Drug Testing And Analysis[JOURNAL]
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200 papers
Carbon Isotope Ratio Analysis of Urinary Steroids Following Extensive Cleanup and Formylation.
Sobolevsky T, Cittan M, Ahrens E
Drug Test Anal · 2026 Jan · PMID 41267339 · Publisher ↗
Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is the method of choice to detect the abuse of synthetic forms of naturally occurring steroids for doping control purposes. GC-C-IRMS relies on a...
Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is the method of choice to detect the abuse of synthetic forms of naturally occurring steroids for doping control purposes. GC-C-IRMS relies on a multistep sample cleanup to ensure each target analyte peak is chromatographically pure before combustion. To achieve that, liquid-liquid or solid phase extraction (SPE) is commonly used in combination with preparative liquid chromatography (LC). In this work, a procedure for isolation, purification, and analysis of endogenous steroids by GC-C-IRMS was developed and validated. The key elements were successive application of strong cation and strong anion exchange SPE with enzymatic hydrolysis in between to strip the ionic species from urine and decrease matrix complexity prior to LC cleanup; preparative two-dimensional LC, where only the testosterone fraction required secondary purification (40 min total run time per sample); and derivatization of selected fractions with formic acid to yield formate esters, followed by GC-C-IRMS analysis. Formylation afforded excellent separation between 5α- and 5β-androstanediols and simplified the detection of the endogenous reference compound pregnanetriol by converting it to a volatile artifact, tentatively identified as 3α,20-diformoxy-17-methyl-18-nor-5β,17α-pregn-13-ene. The overall method performance benefited from the customization of the GC-C-IRMS combustion interface, which improved robustness and facilitated the transfer of sample components into the hot zone of the oxidation reactor, minimizing peak tailing. The former was achieved by keeping the oxygen flow through the reactor at all times, obviating the need for periodic oxidation, and the latter-by implementing a direct capillary-in-capillary connection of the chromatographic column to the oxidation reactor.
HS-FET-GC/MS-Method Development and Validation for Analysis of 45 Terpenes-Creating a Complementary Tool for Comprehensive Profiling of Cannabis Flowers in Forensics.
Hundertmark M, Germerott T, Wunder C
Drug Test Anal · 2026 Jan · PMID 41265476 · Full text
Cannabis sativa is one of the oldest and most versatile plants with many facets ranging from intoxicant to medicine. Legalisation of medicinal cannabis leads to an increasing complexity of specific forensic questions to...
Cannabis sativa is one of the oldest and most versatile plants with many facets ranging from intoxicant to medicine. Legalisation of medicinal cannabis leads to an increasing complexity of specific forensic questions to distinguish between recreational and medicinal use, for example, in context with participation in road traffic. Hence, there is a recent interest in finding objective markers that enable the differentiability of cannabis flowers. Terpenes, volatile hydrocarbons with a modular construction principle of isoprene subunits, are currently suggested as a second substance class alongside phytocannabinoids for the classification of cannabis material. A headspace full evaporation technique gas chromatography mass spectrometry (HS-FET-GC/MS) methodology was successfully validated according to forensic guidelines for the analysis of 45 terpenes in cannabis flowers including 16 monoterpenes, 16 monoterpenoids, 7 sesquiterpenes and 6 sesquiterpenoids. FET-sampling was developed in detail experimentally, revealing evidence of thermal instability of higher-boiling terpenes. Validation included selectivity, linearity of calibration (ranges 10-2000 μg/g), analytical limits (at least 6 μg/g), accuracy (bias) as well as intraday and interday precision. The use of a retention time index mixture as an internal standard and measurement in SIM-scan mode also allows for the qualitative identification of further terpenes present in cannabis. Application to a set of cannabis strains with similar Δ-THC content demonstrated differences and similarities in their terpene profiles.
Detection of Nandrolone Decanoate and Its Metabolites in Equine Hair After Intramuscular Administration.
So YM, Kwok WH, Yuen SMS … +3 more , Wong COL, Wan TSM, Ho ENM
Drug Test Anal · 2026 Jan · PMID 41261805 · Publisher ↗
This paper describes the detection of nandrolone decanoate and its metabolites in mane hair collected from horses that have been treated with nandrolone decanoate (Deca-Durabolin). The intramuscular administration study...
