Software-assisted automated detection and identification of "unknown" analogues of benzodiazepines in liquid chromatography mass spectrometry analysis.

RATIONALE: Benzodiazepines (BZDs) construct a large group of psychoactive drugs acting as depressants of the central nervous system (CNS) and used in medicine as sedatives and anxiolytic and antiepileptic agents. The illicit use of these materials is a worldwide problem, and for many years, part of the benzodiazepines have been abused as rape drugs. For example, flunitrazepam (Rohypnol) is most commonly linked by media reports to drug-facilitated sexual assaults, more commonly referred to as "date rape." Furthermore, there are growing concerns for other misuses of these drugs. Over the last few years, there was an increase in the number, type, and availability of new psychoactive substances (NPS) belonging to the benzodiazepine group, challenging standard forensic labs to fully identify the chemical structure of new, unknown benzodiazepines. METHODS: This work demonstrates a new application of the automated tool for the detection and identification of benzodiazepine analogues using high-resolution-accurate-mass LC-MS analysis, followed by "Compound Discoverer" (CD) software data processing, to automatically detect various benzodiazepine analogues by picking peaks and compare them to in silico calculated modifications made on a predefined basic backbone. Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component. RESULTS: This method was found to be useful for the automated detection and putative identification of a series of nine "unknown" benzodiazepine analogues, at concentrations in the low ng/mL range. CONCLUSIONS: We hereby present a general demonstration of this powerful tool for the forensic community in the detection and identification of hazardous unknown compounds.

Probe Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantification of Benzodiazepines.

BACKGROUND: Legally prescribed benzodiazepines (BZDs) and designer BZDs are widely misused and must be determined in multiple contexts (eg, overdose, drug-facilitated sexual assaults, or driving under the influence of drugs). This study aimed to develop a method for measuring serum BZD levels using probe electrospray ionization (PESI) mass spectrometry and an isotope dilution approach. METHODS: A tandem mass spectrometer equipped with a probe electrospray ionization source in multiple reaction monitoring mode was used. Isotope dilution was applied for quantification using a deuterated internal standard at a fixed concentration for alprazolam, bromazepam, diazepam, nordiazepam, oxazepam, temazepam, zolpidem, and zopiclone. This method included designer BZDs: clonazolam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, nifoxipam, and pyrazolam. Sample preparation was done by mixing 10 µL of serum with 500 µL of an ethanol/ammonium formate 0.01 mol/L buffer. Complete validation was performed, and the method was compared with liquid chromatography coupled with mass spectrometry (LC-MS/MS) and immunoassays (IC) by analyzing 40 real samples. RESULTS: The analysis time for identification and quantification of the 18 molecules was 2.5 minutes. This method was fully validated, and the limits of quantification varied from 5 to 50 mcg/L depending on the molecule. In the 40 real samples, 100% of molecules (n = 89) were detected by both LC-MS/MS and PESI-MS/MS, and regression analysis showed excellent agreement between the 2 methods (r 2 = 0.98). On IC, bromazepam and zolpidem were not detected in 2 and 1 cases, respectively. CONCLUSIONS: PESI-MS/MS allows serum BZD detection and measurement. Given the isotope dilution approach, a calibration curve was not required, and its performance was similar to that of LC-MS/MS, and its specificity was higher than that of IC.

Green nanostructured liquids for the analysis of urine in drug-facilitated sexual assault cases.

