Estimation of the time of zolpidem intake and differentiation between consumption and external contamination using MALDI-MSI for investigations on single hair samples.

Estimation of drug ingestion time (event time) and distinguishing between drug ingestion and external contamination are important for interpreting hair analysis results in forensics practice. Here, we present a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method for in situ analysis of intact hair. We applied a longitudinal cutting method for a single hair to analysis authentic hair samples from a victim of a drug-facilitated sexual assault (DFSA) case and zolpidem-soaked hair. MALDI-MSI showed that zolpidem-positive segments distributed at 4-6 mm or 6-8 mm from the root in three single hairs of a DFSA victim collected 25 days after the event, at concentrations ranging from 0.1 to 5.7 pg mm-1, in agreement with the results from segmental analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). The estimation of drug intake time was about 20-30 days before sampling, which was consistent with the known time of drug intake. This MALDI-MS method allows imaging analysis of trace substances in a single hair and can realize the intuitive reflection of drug taking time. In addition, zolpidem applied by soaking was mainly distributed on both sides of the longitudinal hair shaft, whereas ingested zolpidem was found only in the middle of the hair shaft of the DFSA victim. The MALDI-MS images of unwashed and washed hair suggested that the amount of externally applied drug was decreased by washing, it was still present on surface layer (cuticle) sides although. Visualization using MALDI-MSI could therefore distinguish between drug ingestion and contamination by reflecting the distribution and deposition site of the drug in hair.

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.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique.

OBJECTIVES: To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments. METHODS: Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode. RESULTS: The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm. CONCLUSIONS: The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Micro-segmental analysis of the entry pathway and distribution of zolpidem in hair from different scalp regions after a single dose.

Introduction: Hair testing is well established for the assessment of past drug exposure; however, more research is needed to understand drug incorporation mechanisms and drug entry pathways into hair. Method: In this study, a micro-segmental LC-MS/MS method was used to analyze a 0.4 mm segment of hair after a single oral administration of zolpidem. Five single hairs were plucked at 1 day, 3 days, 7 days, and 28 days after administration from the vertex posterior of three subjects, and 5 single hairs were also plucked from the parietal, left temporal, and right temporal regions of the head at 28 days. Results and discussion: Proximal S1 (0-0.4 mm) in hair plucked at 1 day had the highest level of zolpidem at 1.5-2.4 pg/mm; much lower concentrations (< 1 pg/mm) were detected at proximal S2-S8 (0.4-3.2 mm). The drug concentration decreased gradually in S1 for 7 days after drug intake and disappeared by 28 days, suggesting that the drug from the bloodstream initially combined with the hair follicle and then gradually moved to the hair tip as the hair grew. The zolpidem concentration-hair segment profiles exhibited a large peak (root side) and a small peak (tip side) for the four sampling times in all three subjects, indicating that drug incorporation in the hair bulb occurred mainly from the blood but probably also entered the hair through sweat and sebum. Zolpidem was also detected in all hairs from the vertex posterior in all three subjects but was not detected in 1 hair from the parietal region and 2 hairs from the left temporal region. The consistency in drug detection, drug concentration level, and peak position was better in hair from the vertex posterior than from the other three regions, indicating that the vertex posterior is a suitable sampling region for estimating drug intake.

Determination of 5 synthetic cannabinoids in hair by Segmental analysis using UHPLC-MS/MS and its application to eight polydrug abuse cases.

In recent years, an increasing number of new synthetic cannabinoids (SCs) have appeared in the drug trade market. A UPLC-MS/MS method was developed to simultaneously identify five synthetic cannabinoids in 1 cm segment hair samples. The method was fully validated and confirmed to have good selectivity, accuracy, and precision, as well as satisfactory linearity within the calibrated range. The limit of quantification (LOD) was 0.5 pg/mg, and the lower limit of quantitation (LLOQ) was 1 pg/mg, with intraday and interday accuracies (bias) ranging from - 9.6-13.7%. The validated method was successfully used for qualitative and quantitative analysis of five SCs in authentic hair samples of eight SC abusers. SCs were detected in 8 cases at concentrations ranging from 1.5 to 632.9 pg/mg.