This paper describes the detection of nandrolone decanoate and its metabolites in mane hair collected from horses that have been treated with nandrolone decanoate (Deca-Durabolin). The intramuscular administration study of nandrolone decanoate in three Thoroughbred castrated horses (each received 800 mg weekly for 3 consecutive weeks) was previously conducted to investigate its metabolism and detection time in plasma and urine for doping control purposes. In this work, segmental analysis of the post-administration hair has revealed that (i) nandrolone decanoate and its metabolites, nandrolone and 4-estrene-3,17-dione, could be detected in horse mane after intramuscular administration, and (ii) the spreading of these substances along the entire hair strand was observed, which suggested the involvement of sweat or sebum incorporation.
Further Insights Into the Metabolism of LGD-4033 in Human Urine. Part 2. A New Minor Metabolite With Antagonistic Activity on the Androgen Receptor Can Indicate Recent Substance Intake.
Angelis YS, Sakellariou P, Keiler AM … +8 more , Thevis M, Thomas A, Lam K, Wolber G, Vonaparti A, Voss S, Petrou M, Pitsinos EN
Drug Test Anal · 2026 Jan · PMID 41261783 · Full text
Using a targeted metabolic investigation approach, a new, previously undescribed metabolite, which is a pyrrole derivative of LGD-4033, has been detected and coded as M8. This metabolite can be detected in postadministra...
Using a targeted metabolic investigation approach, a new, previously undescribed metabolite, which is a pyrrole derivative of LGD-4033, has been detected and coded as M8. This metabolite can be detected in postadministration human urine samples up to 6 days after administration. It has also been detected in post-administration samples, mimicking supplement contamination, after repeated 10 μg doses detectable for ≥ 120 h after administration. Given M8's structural similarity to LGD-4033, its androgen receptor (AR) agonist/antagonist properties were studied using in silico molecular docking and functional in vitro AR transactivation assays in the PC3(AR) cell model, alongside other selected LGD-4033 metabolites. The results indicate that M8 can act as a potent AR antagonist, whereas M2c was reconfirmed as a potent AR agonist. Therefore, we propose the inclusion of M2c in ITP doping control methods, as it could be used as an LGD-4033 alternative and may be introduced into the black market. Additionally, the detection of M8, which is an early-stage excreted metabolite, is valuable for estimating sample collection time relative to LGD-4033 intake. When combined with the evaluation of other long-term metabolites like M5b, M5a, M2c, and M2d, M8 detection can aid in distinguishing adverse analytical findings, associated abuse through regular dosing, from unintentional doping caused by certain contamination scenarios or abuse through microdosing.
Metformin in the Horse: Pharmacokinetics and Detection Times Using Monte Carlo Simulations.
Jacobs ME, Blea J, Hardy M … +3 more , McKemie DS, Traynham M, Knych HK
Drug Test Anal · 2026 Jan · PMID 41261763 · Full text
The racehorse industry has strict regulations regarding the detection of prohibited substances in horses. Metformin, a diabetes medication, is a prohibited substance that has been reported in post-race blood and urine sa...
The racehorse industry has strict regulations regarding the detection of prohibited substances in horses. Metformin, a diabetes medication, is a prohibited substance that has been reported in post-race blood and urine samples collected from racehorses. For further characterization of the disposition of metformin, 12 Thoroughbred horses were administered metformin orally and intravenously in a randomized, balanced, two-way crossover design. Serum and urine samples were collected, and drug concentrations determined via liquid chromatography-tandem mass spectrometry. The serum data were analyzed using both noncompartmental analysis and a population pharmacokinetic model. Metformin concentrations were below the LOQ (0.25 ng/mL) in six of 12 horses by Day 11 postadministration, and below the LOQ for all horses on Day 25. The maximum serum concentration of metformin (mean ± SD) was 941.0 ± 467.8 ng/mL, and the mean terminal t was 85.8 ± 15.1 h. Based on Monte Carlo simulations the time that serum metformin concentrations fell below the proposed HISA Anti-Doping and Medication Control (ADMC) minimum reporting level (MRL; 0.5 ng/mL) in a simulated population of 1000 Thoroughbred horses was 475 hrs (~20 days). Metformin concentrations in urine fluctuated significantly between and within individual horses, and there was not a consistent relationship between blood and urine samples across time points. Results of the present study demonstrate a prolonged detection time; thus, a prolonged withdrawal time is needed to prevent a positive finding following exposure to metformin. Additionally, these results suggest that blood is the preferred matrix for regulatory purposes due to the inconsistent elimination in urine.
PEth Cut-Off Thresholds for Hazardous Alcohol Consumption Based on a Drinking Study.
Walther L, Stenton J, Hansson T … +3 more , Blomgren A, Andersson A, Isaksson A
Drug Test Anal · 2026 Jan · PMID 41253128 · Full text
This study aimed to explore how PEth and other commonly used alcohol biomarkers (CDT, AST, ALT, and GGT) respond to regular consumption of what has been generally considered to correspond to low to moderate amounts of al...
