Drug Test Anal [JOURNAL]
Sun
200 papers
Analytical Methods for the Quantification of Drugs and Their Metabolites in Human Hair: A Systematic Review From 2010 to 2025.
Elluard M, Miri L, Caron N … +1 more , Bussières JF
Drug Test Anal · 2026 May · PMID 42178156 · Publisher ↗
Hair has emerged as a valuable biological matrix for documenting long-term or cumulative drug exposure. Although hair analysis is well established in forensic toxicology, its potential application to monitor occupational...
Hair has emerged as a valuable biological matrix for documenting long-term or cumulative drug exposure. Although hair analysis is well established in forensic toxicology, its potential application to monitor occupational exposure to hazardous drugs among healthcare workers remains unexplored. This systematic review aimed to describe analytical methods used for drug quantification in human hair and to assess the feasibility of applying this approach to hazardous drug exposure monitoring. A systematic search of PubMed, Embase, and CINAHL was conducted for studies published between January 2010 and March 2025 using the terms hair, matrix, drug, and analysis. Eligible studies reported quantitative measurements of drugs or metabolites in human hair. Data on study characteristics, analytical methods, and results (LOD, LOQ, concentration range in ng/mg) were extracted and descriptively analyzed. One hundred thirteen studies were included, mainly from Europe, most addressing forensic toxicology (77/113). The vertex posterior region was the preferred sampling site. Liquid-liquid extraction (LLE) with methanol and LC-MS/MS were the predominant analytical approaches, providing the lowest LODs and LOQs. A total of 233 analytes (152 drugs and 81 metabolites) were identified, mainly antidepressants, opioids, and antipsychotics. Only one study assessed a hazardous drug (soluble platinum compounds): Exposed workers showed median platinum levels of 3.24 versus 2.17 pg/mg in unexposed staff and 213 pg/mg in treated patients. Hair analysis is a promising, noninvasive approach for documenting long-term exposure to drugs. However, further studies are needed to validate its use for occupational biomonitoring of hazardous drugs in healthcare settings.
The Evolution of Precision Anti-Doping From UGT2B17 Polymorphism Insights to Integrated Detection Frameworks.
Cheng J, Li Z, Zheng T … +4 more , Ma R, Xiang X, Deng X, Liu B
Drug Test Anal · 2026 May · PMID 42175606 · Publisher ↗
Screening for endogenous anabolic-androgenic steroid abuse has long relied on the urinary testosterone/epitestosterone (T/E) ratio. However, this metric is significantly influenced by genetic polymorphisms such as copy n...
Screening for endogenous anabolic-androgenic steroid abuse has long relied on the urinary testosterone/epitestosterone (T/E) ratio. However, this metric is significantly influenced by genetic polymorphisms such as copy number variations in the UGT2B17 gene. Because the distribution of this genetic heterogeneity varies across ethnic populations, individuals with the del/del genotype face a higher risk of being missed in conventional testing. Drawing on current research progress, this paper proposes several potential strategies: integrating genotyping data into the athlete biological passport to calibrate individual metabolic baselines, employing alternative urinary markers such as the androsterone to etiocholanolone ratio and 5α-diol-3-glucuronide, and utilizing blood-based testing methods, including direct detection of intact steroid esters in dried blood spots to circumvent interference from the UGT2B17-dependent metabolic pathway. In addition, this paper attempts to construct a tiered decision-making framework that combines genotype status with marker selection, advancing testing strategies from single-biomarker thresholds toward multiomics and machine learning models. By reviewing current research across genetics, metabolomics, and artificial intelligence, this paper aims to provide analytical perspectives and potential solutions for addressing the impact of genetic polymorphisms on doping detection.
Characterization of Novel Metabolites of the HIF Stabilizer JNJ-42041935 in Rats Using LC-HRMS for Doping Control Purposes: A Pilot Study.
Hou J, Yang S
Drug Test Anal · 2026 May · PMID 42168136 · Publisher ↗
Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) stimulate erythropoiesis and enhance performance, and were banned by the World Anti-Doping Agency in the year 2011. JNJ-42041935, a potent and selective P...
Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) stimulate erythropoiesis and enhance performance, and were banned by the World Anti-Doping Agency in the year 2011. JNJ-42041935, a potent and selective PHD inhibitor, was administered orally to Sprague-Dawley rats, and its metabolites were tentatively characterized using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in positive and negative ionization modes. Eleven metabolites were identified, with Phase I reactions including monohydroxylation, dechlorination, and decarboxylation, and Phase II reactions comprising methylation, glucuronidation, sulfation, glycine, and taurine conjugation. These results provide insights into JNJ-42041935 biotransformation and support the development of analytical methods for HIF-PHI detection in anti-doping control. Future studies will continue to identify and characterize the valuable metabolites of JNJ-42041935, with a view to providing a more comprehensive understanding of its in vivo metabolic profile and developing improved detection methods.