In this work, we optimize and validate a simple, time-saving, and environmentally friendly sample preparation method based on supramolecular solvents (SUPRAS), green nanostructured liquids, for the extraction of selected drug-facilitated sexual assault (DFSA) substances from human urine. The methodology was fast and simple (stirring, centrifugation, and dilution). Cubosomic SUPRAS were formed by the addition of 1,2-hexanediol (200 µL) to 1.0 mL of human urine containing 1 M Na2SO4. SUPRAS extracts were analyzed by LC-MS/MS. The method was fully validated for 23 DFSA compounds including 10 benzodiazepines, 1 z-hypnotic drug, 5 amphetamine derivatives, 3 cocaine metabolites, and 4 miscellaneous compounds. Extraction efficiency varied between 79 and 119%, and matrix effects were acceptable (-14.3/+21.5) for 87% of the compounds. Method detection and quantification limits ranged from 0.003 to 0.75 ng/mL and from 0.01 to 2.50 ng/mL, respectively. These values were low enough for the established minimum required performance limits (MRPL) of these substances. This simple and green method has a great potential to be implemented for the monitoring of illegal drugs involved in DFSA cases by forensic laboratories.

Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS.

OBJECTIVES: To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring. RESULTS: The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 µg/mL and 252.14 ng/mL, respectively. CONCLUSIONS: This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

Impact of Heat Inactivation of Blood Samples on Therapeutic Drug Monitoring of 5 Second-Generation Antipsychotics and Their Metabolites.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 outbreak has been classified as a pandemic. Because many coronaviruses are heat sensitive, heat inactivation of patient samples at 56°C before testing reduces the risk of transmission. The aim of this study is to assess the impact of heat inactivation of patient blood samples on plasma concentrations of 5 second-generation antipsychotics and their metabolites. METHODS: Blood samples were collected during routine clinical therapeutic drug monitoring examination between April 3, 2021, and April 19, 2021. Samples were divided into 2 groups: group A, noninactivated raw sample, and group B, inactivated samples. Inactivation was performed by a 30-minute incubation at 56°C. The levels of the 5 drugs and their metabolites before and after sample heat inactivation were measured using liquid chromatography-tandem mass spectrometry and compared. Furthermore, correlation and Bland-Altman analyses were conducted. RESULTS: No statistically significant difference was observed between the levels of the 5 drugs and their metabolites (ie, risperidone, 9-OH-risperidone, aripiprazole, dehydroaripiprazole, olanzapine, quetiapine, norquetiapine, clozapine, and norclozapine) in the noninactivated group A and the inactivated group B ( P > 0.05). Each drug's concentration values in inactivated and noninactivated treatments correlated (Spearman rs > 0.98; P < 0.001). The results of the noninactivated treatment methods and samples alone showed good consistency via Bland-Altman analysis. CONCLUSIONS: Blood sample heat inactivation had no significant effect on the therapeutic drug monitoring of 5 second-generation antipsychotics and their metabolites. This inactivated treatment method should be recommended to effectively protect laboratory staff from virus contamination.

Fully Automated Synthesis of Novel TSPO PET Imaging Ligand [18F]Fluoroethyltemazepam.

INTRODUCTION: Benzodiazepines, including temazepam are described as TSPO antagonists. In fact, TSPO was initially described as a peripheral benzodiazepine receptor (PBR) with a secondary binding site for diazepam. TSPO is a potential imaging target of neuroinflammation because there is an amplification of the expression of this receptor. OBJECTIVES: Herein, we developed a novel fluorinated benzodiazepine ligand, [18F]Fluoroethyltemazepam ([18F]F-FETEM), for positron emission tomography (PET) imaging of translocator protein (18 kDa). METHODS: [18F]F-FETEM was radiolabelled with an automated synthesizer via a one-pot procedure. We conducted a [18F]F-aliphatic nucleophilic substitution of a tosylated precursor followed by purification on C18 and Alumina N SPE cartridges. Quality control tests was also carried out. RESULTS: We obtained 2.0-3.0% decay-uncorrected radiochemical activity yield (3.7% decay-corrected) within the whole synthesis time about 33 min. The radiochemical purity of [18F]F-FETEM was over 90% by TLC analysis. CONCLUSIONS: This automated procedure may be used as basis for future production of [18F]F-FETEM for preclinical PET imaging studies.

Pitfalls of toxicological investigations in hair, bones, and nails in extensively decomposed bodies: illustration with two cases.

