal [JOURNAL]
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200 papers
Correlation of Androstenedione and Testosterone Measurements by LC-MS/MS and GC-qTOF.
Martínez Brito D, Leogrande P, Jardines D … +2 more , de la Torre X, Botré F
Drug Test Anal · 2026 Jul · PMID 42394135 · Publisher ↗
This work assessed the correlation between the measurements of A4 and T in serum obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-high resolution mass spectrometry (GC-qTOF). B...
This work assessed the correlation between the measurements of A4 and T in serum obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-high resolution mass spectrometry (GC-qTOF). Both procedures using LC-MS/MS and GC-qTOF instruments were validated using the 6PLUS1Multilevel Serum Calibrator Set MassChrom Steroid Panel 2. Positive and negative quality controls and human serum samples were used to check the correlation between these two approaches. Correlation between T and A4 concentrations measured by GC-qTOF and LC-MS/MS was found to be adequate when correlation tests, regression analysis, and Bland-Altman tests were applied. Although the LOQs required in the WADA technical documents for both analytes were not reached under the described assay conditions, the advantages provided by high-resolution mass spectrometry for the detection and quantification of steroids should not be discarded. The technical document for the identification of substances using mass spectrometry instruments does not include acceptance criteria when using HRMS platforms but should be considered due to the recent increase in its use in anti-doping laboratories.
Reproducibility of Serum Androgen Concentrations by Liquid Chromatography Mass Spectrometry in Healthy Male and Female Athletes.
Handelsman DJ, Bermon S
Drug Test Anal · 2026 Jun · PMID 42374920 · Publisher ↗
Detection of androgen doping relies on mass spectrometry-based methods to detect natural endogenous and exogenous androgens in urine and serum. To distinguish exogenous administration from natural variation in endogenous...
Detection of androgen doping relies on mass spectrometry-based methods to detect natural endogenous and exogenous androgens in urine and serum. To distinguish exogenous administration from natural variation in endogenous serum androgens requires a robust quantitative basis in the variability of serum androgen measurements. The present study therefore aimed to use extensive data from serial antidoping testing of elite male and female athletes to define the reproducibility of serum androgen profiles in male and female athletes. The present study analyzed 5516 samples from 1689 athletes for serum T, DHT, and A4 concentrations and calculated the T/DHT and A4/T ratios from 889 male and 778 female athletes, excluding samples from pregnant, doped, or XY Disorders of Sexual Development (DSD) individuals. As well as the median and interquartile range, the coefficient of variation (CV) and its 95% confidence limits were calculated from three, six, or nine replicate within-person serum samples (309, 146, and 83 for females and 276, 122, and 65 for males, respectively). Variability was greater for females than males for serum T, DHT, and T/DHT ratio (p < 10) but not significantly different for serum A4 and for A4/T ratio. These data may provide a basis for enhanced detection of T doping and for diagnosis of XY DSD disorders, 5α reductase 2, and 17β hydroxysteroid dehydrogenase 3 deficiencies.
Isolation and Characterization of New Tadalafil Analogues in Male Sexual Herbal Supplements.
Göker H, Doganc F, Gumustas M … +1 more , Saklica E
Drug Test Anal · 2026 Jun · PMID 42350094 · Publisher ↗
New tadalafil analogues were found to be added illegally to an herbal supplement marketed for the enhancement of sexual function. The compounds were separated using flash column chromatography. The structures of the dete...
New tadalafil analogues were found to be added illegally to an herbal supplement marketed for the enhancement of sexual function. The compounds were separated using flash column chromatography. The structures of the detected tadalafil analogues were elucidated by HPLC-DAD/MS and nuclear magnetic resonance (NMR) spectroscopy. A protonated molecule at m/z 450.16 was detected by LC-MS, corresponding to a molecular formula of CHNO for both of the diastereomeric compounds. These unknown compounds (1 and 2) were identified as tadalafil analogues containing an additional dihydroxyethyl group and were named N-(2,3-dihydroxypropyl)nortadalafil.
Comparison of RNA Extraction Method for Human Whole Blood: Assessing the Quality, Quantity, and Impact of RBC Lysis Across TRIzol, Invitrogen, and Qiagen Systems.