Investigation of Metformin Contamination in Horses: Implications for Environmental Exposure and Doping Control.
Jacobs ME, Blea J, Hardy M … +3 more , McKemie DS, Traynham M, Knych HK
Drug Test Anal · 2026 Jul · PMID 42165703 · Full text
Metformin is a diabetes medication with minimal therapeutic efficacy in veterinary medicine. However, recently there have been several adverse analytical findings in regulatory samples collected from racehorses. Metformi...
Metformin is a diabetes medication with minimal therapeutic efficacy in veterinary medicine. However, recently there have been several adverse analytical findings in regulatory samples collected from racehorses. Metformin is commonly prescribed to humans, is excreted unchanged in urine, and is resistant to environmental degradation, raising concerns with respect to environmental contamination. In this study, six horses were exposed to shavings contaminated with metformin-positive urine at concentrations of 10, 135, and 270 μg/mL. Blood (plasma, serum, whole blood, and red blood cells) and urine samples were collected for 168 h post-exposure for determination of metformin concentrations. All horses had detectable metformin concentrations in blood samples at least once during the sample collection period. Metformin concentrations in urine samples greatly exceeded those in blood and were detectable as early as 4 h post-contamination. The presence of metformin in these samples raises the possibility of metformin exposure through contamination, especially given the housing conditions at racetracks. The potential for exposure through contamination, resulting in adverse analytical findings, should be considered in the regulation of metformin in racehorses.
Comprehensive Profiling of 6α-Chloro-Testosterone Metabolism in Human Urine Using Gas Chromatography-Mass Spectrometry.
Martínez Brito D, Colamonici C, Curcio D … +2 more , de la Torre X, Botrè F
Drug Test Anal · 2026 May · PMID 42161412 · Publisher ↗
According to its structure, 6α-chloro-17β-hydroxyandrost-4-en-3-one (6-CT) can exhibit both anabolic and antiestrogenic effects, and as such, it is prohibited in sports according to the World Anti-Doping Agency rules. Th...
According to its structure, 6α-chloro-17β-hydroxyandrost-4-en-3-one (6-CT) can exhibit both anabolic and antiestrogenic effects, and as such, it is prohibited in sports according to the World Anti-Doping Agency rules. The aim of this paper was to study the metabolism of 6-CT in humans by gas chromatography couple to mass spectrometry in tandem (GC-MS/MS), time-of-flight (GC-qTOF) and isotopic ratio (GC-C-IRMS), to describe changes in the endogenous steroid profile and the influence of 6-CT consumption on the carbon isotope ratio (CIR). One single oral dose of 6-CT (25 mg) was administered to two volunteers, and preadministration and postadministration samples of urine were collected for, at least, 4 days. Halogenated and dehalogenated metabolites were detected considering the accurate mass of the structure, the endogenous steroid profile was quantified and CIR values were measured. All urinary concentrations or areas were adjusted to a specific gravity of 1.020. The results showed halogenated and dehalogenated metabolites of 6-CT, considering the Phase I main reactions of steroids. The potential favoured 5β- reduction pathway allowed the formation of 5β- metabolites that are known to have weak or null androgenic activity (e.g., 6β-hydroxy-etiocholanolone, 5β-dihydrotestosterone and 5β-androstanediol), but the impact on the steroid profile evaluated in the Athlete Biological Passport (ABP) should be carefully assessed.
Comparison of Dried Blood Spot Sampling Methods for RNA-Based Biomarker Measurement in Anti-Doping.
Bejder J, Schlechten C, Mumenthaler C … +7 more , Salamin O, Kuuranne T, Bonne TC, Graae J, Sørensen H, Nordsborg NB, Leuenberger N
Drug Test Anal · 2026 May · PMID 42161409 · Publisher ↗
Dried blood spot (DBS) sampling offers logistical advantages, but it remains unexplored whether analytical results are comparable across DBS sampling methods. This study investigated whether venous or capillary blood spo...