It is difficult to carry out toxicological investigations in biological samples collected from extensively decomposed bodies and to interpret obtained results as several pitfalls should be considered: redistribution phenomena, degradation of xenobiotics during the postmortem period, contamination by putrefaction fluids, and external contamination. This work aims to present two cases in order to illustrate and discuss these difficulties in this tricky situation. Case#1: the body of a 30-year-old woman was found in a wooded area (1 month after she has been reported missing by her family): hair and a femur section were sampled. Case#2: the decomposed corpse of a 52-year-old man was found in a ditch: hair and nails were sampled. After decontamination steps, toxicological investigations were performed using liquid chromatography with high-resolution mass spectrometry and tandem mass spectrometry detection methods. In case#1, the same drugs or metabolites (benzodiazepines, propranolol, tramadol, acetaminophen, paroxetine, and oxetorone) were detected in hair and in bone specimens. This result combination strongly suggests intakes close to the time of death for three of them (oxazepam, lormetazepam, and propranolol). In case#2, results of toxicological investigations in hair and nails [(hair/nail concentration in ng/mg) nordiazepam (1.12/1.06), oxazepam (0.113/0.042), zolpidem (0.211/< 0.01), hydroxyzine (0.362/< 0.01), and cetirizine (0.872/1.110)] were both consistent with several drug intakes but were not contributory to cause of death determination. In case of positive toxicological results in biological samples collected from extensively decomposed bodies (such as hair, bones, or nails), it is challenging to determine the time, and even more, the level of the dose of exposure(s).

An LC/MS based method to quantify DNA adduct in tumor and organ tissues.

Highly potent DNA damaging agents have become a key class of toxins for antibody-drug conjugate (ADC) based targeted therapy. However, until recently, no quantitative bioanalytical method was available to measure the toxin in the form of DNA adducts. In this work, a novel microwave assisted organic solvent extraction and LC-MS/MS based bioanalytical method was developed to extract and quantify DNA-bound toxin IGN-P1 in tissue samples. Using ADC-1 as the model ADC, the method was orthogonally checked with a radioactive method for the recovery of free toxins from DNA adducts in biological matrices. It was found that the bioanalytical method can achieve a high recovery of the IGN-P1 toxin from DNA adducts. In further assessment, tumor and organ tissue samples collected at multiple time points from in vivo studies after dosing with two other ADCs, ADC-2 and ADC-3, were measured by the method. Given the generic nature of the established bioanalytical method without the need of radiolabels, the methodology could be broadly utilized to quantitatively assess the relationship between DNA adduct levels and pharmacological/toxicological effects.

A scalable platform to identify fungal secondary metabolites and their gene clusters.

The genomes of filamentous fungi contain up to 90 biosynthetic gene clusters (BGCs) encoding diverse secondary metabolites-an enormous reservoir of untapped chemical potential. However, the recalcitrant genetics, cryptic expression, and unculturability of these fungi prevent scientists from systematically exploiting these gene clusters and harvesting their products. As heterologous expression of fungal BGCs is largely limited to the expression of single or partial clusters, we established a scalable process for the expression of large numbers of full-length gene clusters, called FAC-MS. Using fungal artificial chromosomes (FACs) and metabolomic scoring (MS), we screened 56 secondary metabolite BGCs from diverse fungal species for expression in Aspergillus nidulans. We discovered 15 new metabolites and assigned them with confidence to their BGCs. Using the FAC-MS platform, we extensively characterized a new macrolactone, valactamide A, and its hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS). The ability to regularize access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Changes in Opioid-Involved Overdose Deaths by Opioid Type and Presence of Benzodiazepines, Cocaine, and Methamphetamine - 25 States, July-December 2017 to January-June 2018.