Xu Y, Liu X, Zheng X … +1 more , Wu D
Drug Test Anal · 2026 Jun · PMID 42348145 · Publisher ↗
Whole-blood samples are widely utilized in the field of doping control. Owing to the limited volume of whole blood collected from athletes and the fact that Tempus Blood RNA Tube and Paxgene Blood RNA Tube are primarily...
Whole-blood samples are widely utilized in the field of doping control. Owing to the limited volume of whole blood collected from athletes and the fact that Tempus Blood RNA Tube and Paxgene Blood RNA Tube are primarily intended for RNA stabilization and require dedicated extraction workflows, this study aimed to develop a more efficient and cost-effective RNA extraction approach using smaller volumes of EDTA-anticoagulated whole blood. RNA was extracted from human whole blood using three different methods: the TRIzol Reagent, the PureLink RNA Mini Kit (Invitrogen), and the QIAamp RNA Blood Mini Kit (Qiagen). Apparent RNA yield and purity were assessed via a NanoDrop spectrophotometer, and RNA suitability for RT-qPCR analysis was evaluated. The results indicate that red blood cell lysis influenced RNA extraction outcomes. Additionally, clear differences in RNA quality and quantity were observed. Among the three methods evaluated, the QIAamp RNA Blood Mini Kit (Qiagen) tended to yield higher apparent RNA concentrations, purity, and integrity. Therefore, under the exploratory conditions tested, the QIAamp RNA Blood Mini Kit (Qiagen) demonstrated comparatively favorable performance for RNA-based biomarker analysis relevant to doping control.
Exploring the Potential of Population-Optimized Red Blood Cell Antigens and Platelet HLA Typing for Homologous Blood Transfusion Detection in Japanese Athletes.
Momobayashi A, Okano M
Drug Test Anal · 2026 Jun · PMID 42338234 · Publisher ↗
Homologous blood transfusion (HBT) doping, using donor red blood cells (RBCs) to enhance oxygen capacity, remains a persistent challenge for doping control laboratories, especially in specific populations. Current RBC an...
Homologous blood transfusion (HBT) doping, using donor red blood cells (RBCs) to enhance oxygen capacity, remains a persistent challenge for doping control laboratories, especially in specific populations. Current RBC antigen-based flow cytometric detection (double population, DP) has limited sensitivity in genetically homogeneous populations due to common phenotypic overlap. This study aimed to enhance HBT detection by optimizing population-specific RBC antigen panels and incorporating complementary platelet-derived human leukocyte antigen (HLA) markers. Based on antigen-negative frequency, M, N, and Le antigens were evaluated as candidate markers for improved discrimination in the Japanese population. In simulated HBT samples with a 5% mixing ratio, N antigen showed a clearly separated bimodal distribution, indicating strong potential for DP detection. Although Le antigen showed unclear DP, M antigen displayed uniformly high expression, useful for distinguishing expressing populations. In addition, platelet HLA analysis, especially targeting HLA-A24, enabled detection of donor-derived components even with identical RBC antigen phenotypes. Mixed samples containing as low as 1%-5% donor cells produced visually detectable DP signals in the platelet gate. In conclusion, integrating a race-tailored RBC antigen panel with complementary platelet HLA monitoring may provide a potentially improved framework for HBT detection, particularly in ethnically homogeneous populations like Japan. The optimal antigen panel is highly population-specific due to genetic variations, requiring adaptation for other diverse ethnic groups. Future cohort studies are warranted to validate this dual approach and refine detection sensitivity.
HILIC-MS/MS Detection of Meldonium in Meat from Emidonol-Treated Calves and Volunteer Urine After Consumption: An Anti-Doping Perspective.
Postnikov PV, Efimova YA
Drug Test Anal · 2026 Jun · PMID 42337905 · Publisher ↗
A pilot study was conducted to detect meldonium in raw meat samples from young calves slaughtered 15 and 30 days (n = 3 per time-point) after a 15-day course of Emidonol. Meldonium concentration was also assessed in meat...
