Drug Testing and Analysis最新文献

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.

The accidental contamination by the use of transdermal applications of clostebol acetate has been proven by the monitoring of its main urinary metabolite 4-chloro-3α-hydroxy-androst-4-en-17-one (M1). This work is aimed at describing clostebol metabolism in humans and at searching for specific metabolic markers or concentration thresholds allowing for distinguishing between an oral and a transdermal administration, helping to set up adequate criteria to be adopted by the antidoping community when incidental contamination is suspected. Urine samples were collected after the administration of a single dose of clostebol acetate orally (n = 3, males), a single transdermal dose (n = 1, male), and multiple transdermal administrations (n = 3, males, and n = 3, females). After enzymatic hydrolysis, liquid–liquid extraction, and the formation of trimethylsilyl derivatives, the samples were analyzed by gas chromatography coupled to tandem mass spectrometry and time-of-flight mass spectrometry. The metabolism of clostebol after oral and transdermal applications was described. Ten metabolites were detected after oral administration (M1–M10), but only five (M1–M4 and M9) could be detected after transdermal applications under the assay conditions applied. The use of concentrations of any metabolite might be difficult because of the interindividual variability in absorption, metabolism, and/or excretion. Instead, the ratios between M4 and M1 showed plausible results to discriminate between both administrations under the conditions described here. The intentionality of the use of one or other route of administration cannot be assessed.

Since the early 20th century, sampling biological matrices like blood on paper (dried blood spots [DBS]) has been vital in clinical analysis. While DBS microsampling is convenient for small molecules, extensive sample preparation can make LC-MS protein analysis impractical because of the time-consuming steps, especially for low-abundance proteins. Smart sampling, introduced in 2018, simplifies this by integrating sample preparation directly on the sampler. The work presented in this paper aims to compare a newly validated smart sampling method with two other methods: an in-house method based on immunocapture on magnetic beads and a commercial method that uses electrochemiluminescence immunoassay (ECLIA). The performance of the three hCG methods was compared using 21 single-blind serum samples. Linear regression analysis revealed strong correlations (all R² > 0.91) between the actual sample concentrations and the results obtained from all three methods. Immunocapture with magnetic beads showed the strongest linear correlation (R² = 0.974). To assess agreement between the methods, Bland–Altman analysis was conducted. The comparison between smart sampling and magnetic beads showed an average bias of −5.2, with no significant trend in variation across the sample concentration range of 0.5–75 ng/mL. The smart sampling and ECLIA comparison revealed a bias of 0.4 ± 4 ng/mL, indicating even better agreement and consistent results. This paper presents the first-ever comparison of a smart sampling method with existing methods. The results highlight smart sampling as a promising new approach for bioanalysis and boost the technique as a viable alternative in protein biomarker analysis from complex matrices using LC-MS.

Kratom (Mitragyna speciosa), a plant native to Southeast Asia, has long been used for its stimulant and analgesic properties. 7-Hydroxymitragynine (7-HMG) is a potent and selective opioid agonist in vitro and demonstrates a potent opioid effect in living subjects, reversible by naloxone. It has been semi-synthesized into products that are readily available in retail and virtual shops. It is known that 7-HMG has earned the nickname “legal morphine,” and has gained popularity among users seeking pain relief and/or a “high” comparable with prescription opioids. Medicinal chemistry efforts have led to synthetic 7-HMG derivatives such as MGM-15, where stereospecific saturation of the imine N(1)–C(2) double bond increases opioid receptor affinity and activity. Despite its higher in vitro opioid potency, MGM-15 is currently sold in the US for human consumption as a “research chemical” in tablet form, even though there is an absence of this being studied in humans and obviously no FDA approval. In this study, we analyzed commercially available MGM-labeled tablets using UPLC–MS/MS and subsequently evaluated the binding affinities of purified MGM-15 across multiple opioid receptors. Tablets contained an average of 10.9 ± 0.2 mg (10.7 to 11.2 mg) of MGM-15, with no naturally occurring kratom alkaloids or illicit substances detected. MGM-15 shows greater hMOR and hDOR binding affinities than 7-HMG, indicating the potential for higher opioid effects and risks, emphasizing the urgent need for more research to advise regulation and hopefully prevent misuse and harm.

Designer precursors for the synthesis of amphetamine-type stimulants pose a significant challenge to law enforcement. The precursors APAAN (α-phenylacetoacetonitrile) and MAPA (methyl α-acetylphenylacetate) became popular in the previous decade and have since been restricted. Recently, a ring-substituted analog of MAPA used for the synthesis of MDMA (3,4-methylenedioxymethamphetamine) was detected, highlighting the potential for criminal misuse of substituted analogs of these designer precursors. Previous research has characterized the impurity profiles of methamphetamine synthesized from APAAN and MAPA. In this study, the impurity profiles of MDMA and PMMA (p-methoxymethamphetamine) synthesized from ring-substituted analogs of APAAN and MAPA were characterized. In addition, byproducts forming from the conversion of p-methyl and p-fluoro analogs of APAAN and MAPA into analogs of P2P were investigated. MDMA was synthesized from 3,4-methylenedioxy-substituted MAPA (methyl α-acetyl-[3,4-methylenedioxyphenyl]acetate, MAMDPA) via sodium cyanoborohydride reductive amination and hydrogenation with platinum oxide and methylamine. Four impurities originating from MAMDPA were detected in MDMA synthesized via reductive amination, all of which are 3,4-methylenedioxy analogs of the impurities detected in methamphetamine synthesized from MAPA. Synthesis of MDMA via the hydrogenation route only produced one characteristic impurity, which, due to its instability under acidic conditions, is not likely to be present in clandestine MDMA hydrochloride samples. PMMA was synthesized via sodium cyanoborohydride reductive amination of MeO-P2P produced from methoxyl-substituted APAAN (α-p-methoxyphenylacetoacetonitrile, APMPAAN). Five impurities were identified as originating from APMPAAN, two of which are proposed to be the most reliable markers for the use of APMPAAN in the synthesis of PMMA.

Over the last several years, there has been an influx of α₂-agonists into the street drug supply, beginning with the proliferation of xylazine, a potent veterinary sedative. Since 2023, another sedative, medetomidine, has been widely detected. Medetomidine, broadly, encompasses two enantiomers—dexmedetomidine and levomedetomidine—with the dex enantiomer being pharmacologically active and present in human-use pharmaceuticals. In this work, we investigate street drug samples containing medetomidine to better understand the enantiomeric makeup in the illicit supply. Liquid chromatography tandem mass spectrometry (LC–MS/MS) was used to analyze 100 drug product or paraphernalia residue samples. All samples were found to contain a racemic mixture of medetomidine. A subset of samples was analyzed by medetomidine immunoassay test strips, where racemic mixtures were not found to negatively affect results. All samples were also qualitatively analyzed using direct analysis in real time mass spectrometry (DART-MS) to identify commonly co-occurring compounds, which included fentanyl, xylazine, and local anesthetics. Parabens, preservatives found in licit injectable preparations of the drug, were not detected. This work provides a snapshot into the medetomidine makeup of the street drug supply, which needs to be continually monitored given the differences in pharmacology between the two enantiomers.