From 2013 to 2017, the number of opioid-involved overdose deaths (opioid deaths) in the United States increased 90%, from 25,052 to 47,600.* This increase was primarily driven by substantial increases in deaths involving illicitly manufactured fentanyl (IMF) or fentanyl analogs† mixed with heroin, sold as heroin, or pressed into counterfeit prescription pills (1-3). Methamphetamine-involved and cocaine-involved deaths that co-involved opioids also substantially increased from 2016 to 2017 (4). Provisional 2018§ estimates of the number of opioid deaths suggest a small decrease from 2017. Investigating the extent to which decreases occurred broadly or were limited to a subset of opioid types (e.g., prescription opioids versus IMF) and drug combinations (e.g., IMF co-involving cocaine) can assist in targeting of intervention efforts. This report describes opioid deaths during January-June 2018 and changes from July-December 2017 in 25¶ of 32 states and the District of Columbia participating in CDC's State Unintentional Drug Overdose Reporting System (SUDORS).** Opioid deaths were analyzed by involvement (opioid determined by medical examiner or coroner to contribute to overdose death) of prescription or illicit opioids,†† as well as by the presence (detection of the drug in decedent) of co-occurring nonopioid drugs (cocaine, methamphetamine, and benzodiazepines). Three key findings emerged regarding changes in opioid deaths from July-December 2017 to January-June 2018. First, overall opioid deaths decreased 4.6%. Second, decreases occurred in prescription opioid deaths without co-involved illicit opioids and deaths involving non-IMF illicit synthetic opioids (fentanyl analogs and U-series drugs) (5). Third, IMF deaths, especially those with multiple illicit opioids and common nonopioids, increased. Consequently, IMF was involved in approximately two-thirds of opioid deaths during January-June 2018. Notably, during January-June 2018, 62.6% of all opioid deaths co-occurred with at least one common nonopioid drug. To maintain and accelerate reductions in opioid deaths, efforts to prevent IMF-involved deaths and address polysubstance misuse with opioids must be enhanced. Key interventions include broadening outreach to groups at high risk for IMF or fentanyl analog exposure and overdose. Improving linkage to and engagement in risk-reduction services and evidence-based treatment for persons with opioid and other substance use disorders with attention to polysubstance use or misuse is also needed.

Tramadol, Methadone and Benzodiazepines added to Alcoholic Beverages.

AIMS: To search for pharmaceutical additives in illicit alcoholic beverages referred to the laboratory of Legal Medicine Organization in Iran in 2017. METHODS: Hundred beverages were sampled. Ethanol content was determined by gas chromatography with flame ionization detection (GC-FID) and then a liquid-liquid extraction combined with reversed-phase high performance liquid chromatography equipped with a photodiode array detector (PAD) was employed for the qualitative analysis. The analysis was confirmed using gas chromatography coupled with mass spectroscopy (GC/MS). RESULTS: In 15% either one or more of the following were detected: tramadol, methadone, diazepam, oxazepam, flurazepam and alprazolam. Tramadol was found with highest frequency. CONCLUSIONS: The wide availability of addictive pharmaceutical is leading to fortification of alcoholic beverages on some countries. The addition of such depressant additives should be better known because of the potentially fatal consequences of the combination with ethanol, as well as the potential for adverse effects on behavior.

Simultaneous determination of 75 abuse drugs including amphetamines, benzodiazepines, cocaine, opioids, piperazines, zolpidem and metabolites in human hair samples using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 75 abuse drugs and metabolites, including 19 benzodiazepines, 19 amphetamines, two opiates, eight opioids, cocaine, lysergic acid diethylamide, zolpidem, three piperazines and 21 metabolites in human hair samples, was developed and validated. Ten-milligram hair samples were decontaminated, pulverized using a ball mill, extracted with 1 mL of methanol spiked with 28 deuterated internal standards in an ultrasonic bath for 60 min at 50°C, and purified with Q-sep dispersive solid-phase extraction tubes. The purified extracts were evaporated to dryness and the residue was dissolved in 0.1 mL of 10% methanol. The 75 analytes were analyzed on an Acquity HSS T3 column using gradient elution of methanol and 0.1% formic acid and quantified in multiple reaction monitoring mode with positive electrospray ionization. Calibration curves were linear (r ≥ 0.9951) from the lower limit of quantitation (2-200 pg/mg depending on the drug) to 2000 pg/mg. The coefficients of variation and accuracy for intra- and inter-assay analysis at three QC levels were 4.3-12.9% and 89.2-109.1%, respectively. The overall mean recovery ranged from 87.1 to 105.3%. This method was successfully applied to the analysis of 11 forensic hair samples obtained from drug abusers.