A pilot study was conducted to detect meldonium in raw meat samples from young calves slaughtered 15 and 30 days (n = 3 per time-point) after a 15-day course of Emidonol. Meldonium concentration was also assessed in meat samples after cooking before consumption, as well as in urine samples from volunteers after consuming a single 350 g portion of cooked meat. Urine was collected over 48 h. The maximum mean estimated meldonium concentrations in raw meat samples were 5250 ng/g on the 15th day after drug treatment and 3110 ng/g on the 30th day after drug treatment. The mean estimated meldonium concentrations in medium-rare steak cooked for 10 min (15 days after the course) and baked meat were 2960 and 1970 ng/g, respectively; for the "30-day" samples, these values were 1380 and 760 ng/g. The mean maximum estimated urine meldonium concentrations, which peaked at 2-5 h post-consumption, were 386 and 344 ng/mL for the "15-day" meat, and 258 and 219 ng/mL for the "30-day" meat, respectively. Thus, after eating meat samples contaminated with Emidonol, meldonium can be detected in urine 24-28 h post-consumption; during the first 2-14 h, its concentration can be even higher than the WADA MRPL level, which may result in an inadvertent adverse analytical finding for meldonium in sports drug testing.
Development of an Enzyme-Linked Immunosorbent Assay for Pegmolesatide in Doping Analysis: An Initial Testing Method.
Chen J, Niu C, Zhou X
Drug Test Anal · 2026 Jun · PMID 42321008 · Publisher ↗
Pegmolesatide is a novel synthetic erythropoietin-mimetic agent that binds to and activates the erythropoietin receptor (EPOR), thereby stimulating erythropoiesis. Owing to its erythropoiesis-stimulating activity, it has...
Pegmolesatide is a novel synthetic erythropoietin-mimetic agent that binds to and activates the erythropoietin receptor (EPOR), thereby stimulating erythropoiesis. Owing to its erythropoiesis-stimulating activity, it has been included in the latest World Anti-Doping Agency (WADA) Prohibited List as an erythropoietin receptor agonist (S2.1.1). The current analytical approach for pegmolesatide in doping analysis mainly relies on bottom-up nano-liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS) in serum. Although accurate and sensitive, this approach involves extensive sample preparation and time-consuming LC analysis, which limits its suitability for high-throughput initial testing procedure (ITP). In this study, an enzyme-linked immunosorbent assay (ELISA), termed the EPOR-anti-PEG ELISA, was developed as a screening method for pegmolesatide in urine and serum. The assay employs EPOR as the capture reagent to enrich pegmolesatide, followed by detection of its polyethylene glycol (PEG) moiety using the anti-PEG monoclonal antibody RM105. Method validation included assessment of selectivity, limit of detection (LOD), dynamic range, reliability, and carryover. Under optimized conditions, the assay showed an LOD of 1 ng/mL in urine, with an adjusted OD screening cutoff of 0.029. In serum, with an adjusted OD screening cutoff of 0.243, the LOD was 2 ng/mL, which was lower than the limit of identification (LOI) of 7 ng/mL determined by LC-HRMS. Overall, this ELISA provides a streamlined, cost-effective, and high-throughput screening approach for pegmolesatide and may serve as a practical complement to the LC-HRMS method in doping analysis.
Etomidate Analogues in E-Cigarettes: Linking Structural Characterization to Regional Early Warning.
Hsueh PJ, Wei LC
Drug Test Anal · 2026 Jun · PMID 42320993 · Publisher ↗
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Investigations Into the Metabolism and Elimination of Tesofensine in Human Urine.
Krug O, Thomas A, Thevis M
Drug Test Anal · 2026 Jun · PMID 42320973 · Publisher ↗
Tesofensine (NS-2330) is a pharmacologically active compound with weight-reducing effects in obese patients. Although still under regulatory review, it has been marketed online as a dietary supplement promoted for weight...
Tesofensine (NS-2330) is a pharmacologically active compound with weight-reducing effects in obese patients. Although still under regulatory review, it has been marketed online as a dietary supplement promoted for weight management and metabolic enhancement. Due to its impact on body weight, tesofensine could be relevant in competitive sports, particularly in weight-class disciplines and sports where power-to-weight ratio is decisive. It is classified under "S6 stimulants" on the World Anti-Doping Agency's Prohibited List and is prohibited in-competition only, making detailed knowledge of its metabolism and excretion essential for anti-doping purposes. Although the pharmacological effects and elimination of tesofensine and one dealkylated metabolite were described previously, elimination profiles and structural information on additional metabolites have been limited. In this study, in vitro metabolism experiments were conducted, followed by investigation of urinary metabolism and elimination in six volunteers after ingestion of 483 μg tesofensine as a dietary supplement. Urine was collected for up to 600 h, prepared by solid-phase extraction, and analyzed by LC-HRMS. Four principal metabolites were identified: three dealkylated metabolites (M1-M3) and one hydroxylated and glucuronidated metabolite (M4), supported by MS/MS dissociation patterns. The validated analytical method for human urine showed an LOD of 0.01 ng/mL, 34% recovery, and 8% interday imprecision. Marked interindividual variability was observed, with peak concentrations of 1-4 ng/mL after 4-46 h and detection windows up to 500 h. The findings enhance analytical procedures and suggest that recommended dosing is unlikely to result in concentrations constituting an Adverse Analytical Finding (AAF) under currently applicable stimulant minimum reporting levels.
