Analysis on dynamic changes of etizolam and its metabolites and exploration of its development prospect using UPLC-Q-exactive-MS.

As one of the most widely abused designer benzodiazepines in the world, etizolam has been found in many cases in many countries. In this study, UPLC-Q-Exactive-MS was used for the first time to establish a dynamic change model of etizolam and its metabolites in rats. Compared with previous studies, the detection sensitivity and reproducibility of the instrument were higher. In the experiment, we optimized the traditional pharmacokinetic model based on Gauss function. According to the significant difference of etizolam in the plasma elimination phase of rats, a new pharmacokinetic model based on Lorentz function was established to describe the dynamic changes of etizolam more rigorously, which made the error effects lower and the accuracy of the pharmacokinetic parameters was improved. At the same time, the pharmacokinetic parameters of etizolam were compared with four other designer benzodiazepines reported in previous studies in rats, and we found the direct reason for the popularity of etizolam in the NPS market and explored the future development of etizolam for the first time. In addition, 21 metabolites were found through rat experiments to effectively detect etizolam abuse for a long time, of which 4 metabolites had the longest detection window and could be used as long-acting metabolites for experiments, which greatly prolongs the detection window and extends the time range in which etizolam was detected in real cases. This study is the first to conduct a systematic and comprehensive study on the metabolism and pharmacokinetics of etizolam and find out the direct reason for the prevalence of etizolam abuse, and we also discuss the development trend of etizolam in the future market of new psychoactive substances, which is beneficial for forensic experts to assess the trend of drug abuse and can provide reference for relevant drug control and drug treatment.

Gas chromatography properties and mass spectrometry fragmentation of anabolic androgenic steroids in doping control.

Aim: This study aimed to investigate the gas chromatographic properties and mass spectrometric fragmentations of anabolic androgenic steroids (AASs) after trimethylsilylated derivatization. Materials & methods: A total of 113 AASs were analyzed through gas chromatography-mass spectrometry in the full-scan mode. Results: New fragmentation pathways yielding m/z 129, 143 and 169 ions were analyzed. Based on the characteristics of the A-ring, seven classes of drugs were identified and analyzed. Conclusion: The fragmentation pathway of a new classification of 4-en-3-hydroxyl was reported for the first time. The relationship between the chemical structures of AASs and their retention time, along with their molecular ion peak abundance, was also reported herein for the first time.

A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs.

OBJECTIVES: To establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol & S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs. METHODS: A LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid-liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated. RESULTS: R-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5-500 ng/g. Accuracies ranged from -11.9-13.0% for R-(-)-clenbuterol and -10.2-13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6-5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1. CONCLUSIONS: The analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.

Metabolic Profile Analysis of Designer Benzodiazepine Etizolam in Zebrafish and Human Liver Microsomes.

As one of the most widely abused designer benzodiazepines worldwide, Etizolam is characterized by its high addiction potential, low production cost, and difficulty in detection. Due to the rapid metabolism of Etizolam in the human body, the probability of detecting the Etizolam parent drug in actual case samples by forensic personnel is low. Therefore, without detecting the parent drug, analysis of Etizolam metabolites can help forensic personnel provide references and suggestions on whether the suspect has taken Etizolam. This study simulates the objective metabolic process of the human body. It establishes a zebrafish in vivo metabolism model and a human liver microsome in vitro metabolism model to analyze the metabolic characteristics of Etizolam. A total of 28 metabolites were detected in the experiment, including 13 produced in zebrafish, 28 produced in zebrafish urine and feces, and 17 produced in human liver microsomes. The UPLC-Q-Exactive-MS technology was used to analyze the structures and related metabolic pathways of Etizolam metabolites in zebrafish and human liver microsomes, and a total of 9 metabolic pathways were identified, including monohydroxylation, dihydroxylation, hydration, desaturation, methylation, oxidative deamination to alcohol, oxidation, reduction acetylation, and glucuronidation. Among them, metabolites involving hydroxylation reactions (including monohydroxylation and dihydroxylation) accounted for 57.1% of the total number of potential metabolites, indicating that hydroxylation may be the major metabolic pathway of Etizolam. Based on the response values of each metabolite, monohydroxylation (M1), desaturation (M19), and hydration (M16) were recommended as potential biomarkers for Etizolam metabolism. The experimental results provide reference and guidance for forensic personnel in identifying Etizolam use in suspects.