[METHOD FOR QUANTITATIVE DETERMINATION OF PHENAZEPAM IN MIXED SALIVA].

Objective - to develop a method for the quantitative determination of the drug phenazepam in mixed saliva. For the study, samples were obtained mixed saliva in 6 patients in inpatient treatment in a psychiatric hospital. In order to correct the development of the underlying disease, patients took the drug - phenazepam. For comparison, samples of mixed saliva of healthy volunteers (n=6) who did not take medications were studied. A technique has been developed for the quantitative determination of the drug phenazepam in mixed saliva on a Sciex 4500 triple quadrupole liquid chromatography-mass spectrometer with ionization, separation and detection of samples. Allows you to determine the concentration of phenazepam in mixed saliva with an accuracy of from 102 to 109% and can be used to control the removal and dosing of drugs.

Enantiomeric quality control of R-Tofisopam by HPLC using polysaccharide-type chiral stationary phases in polar organic mode.

A novel, fast and economic chiral HPLC method was developed and validated for the resolution of the four isomers of tofisopam. The separation capacity of eleven different chiral columns: six polysaccharide-type including three amylose-based (Chiralpak AD, Chiralpak AD-RH and Chiralpak AS) and three cellulose-based (Chiralcel OD, Chiralcel OJ and Lux Cellulose-4); three cyclodextrin- (Quest-BC, Quest-C2 and Quest-CM) and two macrocyclic glycopeptide antibiotic-type (Chirobiotic T and Chirobiotic TAG) were screened using polar organic or reversed-phase mode. Chiralpak AD, based on amylose tris(3,5-dimethylphenylcarbamate) as chiral selector with neat methanol was identified as the most promising system. In order to improve resolution, an orthogonal experimental design was employed, altering the concentration of 2-propanol, column temperature, and flow rate in a multivariate manner. Using the optimized method (85/15 v/v methanol/2-propanol, 40°C, flow rate: 0.7 mL/min) we were not only able to separate the four isomers but also detect 0.1% S-enantiomer as chiral impurity in R-tofisopam. This is important since the latter is under development as a single enantiomeric agent. Thermodynamic investigation revealed an unusual entropy and enthalpy-entropy co-driven controlled enantioseparation on Chiralcel OJ and on Chiralpak AD column, respectively. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested on a pharmaceutical formulation containing the racemic mixture of the drug. As a further novelty, a separate circular dichroism method was applied for the investigation of the interconversion kinetics of tofisopam conformers, which proved to be crucial for sample preparation and method validation.

Molecularly imprinted polymer based microtiter chemiluminescence array for determination of phenothiazines and benzodiazepines in pork.

In this study, a molecularly imprinted polymer based chemiluminescence array capable of simultaneous determining phenothiazines and benzodiazepines was first reported. Two polymers were coated in different wells of the conventional 96-well microtiter plate as the recognition reagents, and the added analytes competed with a horseradish peroxidase-labeled bi-hapten conjugate to bind the recognition reagents. The light signal was induced by using a highly effective luminol-H2O2-IMP system. The assay procedure consisted of only one sample-loading step prior to data acquisition. Then, the array was used to determine 4 phenothiazines and 5 benzodiazepines in pork simultaneously. The limits of detection for the 9 drugs were in a range of 0.001-0.01 ng/mL, and the recoveries from the fortified blank pork were in a range of 63.5%-94.1%. Furthermore, the array could be reused for 8 times. The detection results for some real pork samples were consistent with an ultra performance liquid chromatography method.