Chemical Characterization of an Emerging Designer Steroid, 6-Bromo-androst-4-en-3,17-dione, via NMR and LC-HRMS and GC-HRMS.
He G, Zhu S, Liu X … +4 more , Wang Z, Zhang L, Lu J, Li J
Drug Test Anal · 2026 Jun · PMID 42312806 · Publisher ↗
A package of raw material, deliberately mislabelled as a muscle-building supplement, was obtained online to contain an unknown steroid. In order to accurately identify the composition of the substance, we used a combinat...
A package of raw material, deliberately mislabelled as a muscle-building supplement, was obtained online to contain an unknown steroid. In order to accurately identify the composition of the substance, we used a combination of advanced techniques: liquid and gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS and LC-HRMS), alongside nuclear magnetic resonance (NMR) spectroscopy. By combining the strengths of these methods, we successfully identified the compound as 6-Bromo-androst-4-en-3,17-dione (6-BrAED). This substance is a designer steroid of testosterone that is not currently on the World Anti-Doping Agency (WADA) prohibited list. As it is unlisted, there is a serious hidden risk that athletes could take it by accident through contaminated supplements.
Identification of Synthetic Urine by Analysis of Stable Carbon and Nitrogen Isotope Ratios and Comparison to Established GC-MS/MS and LC-MS/MS Analysis.
Hülsemann F, Franke L, Wissenbach DK … +2 more , Fußhöller G, Thevis M
Drug Test Anal · 2026 Jun · PMID 42312799 · Publisher ↗
Manipulation of urine samples is a recurring issue in doping control and forensic analyses, for instance through the use of synthetic urine products designed to mimic human urine. This study evaluated analytical approach...
Manipulation of urine samples is a recurring issue in doping control and forensic analyses, for instance through the use of synthetic urine products designed to mimic human urine. This study evaluated analytical approaches for identifying synthetic urine and mixtures of synthetic and authentic urine: gas chromatography tandem mass spectrometry (GC-MS/MS) for analysis of endogenous urinary steroids, elemental analyzer isotope ratio mass spectrometry (EA-IRMS) of carbon and nitrogen, and urea-nitrogen compared to an established liquid chromatography mass spectrometry (LC-MS/MS) method incorporating synthetic urine markers. All methods correctly identified synthetic urine in a double-blind sample set. As an initial testing procedure in doping control, GC-MS/MS identified synthetic urine samples through absence of endogenous steroids. However, EA-IRMS was superior to MS/MS methods in identifying mixtures of synthetic and authentic urine. Synthetic urine isotope ratios of total urinary carbon and nitrogen and/or urea nitrogen (δC ≤ -29.7‰, δN ≤ +0.4‰) were clearly differentiated from authentic urine isotope ratios (δC ≥ -26.1‰, δN ≥ +1.6‰), reflecting the synthetic origin of constituents. Mixtures of synthetic and authentic urine up to 50:50 displayed isotope signatures inconsistent with human urine, enabling reliable detection of adulteration. The LC-MS/MS approach detected mixed samples with high proportions of synthetic urine by combining biomolecule profiling with synthetic urine-specific markers. Overall, the findings demonstrate that EA-IRMS is a complementary tool for identifying synthetic or manipulated urine samples, especially when traditional biomarkers or steroid profiles are inconclusive. The method enhances the reliability of doping control and forensic urine authenticity testing.
Detection of Testosterone Esters in Urine: Evidence of Trace-Level Detectability in Doping Control Samples.
Pecher D, Forsdahl G, Stojanovic B … +1 more , Gmeiner G
Drug Test Anal · 2026 Jun · PMID 42309655 · Publisher ↗
The detection of exogenous testosterone (T) remains a critical challenge in antidoping analysis due to its identical chemical structure to endogenous testosterone. Testosterone is often administered as synthetic esters (...