Detection of 20 endogenous anabolic steroid esters with Girard's Reagent P derivatization in dried blood spots using UPLC-Q-Orbitrap-MS.

The esters of endogenous anabolic steroids are the most frequently used doping agents for prolonging the half-life of exogenously ingested endogenous anabolic steroids. As a cost- and time-saving matrix, dried blood spots (DBSs) are valuable for directly detecting endogenous anabolic steroid esters in blood and for providing conclusive evidence of their abuse. In this study, a method for simultaneous detection of 20 endogenous anabolic steroid esters in DBSs based on ultra-high performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) with parallel reaction monitoring (PRM) was developed and validated, and 10 of these esters were analyzed in DBSs for the first time. This method analyzes the greatest number and types of endogenous anabolic steroid esters of any current method using DBSs. Girard's Reagent P (GRP) was used for the derivatization of endogenous anabolic steroid esters in a DBS matrix for the first time, and the conditions of the derivatization reaction were optimized to achieve a higher sensitivity compared to previous methods. Selectivity, limit of detection (LOD), extraction recovery, precision (intra- and inter-), matrix effects, and carry-over were analyzed to validate the method. The LODs were lower and the recoveries were higher than those of previous studies. The relative standard deviation of the intraday precision was below 20% and the interday precision was below 35%. A product ion analysis of GRP nandrolone ester, GRP boldenone ester, GRP dehydroepiandrosterone acetate, and GRP androstanolone ester derivatives was performed, and the structures of the fragment ions were proposed for the first time.

Metabolic profiles and screening tactics for MDMB-4en-PINACA in human urine and serum samples.

MDMB-4en-PINACA (Methyl 3,3-dimethyl-2-[1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido] butanoate) is a potent agonist of the CB1 receptor. In 2021, it was one of the most common synthetic cannabinoid receptor agonists (SCRAs) seized by the Beijing Drug Control Agency. MDMB-4en-PINACA can be hard to detect in biological specimens because of ester hydrolysis. In this work, a highly sensitive liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed for the detection of MDMB-4en-PINACA metabolites in urine, serum, and hair samples. Metabolites from authentic samples were compared with those from human liver microsomes (HLMs) in vitro and in zebrafish in vivo. A total of 75 metabolites, including 44 previously unreported metabolites, were identified from urine samples. We found that 11 metabolic pathways were involved in MDMB-4en-PINACA metabolism, including acetylation, a novel metabolic pathway for SCRAs. Our results revealed that ester hydrolysis and hydroxylation were to the major metabolic pathways involved in MDMB-4en-PINACA metabolism. Using serum samples, we detected 9 metabolites along with the parent drug. Only the parent drug was detected using hair samples. The existence of ADB-4en-PINACA makes the currently used biomarkers for MDMB-4enPINACA not very specific for the intake of MDMB-4en-PINACA. Therefore, based on the identified metabolites and their structural features, we propose more sensitive screening tactics for MDMB-4en-PINACA using urine and serum samples.

Identification and characterization of higenamine metabolites in human urine by quadrupole-orbitrap LC-MS/MS for doping control.

Higenamine is an alkaloid found in aconite, Annona squamosa, nanzhu (sacred bamboo), and other plants. It can be used to treat coughing, asthma, heart failure, and erectile dysfunction as well as aid in weight loss. It is also a banned substance in and out of competition as defined by the World Anti-Doping Agency (WADA). In this work, higenamine metabolic profiles were investigated in detail. Two healthy volunteers (one male and one female) took a higenamine tablet (5 mg), and urine samples were collected for two weeks. Solid-phase extraction (SPE) without enzymatic hydrolysis was used to clean the urine samples, and the urine extracts were then analyzed by liquid chromatography-quadrupole-orbitrap mass spectrometry (quadrupole-orbitrap LC-MS/MS) with accurate mass measurements. Higenamine and 32 metabolites were detected: 6 methylated, 10 sulfated and 16 glucuronidated metabolites. The chemical structures were elucidated by their fragmentation patterns, and accurate molecular formula determination was obtained for these newly reported metabolites. Three metabolic pathways containing methylation, glucuronidation, sulfation, and combinations of these were provided with methylation as the main metabolic pathway. The post-dose detection windows within urine of all 32 metabolites were compared with that of the parent drug, and a new potential biomarker (M7) was suggested for higenamine misuse. All urine samples were processed by two sample preparation methods: the dilute-and-shoot (DS) procedure and acid hydrolysis followed by SPE, and the time periods for a higenamine positive trails of two methods were compared. Although the DS method used to process the urine samples of athletes in the most of WADA-accredited laboratories to detect only free higenamine, acid hydrolysis followed by SPE is preferable and offers routine analysis to avoid false-negative results.