Dried blood spot (DBS) sampling offers logistical advantages, but it remains unexplored whether analytical results are comparable across DBS sampling methods. This study investigated whether venous or capillary blood spotted on cellulose cards or collected with the Tasso-M20 device provides comparable results for the anti-doping RNA-based biomarkers of erythropoiesis, 5-aminolevulinic acid synthase (ALAS2), and carbonic anhydrase 1 (CA1). In the population of Swiss athletes (n = 12), DBS samples (venous-cellulose and Tasso-M20) were collected in routine anti-doping procedures. In healthy volunteers (n = 12), DBS samples (venous-cellulose, capillary-cellulose and Tasso-M20) were collected at baseline, after 1 week and 10 days post a 3-week recombinant human erythropoietin (rEPO) administration study. ALAS2 and CA1 mRNA were quantified using RT-qPCR, and agreement between matrices was assessed via Passing-Bablok regression. ALAS2 and CA1 expression showed strong linear agreement (r ≥ 0.96) across matrices. Passing-Bablok regression indicated no constant or proportional bias for ALAS2 across all comparisons. For CA1, no bias existed between venous-cellulose and Tasso-M20 in the athlete population, whereas a proportional bias of ~9%-22% was observed when comparing Tasso-M20 DBS with venous- or capillary-cellulose DBS in the rEPO study. No significant differences in relative RNA expression were observed across matrices at any timepoint in the administration study. ALAS2 and CA1 are reliably quantified across venous- and capillary-cellulose-based DBS and Tasso-M20 DBS samples. Strong agreement and minimal bias, with the modest CA1 bias being small relative to expected biological or treatment-induced variability, support their use for longitudinal monitoring, enabling less invasive, flexible, and decentralized sampling in research and anti-doping.
Elimination Profile of Deflazacort and Metabolites in Urine and Plasma Following Oral Administration: Relevance to Sports Drug Testing.
Coll S, Bressan C, Monfort N … +3 more , Aldea-Perona A, Carbó ML, Ventura R
Drug Test Anal · 2026 Jul · PMID 42155981 · Full text
Deflazacort (DEF) is a glucocorticoid prohibited in sports competitions when orally administered. This study aimed to assess the urinary and plasma excretion profiles of DEF and its metabolites after a single oral admini...
Deflazacort (DEF) is a glucocorticoid prohibited in sports competitions when orally administered. This study aimed to assess the urinary and plasma excretion profiles of DEF and its metabolites after a single oral administration to verify the World Anti-Doping Agency's minimum reporting level (MRL) of 30 ng/mL to detect administration of DEF. DEF was orally administered to eight healthy male participants (30 mg). Urine and plasma samples were collected before and after administration, and DEF and its metabolites were quantified using liquid chromatography-tandem mass spectrometry. Urinary concentrations confirmed that DEF is rapidly metabolized to 21-desacetyl-deflazacort (DES), with minimal detection of unchanged DEF. DES and the 6-hydroxy metabolites, 6βOHDES and 6αOHDES, were present in most urine samples during the first 48 h post-administration. DES peaked early (0-4 h) and declined below 30 ng/mL after 24 h, while 6βOHDES peaked later (4-8 h) and remained above 30 ng/mL until 36-48 h. 6αOHDES was detected at lower concentrations and cleared rapidly. In plasma, DES and 6βOHDES were detected within 8 h, with DES showing higher peak levels and longer half-life. DEF itself was not detected in plasma. Pharmacokinetic modeling showed good agreement between urinary and plasma excretion data. Cortisol levels were suppressed post-dose, indicating systemic GC activity, but recovered by 72 h in most subjects. The findings support the use of DES as the primary urinary marker for DEF detection, confirm the suitability of the MRL of 30 ng/mL and support the recommendation of a 3-day washout period due to interindividual variability.
Assessment of Abuse Potential-Related Effects of Oral Dried Kratom Leaf Powder in Healthy Normal Participants Following Single and Multiple Daily Doses.
Coe M, Huestis M, Wang D … +3 more , Hudzik T, Bothmer J, Henningfield J
Drug Test Anal · 2026 Jul · PMID 42144893 · Full text
Kratom is increasingly used in the United States for energy and self-management of pain and mood. While its active alkaloid, mitragynine, has partial μ-opioid receptor agonist activity, clinical data on its abuse potenti...
Kratom is increasingly used in the United States for energy and self-management of pain and mood. While its active alkaloid, mitragynine, has partial μ-opioid receptor agonist activity, clinical data on its abuse potential remain limited. This study evaluated abuse-related outcomes following single and multiple doses of dried kratom leaf powder in healthy kratom-naive adults in a randomized, double-blind, placebo-controlled study. Healthy participants (n = 116) received a single oral dose or 15 consecutive daily doses of Mitra-Leaf kratom capsules (4 cohorts: 500-4000 mg dried leaf powder; equivalent to 6.65-53.2 mg mitragynine; n = 12-13 per cohort, n = 49 total) or placebo (n = 67). Subjective effects were assessed using the Drug Effects Questionnaire (DEQ). Opioid withdrawal symptoms were evaluated using the Subjective Opioid Withdrawal Scale (SOWS) and Clinical Opiate Withdrawal Scale (COWS). Abuse-related treatment-emergent adverse events (ARTEAEs) were recorded. DEQ responses were dose-related, except between the middle two dose levels with statistically significant increases in maximum effect and area-under-the-curve compared to placebo observed only at the highest single dose. DEQ responses decreased following repeated dosing. No participants met the threshold for clinically meaningful opioid withdrawal based on COWS or SOWS. ARTEAEs occurred more frequently at higher doses but were generally mild and resolved without sequelae. No serious adverse events or deaths were reported. Oral administration of kratom at tested doses was well tolerated and associated with modest, dose-related subjective effects, minimal withdrawal symptoms, and low ARTEAE rates. Findings support further research on kratom's safety and therapeutic potential, while emphasizing the importance of dose control and product standardization.