The detection of exogenous testosterone (T) remains a critical challenge in antidoping analysis due to its identical chemical structure to endogenous testosterone. Testosterone is often administered as synthetic esters (T-esters), which are not naturally produced by the human body and serve as unequivocal evidence of doping. Although T-esters are traditionally detected in blood samples, their presence in urine has been largely unexplored due to their apolar nature and rapid hydrolysis. In this study, we developed and validated an ultrasensitive LC-MS/MS method capable of detecting T-esters in urine at sub-pg/mL concentrations. The method employs derivatization with Girard P reagent to enhance sensitivity and selectivity. Application of the method to doping control urine samples revealed the presence of T-esters in cases with elevated testosterone/epitestosterone (T/E) ratios, providing what is, to our knowledge, the first evidence of trace-level detectability of T-esters in human urine. This novel approach offers a complementary tool for identifying exogenous testosterone use.
Towards a Comprehensive Screening Approach for Detecting Gene Doping in Racehorses and Sport Horses by Targeting Common and Intrinsic Plasmid Sequences.
Paz A, Dhorne-Pollet S, Loup B … +4 more , André F, Garcia P, Barrey E, Bailly-Chouriberry L
Drug Test Anal · 2026 Jun · PMID 42285585 · Publisher ↗
The evolution of biotechnology and gene therapy has unfortunately facilitated the emergence of gene doping. Those misused applications are prohibited in equestrian sports (International Equestrian Federation (FEI)) and i...
The evolution of biotechnology and gene therapy has unfortunately facilitated the emergence of gene doping. Those misused applications are prohibited in equestrian sports (International Equestrian Federation (FEI)) and in horseracing (International Federation of Horseracing Authorities [IFHA]), reflecting major concerns regarding horse welfare and the integrity of competitions. Among the biotechnological tools that could be misused, eukaryotic expression plasmids represent a particular threat due to their ease of production, customisable design and ability to carry a wide range of performance transgenes. Current detection methods are mainly based on the identification of these transgenes, but if the sequence is unknown or modified, administration may not be detected. The aim of this work was to overcome this limitation by developing an untargeted transgene screening approach in equine plasma. A database of 349 mammalian expression plasmids was compiled, allowing identification of four conserved targets: the ampicillin resistance gene (AmpR), the cytomegalovirus promoter (pCMV) and two unannotated sequences between the features, which together covered 100% of the plasmids database. A set of PCR hydrolysis probe assays targeting these sequences has been developed and exhibited excellent efficiency, linearity and robustness. The method was able to discriminate, in plasma, 100% of suspicious samples from background noise down to 1200 copies/mL. Following plasmid administration to a horse, all four targets remained detectable up to 48 h using qPCR and dPCR, demonstrating the proof of concept. Overall, this work presented a sensitive, reliable and transgene-independent method that strengthens plasmid detection and reinforces gene doping control in equine athletes.
Investigation of the In Vitro and In Vivo Metabolism and μ-Opioid Receptor Affinity of the Nitazene N-Pyrrolidino Fluetonitazene.
Zemp S, Alluisetti G, Weinmann W … +4 more , Schrag B, Sporkert F, Leclerc M, Grafinger KE
Drug Test Anal · 2026 Jun · PMID 42229976 · Publisher ↗
N-pyrrolidino fluetonitazene (N-pyrrolidino-4'-(2-fluoroethoxy) nitazene) also known as fluetonitazepyne, is the fluorinated analogue of etonitazepyne, belonging to the group of new synthetic opioids (NSOs), which are am...