This study aimed to explore how PEth and other commonly used alcohol biomarkers (CDT, AST, ALT, and GGT) respond to regular consumption of what has been generally considered to correspond to low to moderate amounts of alcohol over a 2-week period. A total of 21 voluntary participants (aged 31-69 years) took part in a 2-week drinking study. Group 1 (n = 11) consumed one glass of wine daily (16 g of alcohol), close to the present Swedish limit for hazardous alcohol consumption, while Group 2 (n = 10) consumed two glasses daily (32 g of alcohol). Alcohol biomarkers were measured at baseline and at three further occasions during the study. After 1 week of alcohol consumption, all participants had measurable concentrations (> 0.005 μmol/L, ≈3.5 ng/mL) of both PEth-homologues (PEth 16:0/18:1 and PEth 16:0/18:2). After 1- and 2-week periods, significant differences in PEth levels were observed between Group 1 and Group 2. The correlation between the two PEth-homologues was strong and increased as the study progressed. In contrast, other biomarkers showed little to no change during the study period. Both PEth-homologues appear capable of identifying hazardous alcohol consumption. The current Swedish reporting threshold for PEth 16:0/18:1 (0.05 μmol/L, ≈35 ng/mL) demonstrates high specificity but low sensitivity in identifying hazardous alcohol consumption involving regular/daily intake. The sensitivity of the other biomarkers is insufficient for detecting alcohol consumption at this level.
Oxidative Processes to Transform and Degrade Amphetamine-Type Stimulants: Alternatives to Incineration.
Mercieca AL, Alonzo M, Chadwick S … +1 more , McDonagh AM
Drug Test Anal · 2026 Jan · PMID 41248988 · Publisher ↗
Although incineration is currently the primary method for the disposal of seized illicit drugs, alternative methods for the disposal of illicit drugs may be necessary to provide safer and more accessible alternatives. Ch...
Although incineration is currently the primary method for the disposal of seized illicit drugs, alternative methods for the disposal of illicit drugs may be necessary to provide safer and more accessible alternatives. Chemical oxidation processes have been identified as a promising alternative method to degrade illicit drugs. Using commercially available reagents and established industry processes, chemical degradation holds potential as an alternative drug disposal technique. This study investigated the oxidants ozone, sodium hypochlorite, trichloroisocyanuric acid, hydrogen peroxide, OXONE, sodium percarbonate, and peracetic acid for their potential to degrade illicit amphetamine-type stimulants using β-phenethylamine (PEA) as an exploratory analog. Oxidants and conditions that showed the highest degradation efficiency with PEA were applied to methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyamphetamine (MDA). Transformation products were identified using gas chromatography-mass spectrometry, and degradation was quantified using a fit-for-purpose method via liquid-chromatography quadrupole time-of-flight mass spectrometry. Of the oxidants explored, ozone performed the best, leading to high degradation efficiencies of methamphetamine (95%), amphetamine (86%), MDMA (100%), and MDA (100%) after 72 h of exposure. Sodium hypochlorite was also highly effective for the degradation of methamphetamine and amphetamine, while trichloroisocyanuric acid was particularly effective for MDMA and MDA. All the major transformation products of degradation were tentatively identified, with only one of 10 listed as a controlled, scheduled, or restricted substance. This research demonstrates how chemical degradation can provide a novel alternative to incineration for the destruction of amphetamine-type stimulants, providing a sustainable, long-term, and accessible method of illicit drug disposal.
Identification of Metabolites for the Novel 5α-Reductase Inhibitor Epristeride In Vitro and Its Potential Impact on Doping Testing.
Li Z, Liu B, Wang Y … +5 more , Cheng J, Aguilera R, Deng X, Chen Q, Chen P
Drug Test Anal · 2026 Jan · PMID 41242714 · Publisher ↗
Epristeride, a novel noncompetitive inhibitor of Type II 5α-reductase, has emerged as a potential therapeutic alternative for benign prostatic hyperplasia (BPH). Given that other 5α-reductase inhibitors, such as finaster...