Academic Performance Pressure and Methylphenidate Consumption: Insights From Wastewater-Based Epidemiology.
Van Wichelen N, Boogaerts T, Van Hal G … +4 more , Fransen E, Gys C, Covaci A, van Nuijs ALN
Drug Test Anal · 2026 Jul · PMID 42126953 · Publisher ↗
Methylphenidate (MPH), a first-line treatment for attention-deficit/hyperactivity disorder (ADHD), is increasingly debated for its use for cognitive enhancement by university/college students without an ADHD diagnosis. H...
Methylphenidate (MPH), a first-line treatment for attention-deficit/hyperactivity disorder (ADHD), is increasingly debated for its use for cognitive enhancement by university/college students without an ADHD diagnosis. However, current knowledge relies on self-reported data, while consumption-based data remain limited. This wastewater-based epidemiology study assessed MPH consumption at population level by measuring ritalinic acid (a urinary MPH consumption biomarker) in influent wastewater samples (n = 679) across 2021 and 2022 in two Belgian cities, Leuven and Brussels. Afterwards, these data were triangulated with available survey data collected by students. In Leuven, where university/college students comprise 55% of the population, MPH consumption doubled during exam periods and rose markedly during exam preparation periods. In Brussels, where students comprise only 7%, fluctuations were minimal. These findings suggest a strong link between academic performance pressure and MPH use, supported by survey data. Given associated health risks, targeted pharmacovigilance and public health interventions among students are needed.
Determination of THC and THC-COOH in Dried Capillary Blood Spots and Comparison to Venous Blood of Recreational Cannabis Consumers in a Pilot Study.
Bantle M, Schirmer W, Bruegger J … +5 more , Wüthrich T, Längin A, Zwicker L, Auer R, Weinmann W
Drug Test Anal · 2026 Jul · PMID 42120005 · Full text
With increased sensitivity of instrumentation, more substances can be analysed using capillary dried blood spots (DBS), and therefore, minimal invasive sample collection can be performed. However, for cannabinoids such a...
With increased sensitivity of instrumentation, more substances can be analysed using capillary dried blood spots (DBS), and therefore, minimal invasive sample collection can be performed. However, for cannabinoids such as (-)-trans-Δ-tetrahydrocannabinol (THC), low extraction efficiencies have been reported in previous studies. In this study, a novel extraction method was developed comprising an extraction with dimethyl sulfoxide followed by methanolic extraction, yielding extraction efficiencies > 80% for both THC and its metabolite 11-nor-9-carboxy-Δ-tetrahydrocannabinol (THC-COOH). The linear ranges of the LC-MS/MS method are 0.5-20 ng/mL for THC and 1.25-100 ng/mL for THC-COOH with good precision and accuracy. Concentrations of THC and THC-COOH in DBS from capillary blood were compared to venous blood concentrations based on 159 blood samples retrieved from a study including recreational cannabis users. THC-COOH concentrations in DBS were in close agreement with venous blood concentrations with a median capillary DBS/venous blood ratio of 1.09 (mean 1.10 ± 0.21), whilst THC concentrations in capillary blood were higher than venous blood concentrations with a median ratio of 2.11 (mean 16.6 ± 87.9) (after exclusion of outliers: 1.84 [mean 2.57 ± 1.93]), and the THC concentration ratio capillary DBS/venous blood showed a larger variability, ranging from 0.75 to 722 (0.75 to 8.18 after exclusion of outliers).
Advancing PFAS Biomonitoring in Hair: Rapid Analytical Method and Global Comparison of US Exposure.
Ameer A, Allen J, Acosta T … +2 more , Concheiro-Guisan M, Pego AMF
Drug Test Anal · 2026 Jul · PMID 42108384 · Publisher ↗
Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in a wide range of consumer and industrial products. Their chemical stability and resistance to degradation lead to environmental persiste...
Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in a wide range of consumer and industrial products. Their chemical stability and resistance to degradation lead to environmental persistence, raising concerns about human exposure and health risks. Although PFAS have been extensively monitored in ecological and traditional biological matrices, limited research has studied their presence in human hair. This study aimed to evaluate PFAS exposure in a New York population and contribute to the growing evidence supporting hair as a viable matrix for PFAS biomonitoring. A quantitative LC-MS/MS method for PFOA, PFNA, and PFHpA in human hair was validated according to ANSI/ASB Standard 036 guidelines, demonstrating linearity across a calibration range of 0.3-25 pg/mg. Real-case hair samples (n = 26), collected from individuals living in New York, were analyzed using LC-MS/MS. PFHpA was detected in 100% of the samples, with a median concentration of 14.11 pg/mg. In contrast, PFOA and PFNA were detected in 30.8% and 7.7% of samples, respectively, with median concentrations of 0.15 pg/mg for both compounds. The New York cohort had a unique PFAS profile, with lower PFOA and PFNA detection than some Asian cohorts but higher PFHpA levels than European and Asian studies. These findings highlight ongoing human exposure to legacy and emerging PFAS, demonstrating the utility of hair as a tool for monitoring chemical exposure trends. This research underscores the importance of ongoing PFAS surveillance to inform environmental health initiatives, guide regulatory efforts, and protect populations at potential elevated risk due to occupation, geography, or socioeconomic factors.
Segmental Hair Analysis of Diphenhydramine and Cyclizine Following a Single Dose.
Bílek J, Nielsen MKK, Kronstrand R … +1 more , Johansen SS
Drug Test Anal · 2026 Jul · PMID 42070998 · Full text
Diphenhydramine and cyclizine are over-the-counter (in many countries) antihistamines that have considerable sedative effects. Therefore, they can be used in drug-facilitated crimes to incapacitate victims. There is a kn...
Diphenhydramine and cyclizine are over-the-counter (in many countries) antihistamines that have considerable sedative effects. Therefore, they can be used in drug-facilitated crimes to incapacitate victims. There is a knowledge gap regarding detection possibilities and concentration levels of these antihistamines in hair. This study aimed to investigate whether a single dose of diphenhydramine and cyclizine could be quantified in head hair, and for how long. A single-dose study was conducted in which 12 adult volunteers ingested a single dose of diphenhydramine (25 mg) and cyclizine (44 mg). Hair samples were collected before drug intake and at 1, 3, 5, and if possible, 12 months after intake. To quantify a single dose of antihistamines in head hair, a validated LC-MS/MS method with a lower limit of quantification of 1 pg/mg was applied. Both antihistamines and their main metabolites, N-demethyldiphenhydramine and norcyclizine, were detected in all hair samples collected up to 5 months after intake. Twelve months after intake, diphenhydramine was quantifiable in 25% and cyclizine in 62.5% of available hair samples. The concentrations found in hair within 1 year after intake ranged from 0 to 610 pg/mg for diphenhydramine and from 0 to 590 pg/mg for cyclizine. The median decrease in concentration over 2, 4, and 11 months was 43%, 70%, and 100% for diphenhydramine and 40%, 64%, and 98% for cyclizine. The data obtained provided indicative concentration ranges for a single oral dose of diphenhydramine and cyclizine in hair, thereby improving the interpretation of hair analyses in forensic casework.
Analysis and Characterization of Kisspeptin and Its Analogues in Serum and Urine Samples by Liquid Chromatography-High-Resolution Mass Spectrometry for Doping Control Purposes.
Krombholz S, Korsmeier L, Thomas A … +1 more , Thevis M
Drug Test Anal · 2026 Jul · PMID 42057309 · Full text
The use of testosterone-stimulating peptides for doping purposes is prohibited for male athletes by the World Anti-Doping Agency (WADA). Among these substances is kisspeptin (KP-54), its isoforms (KP-14, KP-13, and KP-10...
The use of testosterone-stimulating peptides for doping purposes is prohibited for male athletes by the World Anti-Doping Agency (WADA). Among these substances is kisspeptin (KP-54), its isoforms (KP-14, KP-13, and KP-10), and synthetic receptor agonists such as TAK-448. Thus, they have been included in the WADA Prohibited List in 2024. To enable effective detection of kisspeptin misuse, reliable analytical methods are required. Consequently, this study aimed to develop liquid chromatography-high-resolution mass spectrometry-based (LC-MS) methods for the detection of kisspeptin and its analogues in human serum and urine. In addition, peptide stability in different biological matrices was investigated, and metabolic stability was characterized in vitro. Extraction methods based on cation-exchange solid-phase extraction were optimized, enabling a selective and sensitive LC-MS analysis with limits of identification (LOI) ranging from 0.8 ng/mL (KP-54) to 10 pg/mL (TAK-448). Analysis of a reference population comprising n = 20 serum samples and n = 100 urine samples revealed no detectable signals of endogenous kisspeptins and/or its degradation products. All native kisspeptins investigated showed poor stability in urine and blood; however, several degradation products could be identified. These metabolites could serve as complementary target analytes and improve the detectability of kisspeptins in doping control samples. Overall, the study provides an important foundation for the confirmatory analysis of kisspeptins in doping controls and delivers in vitro insights into their metabolic behavior. Analysis of samples collected after administration of the peptides remains necessary to further assess the detectability and metabolic profiles in authentic samples.