LC-MS-MS Determination of 88 Psychotropic Drugs in 1,865 Hair Samples from Addicts in Drug Abstinence.

The emergence of novel drugs and the continuous expansion of the scope of the types of drugs under control have greatly increased requests for screening of a range of drugs in hair. Here, a multi-analyte method for the detection and quantification of 88 psychotropic drugs in the hair of addicts in drug abstinence was developed and fully validated using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples (25 mg) were washed, cut into pieces, cryogenically ground and extracted in methanol. The extracted analytes were separated on an Allure PFP Propyl column (100 × 2.1 mm, 5 mm inside diameter, Restek, USA) and analyzed by LC-MS-MS in multiple reaction monitoring modes. The limits of detection and the limits of quantification ranged from 0.1 to 20 pg/mg and 0.2 to 50 pg/mg, respectively. The intra- and inter-assay precisions (relative standard deviation (RSD)) of all analyses ranged from 0.9% to 14.9% and 1.9% to 15.9%, respectively. Accuracy values were 100 ± 20%. The extraction recovery of quality control samples ranged from 50.9% to 99.6% for all analytes. The matrix effects for all analytes ranged from 46.8% to 99.7%. The method was successfully used to analyze 1,865 hair samples from addicts in drug rehabilitation at their own communities. Among the samples, 129 cases were positive; the majority of positive cases were from males (78.29%), 92.25% of whom were >35 years old. Traditional drugs, like methamphetamine and opioids, accounted for most positive cases, and 27 of the abstinence cases with a use history of methamphetamine were still positive. In addition to abused drugs, like methamphetamine, morphine and cocaine, the sedative-hypnotic and psychotherapeutic drugs, including clonazepam, alprazolam, estazolam, zolpidem and quetiapine, were detected in 26% of the hair samples, suggesting that these addicts may have insomnia and mental problems such as depression and psychosis, probably due to the long-term effects of drugs and withdrawal reactions. Three synthetic cannabinoids were also detected in four (2.7%) cases. A total of 37 cases were positive for methadone, tramadol and dextromethorphan, reflecting a new trend of alternative drug use when traditional drugs were not easy to obtain during the coronavirus disease 2019 outbreak.

Two DFSA cases involving midazolam clarified by the micro-segmental hair analyses.

PURPOSE: In this study, an analytical procedure to identify trace amounts of drug in hair based on micro-segmental hair analysis was presented. The method also can be used to estimate the time of drug ingestion at daily precision by cutting a single hair into sub-millimeter segments which correspond to daily hair growth. METHODS: A method was established for efficient extraction of midazolam, one of the most frequently detected compound in drug-facilitated sexual assault (DFSA) cases, from each 0.4-mm hair segment and validated by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Moreover, two DFSA cases were used to compare the micro-segmental hair analysis with the 1- cm segmental analysis method. RESULTS: The validation showed a lower limit of quantification of 0.5 pg/mm for midazolam, with intraday and interday accuracies (bias) from - 5.2 to 0.9%. The micro-segmental hair analysis method was applied to proximal 1-cm hair segment including hair bulbs in two DFSA cases. The micro-segmental hair analysis results in case 1 showed midazolam in the S15-S17 (5.6-6.8 mm from hair bulb) in a concentration range from 0.5 to 0.9 pg/mm, and the concentrations of midazolam in all hair micro-segments (0-1 cm from the scalp) in case 2 were from 0.5 to 2.0 pg/mm. CONCLUSIONS: Comparison with the conventional method revealed that micro-segmental hair analysis may enhance the utility of hair drug testing and strengthen probative force in DFSA cases.

Time course of estazolam in single-strand hair based on micro-segmental analysis after controlled oral administration.