Epristeride, a novel noncompetitive inhibitor of Type II 5α-reductase, has emerged as a potential therapeutic alternative for benign prostatic hyperplasia (BPH). Given that other 5α-reductase inhibitors, such as finasteride and dutasteride, are already monitored for their potential impact on doping control, comprehensive metabolic studies of epristeride are crucial for antidoping. This study investigates the metabolic pathways and metabolites of epristeride using in vitro microsome models, offering preliminary insights into the pharmacokinetics of this drug. Metabolite profiling was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS), with data acquisition facilitated by Xcalibur 4.2 software and metabolite identification facilitated by Compound Discoverer 3.3. By employing network pharmacology, the potential targets of epristeride are predicted. The binding energy is calculated using AutoDock Vina software to predict its impact on steroid metabolism. The study proposed three primary metabolites of epristeride: two Phase I oxidation products (M1 and M2) and one Phase II glucuronidation product (M3). Pathway analysis revealed that among the five CYP450 isoforms examined, CYP3A4 played a dominant role. The docking results tentatively elucidated five key target proteins (ESR1, CYP19A1, STAT3, AKR1C3, and CYP17A1) with low binding energies, indicating stable interactions. Notably, Phase I metabolites (M1 and M2) showed significant binding potential with these targets, whereas the Phase II metabolite (M3) exhibited lower binding stability. These findings provide a detailed understanding of epristeride's metabolic pathways and its potential biological impacts, offering valuable insights for monitoring its presence as a confounding factor in doping control.
Clenbuterol, but not Inhaled Formoterol, Upregulates the Sarcomere Stabilizer KLHL41 to a Similar Extent as Resistance Training in Human Skeletal Muscle.
Hostrup M, Moesgaard L, Thomassen M … +2 more , Deshmukh A, Jessen S
Drug Test Anal · 2026 Jan · PMID 41187933 · Publisher ↗
Beta-adrenergic agonists are widely used for bronchial relief in respiratory conditions such as asthma and exercise-induced bronchoconstriction. However, this drug class has also been shown to have muscle anabolic proper...
Beta-adrenergic agonists are widely used for bronchial relief in respiratory conditions such as asthma and exercise-induced bronchoconstriction. However, this drug class has also been shown to have muscle anabolic properties. Kelch-Like Family Member 41 (KLHL41) is a sarcomeric protein implicated in muscle remodeling and hypertrophy. In this study, we examined the effects of oral clenbuterol, therapeutic inhaled formoterol, and resistance training on KLHL41 protein abundance in human skeletal muscle. KLHL41 levels were measured by immunoblotting in vastus lateralis muscle biopsies from healthy adults following 2 weeks of oral clenbuterol administration, 6 weeks of inhaled formoterol at therapeutic doses, or 8 weeks of resistance training. We also assessed sex differences and the effects of acute versus prolonged interventions. Prolonged oral clenbuterol administration significantly increased KLHL41 abundance compared to placebo (p < 0.001), with a magnitude similar to that observed after resistance training (p < 0.01), whereas therapeutic inhaled formoterol had no effect on KLHL41 levels. Neither acute clenbuterol administration nor a single resistance training session altered KLHL41 abundance, and no sex differences were observed in baseline KLHL41 levels. These findings indicate that beta-adrenergic stimulation via oral clenbuterol, but not therapeutic inhalation of formoterol, promotes sarcomeric remodeling through KLHL41-related pathways similar to those activated by resistance training. The distinct effects of these agents on KLHL41 support current anti-doping regulations prohibiting clenbuterol use and highlight KLHL41 as a potential molecular marker of skeletal muscle adaptation to hypertrophic stimuli.
Toxic Tear Gas 2-Chloroacetophenone (CN) Forms Adducts With Endogenous Plasma Thiols In Vitro Valuable as Biomarkers of Exposure.
Sieber PH, Steinritz D, Worek F … +1 more , John H
Drug Test Anal · 2026 Jan · PMID 41187790 · Full text
As the tear gas of the highest toxicity, 2-chloroacetophenone (CN) poses a potential threat for exposed individuals. Several fatality cases following exposure to CN are documented, but an unambiguous identification of CN...
As the tear gas of the highest toxicity, 2-chloroacetophenone (CN) poses a potential threat for exposed individuals. Several fatality cases following exposure to CN are documented, but an unambiguous identification of CN exposure is still missing. Thus, we herein present the identification of in vitro reaction products between CN and endogenous molecules useful as biomarkers. After incubation of human plasma with CN, diverse acetophenone (AcPhen)-adducts were formed with the small molecule thiols homocysteine (HCys), glutathione (GSH), and cystine (Cys-Cys). All adducts were detected by microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (μLC-ESI MS/HRMS) working in the parallel reaction monitoring (PRM) mode and were characterized as potential biomarkers of CN exposure. Time- and concentration-dependent adduct formations were investigated, and the stability of AcPhen-adducts in plasma during 4 freeze-and-thaw cycles and in the autosampler was tested. The limit of identification (LOI) of identified AcPhen-adducts in vitro was found at about 6 μM (concentration of CN in plasma) but showed quite limited in vitro stability. The AcPhen-adducts herein presented might be beneficial for future studies addressing CN exposure in vivo.