A Comparison of Three Analytical Procedures for Oxandrolone Long-Term Metabolite Determination in Urine Samples.
Stojanovic B, Pfeffer S, Gmeiner G
Drug Test Anal · 2026 Jul · PMID 42026877 · Publisher ↗
In contemporary antidoping analysis, doping control laboratories are confronted with a wide range of evolving analytical challenges. Continuous methodological refinement is required in this dynamic field, particularly wi...
In contemporary antidoping analysis, doping control laboratories are confronted with a wide range of evolving analytical challenges. Continuous methodological refinement is required in this dynamic field, particularly with respect to analytical strategies for the detection and confirmation of prohibited substances. The present study focuses on the comparative evaluation of three analytical strategies for the determination of long-term metabolites (LTMs) of the anabolic androgenic steroid (AAS) oxandrolone (Oxa) in human urine. The identification and implementation of LTMs in routine doping control have substantially enhanced the detection capabilities of antidoping laboratories, leading to an increased number of adverse analytical findings, particularly for AAS compounds. The primary objective of this work was to develop and validate novel confirmatory analytical procedures for Oxa LTMs and to compare their performance with that of an already established method. The investigated approaches comprise the following: (1) gas chromatography-mass spectrometry (GC-MS) following enzymatic hydrolysis of phase II metabolites and subsequent silylation, (2) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) of methylated phase II metabolites, and (3) LC-HRMS analysis of hydrolyzed phase II metabolites. The first two approaches are reported here for the first time in the context of LTM analysis in antidoping applications. These methodologies represent innovative analytical strategies that may be transferable to other target analytes and thereby contribute to further improvements in sports drug testing.
Recreational Drug Use at a Music Festival: A Dual Approach Using Hair Biomarkers Analysis and Participant Self-Reported Drug Use.
Simão AY, Rosado T, Barroso M … +2 more , Andraus M, Gallardo E
Drug Test Anal · 2026 Jul · PMID 42025208 · Full text
The growing prevalence of substance use and its associated health consequences highlights the need for reliable approaches to assess consumption patterns and validate self-reported data. This study, conducted at an inter...
The growing prevalence of substance use and its associated health consequences highlights the need for reliable approaches to assess consumption patterns and validate self-reported data. This study, conducted at an international music festival in Portugal, aimed to characterise substance use by integrating objective toxicological findings with self-reported information. Quantitative hair analysis was combined with survey data from 249 participants recruited in 2022 and 2023. Hair samples were analysed by liquid chromatography-tandem mass spectrometry to detect psychoactive substances and metabolites. Self-reported use was assessed across multiple timeframes, from same-day consumption to use within the previous year. Alcohol (96%) and cannabinoids (90%) were the most frequently self-reported substances overall, based on lifetime self-reported use. Overall, 50% of participants tested positive for at least one compound in the analysed hair samples, with cocaine, MDMA and ketamine being the most commonly detected substances (24.5%, 24.1% and 22.9%, respectively). Some participants who denied consumption tested positive, particularly for MDMA and ketamine. Self-reported non-use was inversely associated with hair positivity for MDMA (OR = 0.25, 95% CI: 0.09-0.65) and ketamine (OR = 0.19, 95% CI: 0.09-0.40), compared with self-reported users. Discrepancies were also observed for cannabinoids, highlighting limitations of self-reported data. Strong correlations were identified between cocaine and benzoylecgonine, and between cannabis-related analytes (cannabidiol and THC), supporting the consistency of the toxicological results. Integrating self-reported data with objective biological measures improved data reliability, revealed polydrug use patterns and supported substance use monitoring in high-risk populations such as music festival attendees in this high-exposure festival setting.
A Novel Workflow for Fast Elucidation of Drug Metabolites for Screening-Combining In Silico Metabolite Prediction With Trapped Ion Mobility QTOF-MS.
Zschiesche A, Schneider B, Nordhorn I … +3 more , Baessmann C, Huppertz LM, Kempf J
Drug Test Anal · 2026 Jul · PMID 42021506 · Full text
Urine is one of the preferred matrices for standard toxicological analysis, which makes the inclusion of drug metabolites in targeted and untargeted screening mandatory. Mass spectrometry is key for substance identificat...