The mechanism of estazolam incorporation into hair was investigated by studying the time course of estazolam along single-strand hair after two oral administration of estazolam at 28 days interval. Estazolam in single hair segments 0.4 mm in length was verified and quantified by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The distributions of estazolam within a strand of hair (collected at 12 h, 28 days, and 56 days post-administration) were visualized by micro-segmental analysis. The highest estazolam concentration (1.5-9.9 pg/mm) was detected in the hair bulb region (S1), and it then decreased through the hair shaft to the distal end, with a small fluctuation (0.3-3 pg/mm) near the junction of the hair roots and shafts (S4-S7) 12 h after drug intake. These findings suggested that the incorporation of estazolam occurred in two regions, mainly in the hair bulb and to a lesser extent in the upper dermis zone. Models using internal temporal markers (TIMs) and temporal intervals (TIs) were constructed to estimate the day of estazolam ingestion. The estimation accuracy was within an average error of 1.7 mm and 3.0 mm between the calculated and actual positions, based on the TIMs and TIs 56 days after estazolam intake. These findings can help in further elucidation of the drug incorporation mechanism, which is crucial for interpreting hair analysis results used to reveal individual drug-use history.

Metabolism of 4F-MDMB-BICA in zebrafish by liquid chromatography-high resolution mass spectrometry.

In this study, in vivo metabolic studies of the synthetic cannabinoid 4F-MDMB-BICA were investigated using zebrafish models. The metabolites were identified and structurally illustrated by liquid chromatography-high resolution mass spectrometry. Fourteen phase-I metabolites and four phase-II metabolites were generated from zebrafish. The main metabolic pathways of the phase-I metabolism included N-dealkylation, N-dealkylation combined with hydroxylation, amide hydrolysis, oxidative defluorination, oxidative defluorination to butyric acid, acetic acid formation at the indole side chain, hydroxylation, ester hydrolysis followed by hydroxylation, dehydrogenation, dehydrogenation, and N-dealkylation, and oxidative defluorination subsequently combined with dehydrogenation. The main biotransformations of the phase-II metabolism were glucuronidation and sulfation. Two phase-I metabolites (A1 and A11) and four phase-II metabolites (A2, A3, A4, and A12) were reported for the first time. A14, which was confirmed in human biological samples, was detected only in zebrafish samples but not found in human liver microsome incubation study. The current study indicates that the zebrafish model is a promising tool for elucidating the metabolism of NPS in the future.

Rapid Identification of MDMB-CHMINACA Metabolites Using Zebrafish and Human Liver Microsomes as the Biotransformation System by LC-QE-HF-MS.

MDMB-CHMINACA is a newly synthetic cannabinoid, which scoped in NMS Lab, USA. Since there are currently no published data on MDMB-CHMINACA metabolism, we aimed to identify its biotransformation pathways and major metabolites. Liquid chromatography Q-exactive HF hybrid quadrupole-orbitrap mass spectrometry (LC-QE-HF-MS) using full scan positive ion mode and targeted MS-MS (ddms2) techniques with accurate mass measurement were employed to analyze the metabolic sites and pathways. An in vivo metabolic animal model of zebrafish was established to verify the metabolic pathways of MDMB-CHMINACA obtained from human liver microsomal experiment in vitro. The results showed that 29 metabolites were generated in the zebrafish animal model and human liver microsomes model. Biotransformations mainly occurred at the cyclohexylmethyl tail of the compound, minor reactions also occurred at the tert-butyl chain and no reaction was analyzed at the indazole ring. We recommend M1 group (MDMB-CHMINACA ester hydroxylation) and M2 group (MDMB-CHMINACA monohydroxylation) as the potential poisoning markers to document MDMB-CHMINACA intake in clinical and forensic cases. Additionally, this study provides preliminary information regarding the metabolism of MDMB-CHMINACA that will guide analytical standard manufacturers to better provide suitable references for further studies on newly encountered designer drugs.

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.