Characterization and Identification of Commercial Synthetic Urine Products.
Kyle PB, Bell MC, Shalaby A … +1 more , Chilcutt BM
Drug Test Anal · 2026 Jan · PMID 41163476 · Publisher ↗
Synthetic products are marketed for specimen substitution and can be difficult to identify because many contain creatinine, urea, and pH buffers designed to meet acceptance criteria of common specimen validity tests. Met...
Synthetic products are marketed for specimen substitution and can be difficult to identify because many contain creatinine, urea, and pH buffers designed to meet acceptance criteria of common specimen validity tests. Methods involving DNA analysis and liquid chromatography-mass spectrometry have been successful in detecting synthetic products, but of these require significant sample preparation, lengthy analyses, and/or complex technical analysis. The aims of this study were to characterize the properties of commonly available synthetic urine products in order to determine new methods of detection, or provide information that might help others determine methods to detect synthetic urine products. Physical evaluations, clinical urinalysis, mass spectrometry, and microscopic analysis were used to evaluate 10 synthetic urine products, and results were compared to authentic human specimens. Two of 10 synthetic products exhibited lasting bubbles or foam after shaking. None of the synthetic products contained cells or formed biological elements. In contrast, 99% of 1127 outpatient specimens and 98% of the 250 pain management specimens evaluated in this study contained biological elements. The automated microscopy analyzer used in this study exhibited 100% sensitivity, 98% specificity, required no specimen pretreatment, provided results in less than 2 min, and was interfaced to the laboratory information system for rapid and accurate reporting. Given that normal human urine contains over 1 million cells per liter, microscopic analysis may provide an effective method to differentiate synthetic products from authentic urine. The shake test followed by microscopic analysis should provide an efficient and effective combination to detect synthetic urine products.
Mapping of the Pharmacological Effect of Prohibited Substances and Methods to Athletic Physiological Characteristics.
Hayward G, Gaborini L, Robinson N … +2 more , Stuart M, Mottram D
Drug Test Anal · 2026 Jan · PMID 41147730 · Publisher ↗
This study presents a novel physio-pharmacological framework for assessing the potential performance-enhancing effects of substances and methods included in the 2022 World Anti-Doping Agency (WADA) Prohibited List across...
This study presents a novel physio-pharmacological framework for assessing the potential performance-enhancing effects of substances and methods included in the 2022 World Anti-Doping Agency (WADA) Prohibited List across 160 Olympic sport disciplines. An expert panel evaluated 31 consolidated pharmacological categories for their capacity to enhance performance across six core physiological athletic demands. Using a calibrated scoring function and bilinear interpolation, we mapped these effects to each sport's dominant physiological profile. Findings confirmed the well-known ergogenic effects of anabolic agents and erythropoietins for power and endurance-based sports, respectively, including the general ergolytic effects of narcotics and cannabinoids. Moreover, the methodology can be used to evaluate any new sport or substance given their respective physiological demands. The study underscores the need for targeted anti-doping strategies, suggesting that risk assessment and test distribution planning should be aligned with sport-specific physiological demands and the relative performance advantages conferred by different doping strategies. This summary of the current physiological knowledge and pharmacological knowledge has the potential to enhance detection efficiency, optimize resource allocation, and refine prohibited substance screening in elite sport.
A Meta-Analysis of International Flunixin Pharmacokinetics in Horses: Toward Regulatory Harmonization and Individualized Detection Times Using Bayesian Paradigm.
Kuroda T, Knych HK, Noble GK … +7 more , Minamijima Y, Leung GN, Nomura M, Mizobe F, Ishikawa Y, Kusano K, Toutain PL
Drug Test Anal · 2026 Jan · PMID 41137541 · Full text
Flunixin meglumine is widely used to manage pain and inflammation in horses, and its regulation requires robust pharmacokinetic analysis for harmonization. In this study, we conducted a meta-analysis of flunixin disposit...