Simultaneous detection of 93 anabolic androgenic steroids in dietary supplements using gas chromatography tandem mass spectrometry.

In recent years, anabolic androgenic steroids (AASs) have been frequently detected as undeclared ingredients in dietary supplements, where the adverse analytical findings (AAFs) were obtained from analysis of athletes' urine samples after ingestion. In our present study, a GC-MS/MS method for simultaneous detection of 93 anabolic steroids was developed. The chromatographic and mass spectrometric conditions were optimized, and selective reaction monitoring (SRM) mode was adopted to obtain the necessary sensitivity. The whole sample analysis process was completed within 23 min, and the limit of detection (LOD) was 0.5-4 ng.g-1 for solid samples and 0.1-0.8 ng.mL-1 for liquid samples. This method was verified according to World Anti-Doping Agency (WADA) regulations. In addition, the method was found to be specific, accurate. The developed method was then applied to a routine analysis of more than 300 liquid and solid dietary supplements, and one testosterone-positive sample was found. Three suspected drugs, (4-hydroxyandrostenedione, DHEA, and 6-Br androstenedione) were found in three dietary supplements obtained from the Internet through the pretreatment method of this study. This study provides a high-throughput method for screening and monitoring the ingredients of supplements and their subsequent harm to public health.

Metabolic Profiles of 5F-MDMB-PICA in Human Urine, Serum and Hair Samples Using LC-Q Exactive HF-MS.

In 2020, 5F-MDMB-PICA (5F-MDMB-2201) was one of the most common synthetic cannabinoids (SCs) identified in drugs seized by the Beijing Drug Control Agency, and it was categorized as Schedule II by the United Nations Office on Drugs and Crime in March 2020. It is difficult to detect 5F-MDMB-PICA in biological matrices due to its fast metabolic rate in vivo. In this work, 5F-MDMB-PICA metabolic profiles were investigated by liquid chromatography--quadrupole exactive high field orbitrap mass spectrometer (LC-Q Exactive HF MS), with accurate mass measurements in human urine, serum and pubic hair. To obtain intact metabolites, solid-phase extraction for urine and serum and direct ultrasonic extraction for pubic hair were applied to clean the samples without enzymatic hydrolysis. The differences in 5F-MDMB-PICA metabolism in the three different matrices were compared for the first time to determine the best detection biomarkers for monitoring 5F-MDMB-PICA misuse. Urine samples were determined to be the preferred biological material for identifying 5F-MDMB-PICA abuse. Forty-seven intact metabolites were detected in human urine, the ester hydrolyzed with glucuronidated metabolite in urine samples can be used as the primary biomarker to identify drug misuse. Fifteen metabolites were found in serum samples. Ester hydrolysis was considered to be the major metabolic pathway, and a large number of metabolites were involved with it. Zero metabolites apart from the parent drug were detected in pubic hair samples. Twenty-eight new metabolites and their metabolic pathways were characterized and tentatively identified by LC-QE-HF-MS, and a new potential biomarker (M5 ester hydrolysis + propionic acid) was also identified.

Dried blood spots in doping analysis.

A series of dried blood spot (DBS) detection methods for doping agents have been developed in the last two decades. The DBS technique minimizes invasiveness and reduces storage and shipping costs. Recently, the World Anti-Doping Agency announced the use of DBS for the 2022 Beijing Winter Olympic Games and Paralympic Games owing to the advantages of the DBS application in routine doping control. Therefore the further development of detection methods for doping agents in DBS is important and urgent. This review summarizes five aspects of DBS application in doping analysis: sample collection, storage conditions, pretreatment, instrumentation and validation according to the Prohibited List issued by the World Anti-Doping Agency, and proposes some suggestions for future studies of DBS in doping analysis.

Enantiomeric analysis of clenbuterol in Chinese people by LC-MS/MS to distinguish doping abuse from meat contamination.