N-pyrrolidino fluetonitazene (N-pyrrolidino-4'-(2-fluoroethoxy) nitazene) also known as fluetonitazepyne, is the fluorinated analogue of etonitazepyne, belonging to the group of new synthetic opioids (NSOs), which are among the fastest-growing classes of new psychoactive substances. In the present study, the N-pyrrolidino fluetonitazene metabolism was investigated in vitro using pooled human liver microJsomes (pHLM) and in authentic samples from a forensic postmortem case. Qualitative analysis was performed using liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). Confirmation and semiquantitative analysis of N-pyrrolidino fluetonitazene in urine and blood were carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS). The μ-opioid (MOR) receptor affinity of N-pyrrolidino fluetonitazene was determined using an LC-MS/MS-based competitive binding assay. In total, eight different metabolites for N-pyrrolidino fluetonitazene were tentatively identified in vitro in pHLM incubations of which three (M2 O-dealkylation, M6 oxidative deamination and M8 carboxylation to N-butanoic acid) were also found in the postmortem urine sample. In the blood samples, only one metabolite was found (M9 formed by N-acetylation of 5-aminofluetodesnitazene). This metabolite was only observed in the in vivo samples and was the most abundant metabolite in blood and urine samples. We also detected metabolite M2 (formed by 4'-hydroxylation), which is common in the metabolism of other nitazepyne-type substances. For the confirmation of N-pyrrolidino fluetonitazene, we recommend including M8 and M9 as analytical target compounds, since they are specific N-pyrrolidino fluetonitazene metabolites. Furthermore, we demonstrated that the LC-MS/MS-based MOR receptor affinity assay yields valid results for N-pyrrolidino fluetonitazene that are consistent with those obtained using established radioligand-based methods.
In Vitro Metabolism of Δ-, Δ-, and Δ-THC in Human Hepatocytes: Distinct Δ-THC Biotransformation and Implications for Drug Testing.
Kronstrand R, Green H, Loh M … +2 more , Rüttimann F, Monti MC
Drug Test Anal · 2026 Jun · PMID 42229946 · Publisher ↗
With its widespread recreational and increasing medical use, ∆-tetrahydrocannabinol (∆-THC), the main psychoactive compound in cannabis, is regularly subjected to drug testing in clinical and forensic toxicology. ∆- and...
With its widespread recreational and increasing medical use, ∆-tetrahydrocannabinol (∆-THC), the main psychoactive compound in cannabis, is regularly subjected to drug testing in clinical and forensic toxicology. ∆- and ∆-THC have recently entered unregulated drug markets. Their close structural similarity to ∆-THC poses various analytical challenges, with a high risk of misidentification and misinterpretation of bioanalytical data. This study investigated the in vitro metabolic fate of ∆- and ∆-THC in comparison to ∆-THC using human hepatocytes and high-performance liquid chromatography coupled to high-resolution time-of-flight analysis (HPLC-QToF). A comparable metabolism was observed for ∆-THC and ∆-THC, with the formation of the monohydroxy and carboxy metabolites and their glucuronides. In contrast, ∆-THC was found to be extensively glucuronidated (forming ∆-THC-glucuronide) and monohydroxylated, with only minor formation of a carboxy metabolite. Structural considerations led to the hypothesis that ∆-THC is predominantly hydroxylated at a different site than ∆-THC and ∆-THC. THC isomers should be considered in cannabis drug testing. The differing metabolism of ∆-THC exacerbates the risk of misinterpretation of analytical results.
Comparing the Stability and Detectability of Testosterone Esters in Capillary Serum, Sodium Fluoride-Containing Plasma, and Dried Blood Spots After Testosterone Ester Administration.
Goodrum JM, Nair VS, Crouch AK … +2 more , Eichner D, Miller GD
Drug Test Anal · 2026 May · PMID 42219501 · Publisher ↗
Steroid esters are a frequently used ergogenic aid among athletes, and their direct detection represents an important approach to anti-doping efforts. However, the presence of esterase enzymes in blood complicates these...
Steroid esters are a frequently used ergogenic aid among athletes, and their direct detection represents an important approach to anti-doping efforts. However, the presence of esterase enzymes in blood complicates these efforts. Although dried blood spots (DBS) have emerged as a promising solution to the esterase problem, the limited sample volume creates both analytical and practical challenges. The larger collection volume for liquid blood may allow for higher analytical sensitivity, thus offsetting the effects of esterase activity in practical scenarios. First, to evaluate the effects of blood esterases ex vivo, 10 individuals were administered a single dose of either testosterone propionate or testosterone cypionate. Venous blood was collected into serum separator tubes (SST) serum and NaF-containing tubes and stored at varying conditions. Both esters significantly decreased in serum while no ester loss was observed in the NaF-containing plasma. Next, to compare the detectability across matrices stored at conditions meant to mimic real-world scenarios, the individuals administered testosterone propionate collected capillary serum, capillary NaF-containing plasma, and capillary DBS at various times postadministration. Each sample type was stored at different conditions designed to mimic what may be encountered during real-world shipping scenarios. Similar detectability of testosterone propionate was observed between all NaF, DBS, and SST samples stored under cool conditions, whereas detectability was better in NaF and DBS samples under ambient and warm conditions. These results indicate that for samples not shipped in temperature regulated conditions, DBS and NaF-containing plasma are the preferred matrices.