Flunixin meglumine is widely used to manage pain and inflammation in horses, and its regulation requires robust pharmacokinetic analysis for harmonization. In this study, we conducted a meta-analysis of flunixin disposition using plasma and urine concentration data from 65 horses across four countries to robustly estimate pharmacokinetic parameters in setting screening limits (SLs) for controlling medications in horses. A population (POP) model was developed using nonlinear mixed-effects model analysis. The irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were determined to be 1.9 and 70.2 ng/mL, respectively, with a typical urine-to-plasma ratio (Rss) of 35.9. Using the current International Federation of Horseracing Authorities (IFHA) screening limits (ISLs) (1 ng/mL for plasma; 100 ng/mL for urine), a longer detection time (DT) was observed for plasma than for urine, especially after multiple doses, as plasma ISL corresponds to a slower terminal elimination phase. Increasing the current plasma ISL from 1 to 3 ng/mL-while keeping the current urine ISL at 100 ng/mL-could better align the plasma and urine DTs. As a limitation of this study, both Standardbred and Thoroughbred data were included, and further data collection is needed to fully ascertain potential breed-specific effects. Moreover, this POP model also enabled relatively accurate Bayesian estimation of individual withdrawal times (WTs) from limited data. Clinicians could apply this Bayesian approach to making informed WT recommendations for horses when sufficient data is available. While existing non-POP statistical models remain viable, they may require a more conservative approach to WT estimation than Bayesian methods.
Distinguishing Between Oral and Transdermal Administration Routes of Methyltestosterone Through Human Urine and Dried Blood Spot Analyses for Doping Control Purposes.
Okano M, Watanabe Y, Miyamoto A … +2 more , Ota M, Sato M
Drug Test Anal · 2026 Jan · PMID 41128440 · Publisher ↗
Unintentional doping violations have been reported after secondary skin exposure, including partner contact or contact sports. Methyltestosterone (MT), an anabolic androgenic steroid on the World Anti-Doping Agency Prohi...
Unintentional doping violations have been reported after secondary skin exposure, including partner contact or contact sports. Methyltestosterone (MT), an anabolic androgenic steroid on the World Anti-Doping Agency Prohibited List, is readily available in Japan as an over-the-counter topical hair-growth preparation and as oral tablets. This study evaluated whether transdermal absorption of MT can be distinguished from oral administration using human urine and dried blood spot (DBS) analyses. Ten men received MT once daily for five consecutive days (five oral, five transdermal). The urinary tetrahydro-metabolites 17α-methyl-5α-androstane-3α,17β-diol (5α-THMT) and 17α-methyl-5β-androstane-3α,17β-diol (5β-THMT) were detectable within hours after administration for both routes. Following transdermal administration, 5α-THMT remained detectable up to 97 h after the final administration, whereas, after oral administration, 5β-THMT persisted longer, up to 265 h. Transdermal application produced a marked increase in 5α-THMT, consistent with high 5α-reductase activity in skin, yielding a significantly higher 5α-THMT/5β-THMT ratio than after oral administration. Nonetheless, individuals with inherently high 5α-reductase activity may exhibit elevated ratios even after oral dosing, warranting careful interpretation. In DBS after oral dosing, parent MT was generally detectable up to 24 h, providing a shorter detection window than urinary tetrahydro-metabolites. Overall, combining urine (tetrahydro-metabolites) and DBS (parent MT) analyses enables effective detection of MT use and supports differentiation of administration routes.
CERA Detection and Stability in Blood Versus Urine.
Salamin O, Chappuis J, Bækken LV … +2 more , Kuuranne T, Leuenberger N
Drug Test Anal · 2026 Jan · PMID 41121755 · Full text
Erythropoietin receptor agonists (ERAs), including continuous erythropoietin receptor activators (CERAs), are potent blood doping substances used to enhance endurance performance by stimulating erythropoiesis. While trad...
Erythropoietin receptor agonists (ERAs), including continuous erythropoietin receptor activators (CERAs), are potent blood doping substances used to enhance endurance performance by stimulating erythropoiesis. While traditionally detected through direct analysis of urine or serum samples using sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) and western blotting, the slow urinary elimination of third-generation ERAs like CERA has shifted anti-doping strategies toward serum-based detection. This study compared the detectability and stability of CERA in urine and serum matrices and evaluated the added value of combining direct detection with hematological profiling. Using samples from a controlled CERA administration study and an authentic case example, we assessed CERA detection in serum, urine, and simulated dried blood spot (DBS) matrices (Tasso-M20). Additionally, we conducted stability experiments by incubating spiked matrices at 37°C for up to 72 h. Our results confirmed the superior stability and consistent detectability of CERA in serum and DBS compared with urine. Moreover, hematological alterations such as increased reticulocytes percentage flagged by the Athlete Biological Passport (ABP) supported targeted serum testing, leading to the successful detection of CERA. These findings highlight the importance of systematic blood collection for both direct and indirect detection strategies. Furthermore, DBS samples showed promising analytical performance and resistance to elevated temperature, suggesting their utility as minimally invasive alternatives in anti-doping programs. Overall, our study reinforces the relevance of blood matrices in the detection of CERA and advocates for the broader integration of blood-based strategies in targeting doping practices with ERAs.