Aim: Follow-up investigations are often required for clenbuterol-positive cases. A method to distinguish doping abuse from meat contamination was developed. Materials & methods: A total of 26 volunteers were recruited to ingest clenbuterol contaminated-pork and clenbuterol tablets. Results: For 20 volunteers, after ingestion of contaminated-pork, R-(-)/S-(+)-clenbuterol ratio was <1.0, while the value was >1.0 after taking clenbuterol tablets. However, after taking clenbuterol tablets, some ratio points of the other six volunteers were between 0.9 and 1.0. A case of an abnormal cold and fever, which returned to normal after recovery, was also reported firstly. Conclusion: A change in R-(-)/S-(+)-clenbuterol was reported in the Chinese population initially. A ratio of 0.9 was recommended in doping related cases for the Chinese population.

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS).

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, N-glucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.

Analysis of AMB-FUBINACA Biotransformation Pathways in Human Liver Microsome and Zebrafish Systems by Liquid Chromatography-High Resolution Mass Spectrometry.

In this study, the metabolic profiles of a new illicit drug AMB-FUBINACA were investigated using both human liver microsome and zebrafish models. Liquid chromatography Q Extractive HF Hybrid Quadrupole-Orbitrap mass spectrometry (LC-QE-HF-MS) was employed to analyze the metabolic sites and pathways. AMB-FUBINACA was added to the in vitro liver microsome incubation model to simulate the metabolic processes in human body. The results showed that a total of 17 metabolites were generated in the human liver microsome model; the main metabolic pathways of the phase I metabolism included ester hydrolysis, methylation, ester hydrolysis combined with decarboxylation, hydroxylation, ester hydrolysis combined with indazole ring hydroxylation, etc. while glucuronidation served as the main metabolic pathway of the phase II metabolism. The zebrafish system produced a similar result with 16 of the same 17 metabolites identified. The phase I metabolites M3.1 (ester hydrolysis), M1.2 (alkyl chain hydrolysis) and the phase II metabolite M3.2 (M3.1 glucuronide) were recommended to be the potential poisoning markers.

Oxytrodiflavanone A and Oxytrochalcoflavanones A,B: New Biflavonoids from Oxytropis chiliophylla.

Three previously undescribed biflavonoids, oxytrodiflavanone A (1), and oxytrochalcoflavanones A,B (2,3), were isolated from the aerial part of Oxytropis chiliophylla, together with their putative biosynthetic monomers, i.e., (2S)-5,7-dihydroxyflavanone (4), (2S)-7-hydroxyflavanone (5), and 2',4'-dihydroxychalcone (6). The structures of these compounds were elucidated by a combination analysis of spectroscopic data. The cytotoxic activities of all the isolated compounds against PC-3 human prostate cancer cell line are also presented.

Structurally Diverse Cytotoxic Dimeric Chalcones from Oxytropis chiliophylla.

Ten isomeric cyclobutane- and cyclohexene-containing chalcone dimers, oxyfadichalcones A-G, were isolated from the aerial parts of Oxytropis chiliophylla. These included six new compounds and three pairs of enantiomers that are being reported from natural sources for the first time. The relative configurations were elucidated by spectroscopic data analysis, while the absolute configurations were determined by comparing the experimental and calculated electronic circular dichroism spectra. Quantitative LC-MS analysis of the main dimers from different parts of the plant revealed their characteristic accumulation in the viscous secretion and provided supporting evidence for the hypothesized photochemical biosynthesis. In addition, the cytotoxic activities of all isolates against the PC-3 human prostate cancer cell line are reported.

Doping control analysis of 13 steroids and structural-like analytes in human urine using Quadrupole-Orbitrap LC-MS/MS with parallel reaction monitoring (PRM) mode.

Anabolic androgenic steroids (AASs) and structural-like substances are commonly prohibited substances found in doping control studies that can be difficult to accurately detect. In the present study, 11 AASs and 2 structural-like substances that are commonly detected were examined. Currently, such analytes are detected using low resolution GC-MS/MS or LC-MS/MS, with detection not always possible. Herein, the high resolution Quadrupole-Orbitrap liquid chromatography-tandem mass spectrometry LC-MS/MS system Q Exactive was utilized to increase the specificity. This approach was then combined for the first time with parallel reaction monitoring (PRM) during the screening procedure. The results confirmed high specificity, with the LODs of all 13 analytes being at least 25-fold lower than corresponding MRPLs as defined by WADA. Furthermore, the extraction recoveries were above 70% and the intra- and inter-day precisions were lower than 15%. This approach was successfully applied to analyze over 10,000 samples with no false-positive or false-negative results, thus suggesting that Quadrupole-Orbitrap LC-MS/MS when combined with PRM is an effective method for doping control analysis.