Urine is one of the preferred matrices for standard toxicological analysis, which makes the inclusion of drug metabolites in targeted and untargeted screening mandatory. Mass spectrometry is key for substance identification, but updating methods for emerging substances like new psychoactive substances (NPS) is challenging due to the limited availability of reference standards for metabolites. This is particularly problematic for drugs that are barely or not detectable at all in urine. Insufficient metabolic knowledge and lack of spectral data carry the risk of false negatives. This study evaluates a non-targeted workflow using ultrahigh-performance liquid chromatography-trapped ion mobility spectrometry time-of-flight mass spectrometry (UHPLC-timsTOF-MS) and dedicated processing software (MetaboScape), integrating in silico metabolite prediction (BioTransformer), fragmentation (MetFrag), collision cross-section (CCS) prediction, and library searching. Quetiapine was selected as a model compound. Phase I metabolites were generated via pooled human liver microsomes (pHLMs) and analyzed by UHPLC-timsTOF-MS. Features were extracted and annotated with MetaboScape. The workflow successfully annotated 20 phase I metabolites in the pHLM assay, with 13 confirmed by library matching and 18 by BioTransformer. These metabolites were added to a targeted UHPLC-QTOF-MS method for analysis of 30 quetiapine-positive ante- and post-mortem urine samples from forensic casework. This revealed N-, O-dealkyl and carboxylated metabolites as the most abundant biomarkers in human urine. This integrated approach enables rapid and reliable metabolite detection, supports biomarker discovery, and facilitates routine screening updates, especially for substances without reference standards. Although not intended for exhaustive metabolic characterization, it offers practical applicability in evolving drug landscapes.
Examining Phosphatidylethanol (PEth) Formation Rates Within and Between Subjects in Ex Vivo Whole Blood.
Stark HG, Sanchez JJ, Hill-Kapturczak N … +2 more , Roache JD, Ginsburg BC
Drug Test Anal · 2026 Jul · PMID 42019969 · Full text
Interpretation of phosphatidylethanol (PEth) is complicated by between-subject variation in the PEth concentrations after matched ethanol exposure. Previous research suggests differences in PEth formation rates may expla...
Interpretation of phosphatidylethanol (PEth) is complicated by between-subject variation in the PEth concentrations after matched ethanol exposure. Previous research suggests differences in PEth formation rates may explain this variability but lacks within-subject assessments. This study assesses the extent of variability in PEth formation rates in ex vivo whole blood across a range of ethanol concentrations, with repeated ethanol exposures in blood collected 1 month apart, and the relationship of phosphatidylcholine concentration and PEth formation rate. Whole blood from 10 participants was collected via venipuncture. For each participant, blood was divided into separate samples and incubated with ethanol concentrations ranging 0-236.7 mg/dL at 37°C. Aliquots were removed from each sample every hour for 5 h. PEth 16:0/18:1 concentrations were measured using high performance liquid chromatography with tandem mass spectrometry. PEth formation rates were determined as the slope of linear regressions of PEth concentration over time. The experiment was repeated 1 month later for each participant. The relationship between PEth formation rate and phosphatidylcholine concentration was evaluated with linear regression analysis. PEth formation rate increased with increasing ethanol concentration, with variability between participants, but was consistent within-subject. Different PEth formation rates at the same ethanol concentration were observed in four individuals across two blood collection timepoints. No relationship was observed between phosphatidylcholine concentrations and formation rates, consistent within participants. PEth formation rate varies between individuals and within some individuals. PEth concentrations should be used to monitor an individual's ethanol use over time rather than compared between individuals.
Detection of the GH Analogue Redalsomatropin Alfa in Sports Drug Testing: Immunological Approaches and LC-HRMS/MS.
Walpurgis K, Thomas A, Thelen J … +4 more , Majer B, Al-Jaber M, Abushareeda W, Thevis M
Drug Test Anal · 2026 Jul · PMID 42014927 · Full text
Human growth hormone (hGH) is a peptide hormone exerting different growth-promoting and metabolic functions through binding to the GH receptor (GHR). Due to the presumed lipolytic and anabolic effects, the misuse of reco...