Evaluating the Testosterone-to-Luteinizing Hormone Ratio in Male Anti-Doping Serum Samples: Results From a Transdermal Testosterone Administration Trial and Field Collected Samples.
Goodrum JM, Nair VS, Crouch AK … +2 more , Eichner D, Miller GD
Drug Test Anal · 2026 Jan · PMID 41088841 · Publisher ↗
The serum testosterone-to-luteinizing hormone (T/LH) ratio has previously been suggested as a sensitive marker of testosterone use in an anti-doping setting. When measured with an automated immunoassay platform, this rat...
The serum testosterone-to-luteinizing hormone (T/LH) ratio has previously been suggested as a sensitive marker of testosterone use in an anti-doping setting. When measured with an automated immunoassay platform, this ratio represents a quick and cost-effective screening biomarker to further direct isotope ratio mass spectrometry (IRMS) testing efforts in concurrently collected urine samples to confirm testosterone abuse. To evaluate the practicality of implementing the serum T/LH ratio for routine use, testosterone values in 356 serum samples were compared between a "gold-standard" LC-MS/MS method and an automated immunoassay method. Excellent correlation and minimal bias between the two methods were observed, highlighting the validity of the immunoassay method. Next, a testosterone administration study utilizing a transdermal delivery route was conducted to compare the effectiveness of the serum T/LH ratio to the currently used serum testosterone to androstenedione (T/A4) and urinary testosterone to epitestosterone (T/E) ratios. The serum T/LH ratio was more sensitive than the T/A4 ratio and showed similar sensitivity to the urinary T/E ratio with high interindividual variability. Finally, T/LH analysis was conducted on 626 capillary serum samples collected in the field from male Ultimate Fighting Championship athletes. Of the three samples that showed elevated T/LH ratios in this pool, two of the corresponding urine samples were IRMS positive, one of which showed an unremarkable urinary T/E ratio and relatively normal steroid profile. These results indicate serum T/LH ratio monitoring provides a beneficial addition to the anti-doping tool kit and can improve urinary IRMS testing recommendations.
Equine Metabolism of Voxelotor and Its Impact on Hematological Indices: A Doping Control Perspective.
Kal AKK, Subhahar MB, Philip M … +5 more , Graiban FM, Karatt TK, Mathew B, George RM, Maruthasalam B
Drug Test Anal · 2025 Dec · PMID 41063632 · Publisher ↗
Voxelotor, a therapeutic drug for sickle cell disease, has been reported to elevate serum erythropoietin and hemoglobin levels in healthy individuals. Because of its potential to alter blood parameters, the World Anti-Do...
Voxelotor, a therapeutic drug for sickle cell disease, has been reported to elevate serum erythropoietin and hemoglobin levels in healthy individuals. Because of its potential to alter blood parameters, the World Anti-Doping Agency (WADA) classified voxelotor under category M1 of the 2023 Prohibited List. Despite this classification, little is known about its metabolic behavior in either humans or animals. In this study, the metabolism of voxelotor was investigated in Thoroughbred horses after oral administration. Using liquid chromatography high-resolution mass spectrometry (LC-HRMS), 35 metabolites were detected. Among them, the most prominent pathways included hydroxylation and reductive transformations (Phase I), as well as glucuronidation and sulfonation (Phase II). Notably, several glucuronide conjugates and hydroxylated derivatives were identified as major metabolites, with extended detection times that make them particularly relevant for antidoping surveillance. Blood analyses also revealed changes in red blood cell count, hemoglobin concentration, packed cell volume, and platelet levels. Together, these findings provide practical insight into voxelotor's metabolic profile in equines and highlight specific metabolites useful for doping control. Further studies are needed to better define its hematological impact and to confirm whether the observed clinical effects are drug related.
Prolonged Detection of GHB Intake in Urine: Are We Finally There?
Faldborg KB, Hasselstrøm JB, Kraemer T … +2 more , Andersen CU, Steuer AE
Drug Test Anal · 2025 Dec · PMID 41048112 · Full text
Gamma-hydroxybutyric acid (GHB) has been implicated in drug-facilitated sexual assault (DFSA). However, due to its rapid elimination and the challenge of distinguishing exogenous from endogenous levels, GHB is likely to...