New Potential Biomarker for Methasterone Misuse in Human Urine by Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry.

In this study, methasterone urinary metabolic profiles were investigated by liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. A healthy male volunteer was asked to take the drug and liquid-liquid extraction was employed to process urine samples. Chromatographic peaks for potential metabolites were hunted out with the theoretical [M - H](-) as a target ion in a full scan experiment and actual deprotonated ions were studied in targeted MS/MS experiment. Fifteen metabolites including two new sulfates (S1 and S2), three glucuronide conjugates (G2, G6 and G7), and three free metabolites (M2, M4 and M6) were detected for methasterone. Three metabolites involving G4, G5 and M5 were obtained for the first time in human urine samples. Owing to the absence of helpful fragments to elucidate the steroid ring structure of methasterone phase II metabolites, gas chromatography mass spectrometry (GC-MS) was employed to obtain structural information of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and the potential structure was inferred using a combined MS method. Metabolite detection times were also analyzed and G2 (18-nor-17ß-hydroxymethyl-2α, 17α-dimethyl-androst-13-en-3α-ol-ξ-O-glucuronide) was thought to be new potential biomarker for methasterone misuse which can be detected up to 10 days.

Applications and challenges in using LC-MS/MS assays for quantitative doping analysis.

LC-MS/MS is useful for qualitative and quantitative analysis of 'doped' biological samples from athletes. LC-MS/MS-based assays at low-mass resolution allow fast and sensitive screening and quantification of targeted analytes that are based on preselected diagnostic precursor-product ion pairs. Whereas LC coupled with high-resolution/high-accuracy MS can be used for identification and quantification, both have advantages and challenges for routine analysis. Here, we review the literature regarding various quantification methods for measuring prohibited substances in athletes as they pertain to World Anti-Doping Agency regulations.

New drostanolone metabolites in human urine by liquid chromatography time-of-flight tandem mass spectrometry and their application for doping control.

Drostanolone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Here, we studied drostanolone urinary metabolic profiles using liquid chromatography quadruple time of flight mass spectrometry (LC-QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. The drug was administered to one healthy male volunteer and liquid-liquid extraction along with direct-injection were used to analyze urine samples. Chromatographic peaks for potential metabolites were identified with the theoretical [M-H](-) as a target ion in a full scan experiment and actual deprotonated ions were analyzed in targeted MS/MS mode. Eleven metabolites including five new sulfates, five glucuronide conjugates, and one free metabolite were confirmed for drostanolone. Due to the absence of useful fragment ions to illustrate the steroid ring structure of drostanolone phase II metabolites, gas chromatography mass spectrometry (GC-MS) was used to obtain structural details of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and a potential structure was proposed using a combined MS approach. Metabolite detection times were recorded and S4 (2α-methyl-5α-androstan-17-one-6ß-ol-3α-sulfate) and G1 (2α-methyl-5α-androstan-17-one-3α-glucuronide) were thought to be new potential biomarkers for drostanolone misuse which can be detected up to 24days by liquid-liquid extraction and 7days by direct-injection analysis after intramuscular injection. S4 and G1 were also detected in two drostanolone-positive routine urine samples.

New long term metabolite in human urine for metenolone misuse by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this study, metenolone metabolic profiles were investigated. Metenolone was administered to one healthy male volunteer. Liquid-liquid extraction and direct-injection were applied to processing urine samples. Urinary extracts were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOFMS) using full scan and product ion scan with accurate mass measurement for the first time. Due to the lack of useful fragment ion for structural elucidation, GC-MS instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after hydrolysis, and the EI mass spectrum was always informative in steroidal structure studies owing to more useful fragment ions than the ESI mass spectrum. 16 metabolites including 6 glucuronide and 9 unreported sulfate conjugates were characterized and tentatively identified. All the metabolites were evaluated in terms of how long they could be detected. The sulfate conjugate S6 (1-methylen-5α-androst-3,17-dione-2ξ-sulfate) was considered to be a new long term metabolite for metenolone misuse that could be detected 40 days by liquid-liquid extraction and up to 30 days by direct-injection analysis after oral administration.