Investigations Into the Metabolism and Elimination of Flmodafinil and Fladrafinil for Sports Drug Testing Purposes.
Krug O, Guddat S, Görgens C … +4 more , Walpurgis K, Toma F, Thomas A, Thevis M
Drug Test Anal · 2026 May · PMID 42210629 · Publisher ↗
Flmodafinil (CRL-40,940; 2-[bis(4-fluorophenyl)methylsulfinyl]acetamide) and fladrafinil (CRL-40,941; 2-[bis(4-fluorophenyl)methylsulfinyl]-N-hydroxyacetamide) are structural analogs of modafinil. Their reported stimulat...
Flmodafinil (CRL-40,940; 2-[bis(4-fluorophenyl)methylsulfinyl]acetamide) and fladrafinil (CRL-40,941; 2-[bis(4-fluorophenyl)methylsulfinyl]-N-hydroxyacetamide) are structural analogs of modafinil. Their reported stimulating effects on the central nervous system have contributed to an increasing popularity outside the medical field, particularly for the enhancement of cognitive performance. In recent years, there has been a notable increase in interest within the sporting community regarding substances such as modafinil. Flmodafinil and fladrafinil are subjects of class "S6 stimulants" of the World Anti-Doping Agency (WADA) Prohibited List, and therefore prohibited in-competition. In this study, the elimination profiles of flmodafinil, fladrafinil, and their metabolites in urine and blood were investigated. Six volunteers ingested 20 mg of either flmodafinil or fladrafinil. Urine and blood (DBS) samples were analyzed by means of LC-HRMS, where limits of detection between 0.2 and 4 ng/mL were accomplished. After ingestion of flmodafinil, the metabolites flmodafinil acid and flmodafinil sulfone were detected. Since fladrafinil acts as a prodrug for flmodafinil, these metabolites were likewise detected after ingestion of fladrafinil. It was found that urinary maximum concentrations ranged from 191 to 891 ng/mL for flmodafinil and from 52 to 111 ng/mL for fladrafinil. The maximum concentrations in blood ranged from 45 to 98 ng/mL for flmodafinil and from 11 to 35 ng/mL for fladrafinil. When fladrafinil is administered, a slight offset can be seen until the maximum concentration of flmodafinil is reached, related to the conversion of the prodrug fladrafinil to flmodafinil. In consideration of the relatively long detection windows of both the intact drugs and their metabolites, careful result interpretation is indicated in case of an AAF.
Exploring Cerumen as an Alternative Matrix for Drug Detection and Quantification: Method Development and Application in Clinical Samples.
Kevrekidis DP, Gika HG, Panagiotoglou G … +3 more , Parlapani E, Pavlidis P, Raikos N
Drug Test Anal · 2026 May · PMID 42191567 · Publisher ↗
Cerumen (earwax) is a complex mix of apocrine and holocrine secretions and skin cells. In recent years, it has emerged as an innovative specimen for the detection of xenobiotics, suggesting that it has both clinical and...
Cerumen (earwax) is a complex mix of apocrine and holocrine secretions and skin cells. In recent years, it has emerged as an innovative specimen for the detection of xenobiotics, suggesting that it has both clinical and forensic applications. An LC-MS/MS method was developed for the detection of 33 psychoactive medications in cerumen and urine and applied to samples from 60 psychiatric inpatient volunteers. The history of administration and qualitative results between urine and cerumen were generally consistent, with haloperidol, quetiapine, and nebivolol being detected more frequently in cerumen. In certain cases, drugs were detected both with very recent and without any recent administration, even when they were not in urine, highlighting opportunities for utilization in monitoring both acute and chronic administration and even following the cessation of administration. However, no clear relationship was found between drug concentrations and time of administration or total daily dose in most cases, consistent with challenges of routine collection. Cerumen concentrations were higher among more hydrophilic drugs with lower molecular weight and protein binding and demonstrated the best fit with an inverse regression model. No relationship was found with patient sex. Our results suggest that the disposition of drugs in cerumen is complex, being downstream of insufficiently elucidated pathways through migrating skin cells, ceruminous, and sebaceous glands. The present study is the first to investigate the routine collection of cerumen in a clinical setting and has broad implications for the interpretation of drug testing results with this emerging alternative biological specimen.