Human growth hormone (hGH) is a peptide hormone exerting different growth-promoting and metabolic functions through binding to the GH receptor (GHR). Due to the presumed lipolytic and anabolic effects, the misuse of recombinant 22-kDa hGH in sports is prohibited both in- and out-of-competition, and also synthetic long-acting GH analogues were added to the WADA Prohibited List in 2022. Within this research project, the detectability of redalsomatropin alfa (JR-142) with the immunoassays routinely employed by anti-doping laboratories for rhGH testing was evaluated. The drug candidate represents a recombinant fusion protein of 22-kDa hGH (191 amino acids) and human serum albumin (HSA, 585 amino acids) and is currently undergoing Phase III of its clinical development. Due to the attachment of HSA to the C-terminus of 22-kDa hGH, only "Kit 2" of the routinely employed immunoassay was found to recognize the drug, and the cross-reactivity was significantly reduced when compared to native hGH. As the misuse of redalsomatropin alfa in sports can therefore remain undetected when using this assay alone, the drug was implemented into the existing qualitative initial testing procedure (ITP) for the hGH analogue somatrogon employing affinity purification with GHR-Fc-conjugated magnetic beads for sample extraction and tryptic digestion in combination with LC-HRMS/MS for protein identification. Method validation demonstrated that the assay allows for a sensitive and specific detection of the fusion protein down to concentrations of 50 ng/mL. Moreover, these findings show that this LC-HRMS/MS assay can be further expanded to simultaneously test for multiple long-acting growth hormones (LAGHs).
Identification and Characterization of the Designer Opioid N-Pyrrolidino Fluetonitazene in Nasal Spray.
Picht F, Cepus V, Hohlfeld R … +2 more , Klingbeil J, Weber M
Drug Test Anal · 2026 Jul · PMID 41983957 · Full text
Detection of drugs in non-biological samples serves as a fundamental basis for identifying emerging trends in the field of new psychoactive substances and provides valuable information for optimizing analytical methods w...
Detection of drugs in non-biological samples serves as a fundamental basis for identifying emerging trends in the field of new psychoactive substances and provides valuable information for optimizing analytical methods within the field of chemical analysis. Nitazenes belong to this group of compounds and have been reported to cause poisonings. N-pyrrolidino fluetonitazene, also known as fluetonitazepyne (2-(4-(2-fluoroethoxy)benzyl)-5-nitro-1-(2-(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole), is another emerging synthetic opioid that may contribute to the challenges faced globally. This article describes the identification and characterization of N-pyrrolidino fluetonitazene in a rarely reported medium for opioids: nasal spray. The sample investigated here was collected from a patient treated at the University Medicine Halle following first-time recreational use of a nasal spray and was submitted in an amber plastic bottle containing a small volume of a yellowish viscous liquid. The analytical techniques used for identifying this compound included gas chromatography-mass spectrometry, liquid chromatography-quadrupole time-of-flight mass spectrometry, nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy.
Challenges in the Interpretation of Isolated THC-COOH Detection in Hair: Forensic and Clinical Implications.
Ramirez Fernandez MDM, Cirimele V, Ruyssinckx E … +4 more , Di Fazio V, Jacobs W, Schmit G, Wille SMR
Drug Test Anal · 2026 Jun · PMID 41978365 · Publisher ↗
Cannabis detection in hair typically relies on Δ9-tetrahydrocannabinol (THC), but interpretation is limited by its lipophilicity, external contamination, and washout. The metabolite 11-nor-9-carboxy-THC (THC-COOH) is con...
Cannabis detection in hair typically relies on Δ9-tetrahydrocannabinol (THC), but interpretation is limited by its lipophilicity, external contamination, and washout. The metabolite 11-nor-9-carboxy-THC (THC-COOH) is considered a more specific biomarker, though often present at much lower concentrations than THC. Cases with THC-COOH detected without THC present an interpretative challenge. This study investigated the prevalence, segmental distribution, and significance of hair samples positive for THC-COOH in the absence of THC in a forensic cohort monitored for driving license regranting. Hair (n = 639), blood (n = 198), and urine (n = 221) were collected. Hair was segmented (0-3 cm proximal, 3-6 cm distal) and analyzed using validated UPLC-MS/MS for THC, CBN, CBD, and THC-COOH. Cutoffs were THC ≥ 50 pg/mg and THC-COOH ≥ 0.2 pg/mg. Self-reported use was recorded and compared to biological results using phi (φ) coefficients. Of 639 hair samples, 276 were negative, 207 positive for both THC and THC-COOH, 76 positive for THC-COOH only, and 52 positive for THC only. THC-COOH-only cases included 29 with both segments positive. Median proximal hair concentrations (pg/mg) were THC-COOH 3, THC 340, CBD 270, CBN 170, higher than distal segments. Concordance with self-report improved when THC-COOH was considered when detected alone (φ up to 0.54). Wash tests of THC-COOH-only hair (n = 100) were negative, confirming endogenous origin. THC-COOH detection in hair, even without THC, is valid evidence of cannabis use. Multi-matrix analysis and recognition of THC-COOH as a primary marker improve abstinence monitoring and support updates to forensic guidelines.
About
- Frequency
- Sun
- Papers found
- 200
- RSS feed
- Subscribe