Gamma-hydroxybutyric acid (GHB) has been implicated in drug-facilitated sexual assault (DFSA). However, due to its rapid elimination and the challenge of distinguishing exogenous from endogenous levels, GHB is likely to be significantly underestimated in DFSA cases. Consequently, previous research has focused on identifying biomarkers associated with GHB intake that persist longer than GHB itself. Conjugates of GHB with amino acids and pentose have been proposed as potential candidates, but data on their detection times remain limited. In this study, a randomised, placebo-controlled trial involving 30 healthy volunteers was conducted. Urine samples were collected at regular intervals over 5 days post-administration. Using a validated LC-MS/MS method, we quantified known and proposed GHB biomarkers, aiming to identify those capable of extending the detection window. GHB-amino acid conjugates showed limited utility beyond early time points, whereas a tentatively identified GHB-pentose emerged as a very promising marker. With structural confirmation and further validation, it may allow reliable detection for at least 24 h after intake, representing a significant advance in DFSA investigations.
Prevalence of AAS-Positive Samples at Drug Abuse Laboratory Sweden Between 2014 and 2023 and Sub-Study of Dual Use of AAS and Narcotics.
Bohlin KP, Villén T, Hopcraft O … +2 more , Pohanka A, Ekström L
Drug Test Anal · 2025 Dec · PMID 40992881 · Full text
It is of interest to investigate trends in AAS usage profile. Here we aimed to retrospectively study the prevalence of AAS-positive samples based on 21,172 consecutive analyses from routine AAS testing 2014-2023. Moreove...
It is of interest to investigate trends in AAS usage profile. Here we aimed to retrospectively study the prevalence of AAS-positive samples based on 21,172 consecutive analyses from routine AAS testing 2014-2023. Moreover, 310 urine samples from 2022 to 2023 were reanalyzed for a broader AAS panel as well as for the presence of narcotics. Between 2014 and 2023, the frequency of reported AAS-positive samples varied between 6% and 11%, with no trend discerned. The prevalence of samples containing several AAS also shows a similar distribution. The most common AAS detected were consistently testosterone, nandrolone, and drostanolone. Of the 310 urine samples reanalyzed, 80 male and 6 female samples were positive for AAS. Thirteen of the samples showed T/E 4-10, indicative of testosterone use, with no other AAS. Consequently, 4% of the samples might have been reported as false negatives. Of the AAS-positive samples, amphetamine was found in 10% and 0% of the male and female samples, respectively. Cannabis was more often detected in AAS-positive female samples (50%) than in male samples (25%), whereas cocaine was more commonly detected in male than in female samples (33 versus 17%). The prevalence of cannabis and amphetamine was like previous AAS studies conducted in Sweden, whereas the presence of cocaine in male samples was substantially higher. Co-use of AAS and narcotics is a well-known problem and highlights the importance of preventive actions and education/awareness of AAS.
Identification of Candidate Biomarkers Detected in the Urine of Racehorses After Anabolic Agent Administration: Use of Orthogonal Methods for Structural Elucidation.
Cloteau C, Delcourt V, Loup B … +9 more , Chabot B, Pescher M, Susdorf E, Kaabia Z, Garcia P, Popot MA, Le Bizec B, Dervilly G, Bailly-Chouriberry L
Drug Test Anal · 2025 Dec · PMID 40968575 · Publisher ↗
Biomarker identification by mass spectrometry represents a key step in the workflow of nontargeted metabolomic studies. Given the complexity of the data, this step, which must be carried out by a trained specialist, is t...
Biomarker identification by mass spectrometry represents a key step in the workflow of nontargeted metabolomic studies. Given the complexity of the data, this step, which must be carried out by a trained specialist, is time-consuming, and the biomarkers discovered are not always identified. While this stage is not an obstacle to the development of new screening and classification tools, it is nonetheless crucial to a better understanding of the results obtained. For this reason, the aim of this study was to perform structural elucidation of candidate biomarkers, which had previously been displayed to screen for the administration of anabolic agents in the urine of racehorses and whose robustness had been evaluated. The present study involved a combination of various analytical strategies, including enzymatic hydrolysis, high-resolution mass spectrometry and ion mobility (LC-HRMS, LC-IMS-HRMS), and in vitro experiments. Two candidate biomarkers were identified as phase II metabolites of tebuconazole, belonging to the equine exposome. This identification opens the way to further investigations into the relationship between the presence of this compound and its disruption in horse urine following anabolic agent administration. Overall, the use of orthogonal approaches provided better complementary information on the structure of the compound and ultimately enabled us to identify biomarkers with the highest possible level of confidence.
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