The Effect of Permanent Dye Colour on Illicit Drug and Metabolite Levels in Chemically Treated Hair.
Duda A
Drug Test Anal · 2026 May · PMID 42184960 · Publisher ↗
Hair analysis for drugs of abuse is widely applied in forensic contexts, particularly in family law cases where child protection is a critical consideration. It is recognised that adulteration of hair samples via chemica...
Hair analysis for drugs of abuse is widely applied in forensic contexts, particularly in family law cases where child protection is a critical consideration. It is recognised that adulteration of hair samples via chemical treatments can occur; however, the specific influences of dye colour on parent drug and metabolite levels have yet to be systematically examined. In this study, 100 authentic hair samples positive for tetrahydrocannabinol (THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cocaine (COC) and benzoylecgonine (BZE) were treated with nine permanent dyes and one bleach product. Concentrations were quantified using validated LC-MS/MS methods, and original and post-treatment values were compared statistically via the two-tailed matched pairs t-test. Results demonstrated no consistent influence from dye colour on analyte concentrations. Following treatment, THC and COC concentrations predominantly decreased, consistent with oxidative degradation and reduced binding to the hair matrix. In contrast, THC-COOH and BZE concentrations increased in the majority of samples, suggesting in situ formation from peroxide-mediated oxidation of parent compounds, as well as increased residue release from damaged keratin structures. Bleaching produced the most pronounced changes, with significant decreases in THC, THC-COOH and COC levels and significant increases in BZE. COC was the analyte least affected by treatments overall. These findings indicate that dye colour itself is irrelevant, although chemical treatments can significantly impact toxicological results. Broader implications, such as increased risks of misinterpretation of results from artificially increased or decreased levels (shifted above or below reporting thresholds) and subsequent inaccurate conclusions regarding drug use, must be considered when analysing chemically treated hair samples.
Phase I Metabolism of Novel Phencyclidine Derivative 3-Cl-PCP: In Vitro Studies With Pooled Human Liver Microsomes and Investigation of a Post-Mortem Case.
Kutzler J, Veit T, Keller T … +1 more , Auwärter V
Drug Test Anal · 2026 May · PMID 42178157 · Publisher ↗
Assessing fatalities linked to new psychoactive substances (NPS) often remains challenging. This study investigates a fatal intoxication involving the novel phencyclidine derivative 3-chloro-phencyclidine (3-Cl-PCP), ana...
Assessing fatalities linked to new psychoactive substances (NPS) often remains challenging. This study investigates a fatal intoxication involving the novel phencyclidine derivative 3-chloro-phencyclidine (3-Cl-PCP), analyzing its phase I metabolism aided by pooled human liver microsomes (pHLMs) and supported by in silico models. Postmortem samples (bile, urine, cardiac and femoral blood, and gastric contents) and three syringes were collected from a fatal intoxication case. Quantification was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vitro metabolism studies employed pHLM incubation, with metabolites tentatively identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). To evaluate phase II metabolism, urine and bile samples were measured with and without β-glucuronidase treatment. 3-Cl-PCP concentrations ranged from 610 ng/mL in cardiac blood to 830 ng/mL in femoral blood. Higher levels were detected in urine (1200 ng/mL) and bile (4300 ng/mL). Seven phase I metabolites were tentatively identified in vitro and postmortem, primarily involving hydroxylation of the cyclohexyl (M1-M3) and piperidine (M4-M5) rings. Additionally, piperidine ring-opened carboxyl (M6) and ring-opened alcohol (M7) metabolites were tentatively identified. Post-enzyme urine analysis revealed extensive glucuronidation of metabolites M1-M5, with M4 (hydroxyl piperidine) showing the highest abundance. Bile showed elevated abundance of unconjugated metabolites, particularly M6. Metabolites M4-M7 were detected in all investigated matrices. Unchanged 3-Cl-PCP predominantly accumulated in bile. Urine analysis should prioritize the parent compound along with the metabolites hydroxy cyclohexyl (M3), hydroxy piperidine (M4), and piperidine ring-opened carboxyl (M6) and should include a prior β-glucuronidase cleavage step.
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