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.

Despite the recent success in introducing supramolecular solvents (SUPRAS)-based extraction to drug analysis, its application and robustness in day-to-day regular urine testing have yet to be demonstrated. Moreover, the applicability of SUPRAS in equine doping control testing remains unexplored. In this work, we have successfully developed for the first time a simple, rapid, inexpensive, and environmentally friendly SUPRAS extraction method for analyzing 76 prohibited substances of different classes (selective androgen receptor modulators, hypoxia-inducible factor prolyl hydroxylase inhibitors, angiotensin II receptor antagonists, benzodiazepines, etc.) in hydrolyzed horse urine with liquid chromatography-mass spectrometry (LC/MS) for detection. The developed 1,2-hexanediol-based SUPRAS-LC/MS method has been fully validated, and its applicability and robustness in day-to-day testing of horse urine have also been demonstrated. This work marks a significant milestone in the advancement of green and sustainable drug testing methodology in equine sports, offering a novel approach to address one of the complexities inherent in equine doping control.

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.

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 exvivo 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.

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).

A subject, admitted to an addiction treatment unit, was occupationally exposed to solvent mixtures and paint thinners containing aliphatic alcohols, while being a coachbuilder. He repeatedly tested positive for ethyl glucuronide (EtG) in weekly urine immunoassays performed in a hospital laboratory. However, he denied any alcohol consumption. Therefore, presumptive positive urine samples, as well as chest and pubic hair samples, were submitted to targeted liquid chromatography-high-resolution accurate mass-Orbitrap-mass spectrometry (LC-HRAM-Orbitrap-MS) analysis, after derivatization of analytes with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC). MS acquisition was carried out in full scan positive-ion mode, followed by data-dependent MS2 and parallel reaction monitoring confirmation modes, according to an inclusion list of accurate mass values calculated from the elemental composition of MH+ ion species of several derivatized EtG homologs. All urine samples turned out to be EtG negative. Instead, methyl, propyl, butyl, and hexyl glucuronides were detected at varying levels depending on the exposure and/or inhalation conditions (product types, amounts, etc.), and urine sampling times. As expected, chest hair and pubic hair were EtG negative too, whereas the same other alcohol glucuronides were detected in both matrices, with higher abundances in pubic hair. Cross-reaction of EtG homologs, in this case due to exposure to chemicals containing several aliphatic alcohols, explains the false-positive urine immunoassay results. Furthermore, LC-HRAM-Orbitrap-MS analyses show that EtG homologs, like EtG, can be incorporated and detected in a variety of hair samples.

The steroidal module of the Athlete Biological Passport (ABP) longitudinally monitors five ratios between urinary concentrations of endogenous anabolic and androgenic steroids. Even though it has improved detection of testosterone doping, the interpretation of data from multiple discrete biomarkers is complex. This study sought to create a single score to identify doping rather than relying on the interpretation of each parameter alone. A Bayesian model was used to define an ABP sequence probability for each biomarker to assess the extremity of a measurement relative to the expected levels from ABP. This was used to discriminate between doped and presumed clean individuals based upon pattern classification of biomarkers using classification algorithms. Data were obtained from laboratory-controlled experimental studies as well as routine doping control tests. A laboratory model (where classifier is trained using the laboratory-controlled data only) and a mixed model (where classifier is trained on combined laboratory-controlled and doping control data) were developed and tested on the doping control data. Logistical regression was seen to have the best classification performance across the methods used, with the Abnormal Steroid Profile Score (ASPS) representing the estimated probability from the logistical regression model. Classifier performance produced an AUC of 0.67 and 0.75 when trained on the laboratory model and the mixed model, respectively, with T/E and 5α-Diol/5β-Diol representing the main biomarkers driving the ASPS. These findings demonstrate that the ASPS can discriminate between the doping status of individuals, even if a mixture of steroids, administration methods and doses are used.

The direct blood-alcohol biomarker phosphatidylethanol (PEth), especially its most abundant analogue 1-palmitoyl-2-oleoyl-sn-phosphatidylethanol (PEth 16:0/18:1) has gained increasing relevance in clinical and forensic applications for assessing alcohol consumption. Accurate quantification of PEth is essential to reliably differentiate between abstinence, moderate alcohol consumption and excessive alcohol intake. Measurement accuracy of PEth 16:0/18:1 by well-established liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches such as multiple reaction monitoring (MRM) can be confounded by the presence of the regioisomer 1-oleoyl-2-palmitoyl-sn-phosphatidylethanol (PEth 18:1/16:0) in samples and synthetic reference standards. To address this measurement uncertainty, we conducted a new assessment of the isomeric composition of six currently available reference materials from four suppliers using collision-induced dissociation/ozone-induced dissociation (CID/OzID). Examination of these synthetic compounds found a high degree of regioisomeric purity of > 95%. Thus verified, the relative abundance of two key LC-MS/MS transitions were compared across a range of collision energies for both reference materials and an exemplary set of 10 dried blood spot case samples. These findings suggest a significantly wider range of natural isomer distributions spanning both higher and lower regiochemical composition (88.8%-98.85%) than the reference materials but within a range that would not significantly impact clinical classification.

Distinguishing between intake of the prohibited substance trimetazidine from administration of approved migraine medicine containing the nonprohibited substance, lomerizine, is crucial for anti-doping control laboratories. Investigations in males after lomerizine intake have been conducted leaving lomerizine M6 as the most useful metabolite. To our knowledge, the excretion profile of lomerizine among women has not been studied and leaves a potential gap to be misused by athletes in the case of appeal of a reported adverse analytical finding. We have investigated lomerizine M6 in women to address the gender-specific biology to correctly detect misusage of the prohibited substance trimetazidine within sports. To determine the importance of lomerizine M6 in trimetazidine cases, we analyzed urine samples collected from 12 individuals (eight females and four males) over 144 h, after oral intake of 5-mg lomerizine dihydrochloride. A dilute and shoot method was used, and the samples were analyzed on a LC-HRMS instrument. In all study subjects, trimetazidine was found from 2 h up to 48 h, and lomerizine M6 was detected from 2 h and onward. Lomerizine was only detected above the limit of identification of the method for a few of the subjects. The study demonstrated that by monitoring lomerizine M6, it was possible to determine the origin of trimetazidine in women. The metabolism for lomerizine dihydrochloride does not appear to differ significantly between genders. However, lomerizine M6 concentrations are not always higher than trimetazidine concentrations. Special care should be taken when interpreting trimetazidine concentrations at 1 ng/mL or below.