Quantitative structure -retention relationship modeling of selected antipsychotics and their impurities in green liquid chromatography using cyclodextrin mobile phases.

Applying green chromatography methods is currently one of the challenges in liquid chromatography. Among different strategies, using cyclodextrin (CD) mobile phase modifiers was applied in this paper. CDs can form inclusion complexes with a wide variety of hydrophobic organic compounds and, consequently, affect their retention behavior. CD-containing mobile phases possess complicated complexation and adsorption equilibria so retention is dependent not only on chromatographic parameters and properties of the compound but also on properties of compound-CD complex. Docking study was used to calculate association constants of the selected antipsychotics (risperidone, olanzapine, and their impurities) and ß-CD complexes and predict which part of the molecule structure will most likely incorporate into the ß-CD cavity. Quantitative structure-retention relationship model (QSRR) for selected model substances was built employing artificial neural network (ANN) technique. Reliable QSRR model was obtained using molecular descriptors, complex association constants, and chromatographic factors. The multilayer perceptron network with 11-8-1 topology, trained with back propagation algorithm, showed the best performance. Root mean square error for training, validation, and test was 0.2954, 0.3633, and 0.4864, respectively. The correlation coefficient (R2) between experimentally obtained retention factor values [k(exp)] and values computed or predicted by ANN [k(ANN)] was 0.9962 for training, 0.9927 for validation, and 0.9829 for test, indicating good predictive ability of the model. The optimized network was used for development of green chromatography method for separation of risperidone and its related impurities, as well as olanzapine and its related impurities in a relatively short run time and with low consumption of organic modifier. The developed methods were validated in accordance with ICH Q2 (R1) quideline and all parameters fulfilled the defined criteria. The greenness of the proposed methods has also been demonstrated. Graphical Abstract Complex association constants as inputs of QSRR model in ß-cyclodextrin modified HPLC system and development of green chromatography methods.

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS.

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL-1 and 3.2 to 160 ng mL-1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and - 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.

[Identification of New Designer Benzodiazepine Diclazepam in Drug Facilitated Sexual Assault].

OBJECTIVES: To identify the new designer drugs which are totally unknown and not in the routine testing list by the technologies such as high-resolution mass spectrometry in drug facilitated sexual assault, in order to solve the problem in actual cases. METHODS: The milky fluid from an actual case was extracted and analyzed using LC-QE, ¹H-NMR and GC-MS, respectively. The accurate masses and cluster ions isotope patterns of unknown compound were obtained by LC-QE. The molecular formula was confirmed as C16H12C2N2O based on the protons number of ¹H-NMR. The isomers diclazepam and 4-chlorodiazepam were separated and detected with GC-MS. RESULTS: The new designer benzodiazepine as diclazepam in the milky fluid was identified. The results provided direct evidence for the investigation and qualitative analysis of such cases. CONCLUSIONS: The combined application of various methods, including LC-QE, ¹H-NMR and GC-MS, can be used to detect unknown new psychoactive substances.

Metabolic profile of oxazepam and related benzodiazepines: clinical and forensic aspects.

Anxiolytic drugs, namely benzodiazepines, are the most commonly used psychoactive substances since anxiety disorders are prevalent mental disorders particularly in the Western world. Oxazepam is a short-acting benzodiazepine and one of the most frequently prescribed anxiolytic drugs. It is also the active metabolite of a wide range of other benzodiazepines, such as diazepam, ketazolam, temazepam, chlordiazepoxide, demoxazepam, halazepam, medazepam, prazepam, pinazepam, and chlorazepate. Therefore, relevant clinical and forensic outocomes may arise, namely those related to interference in driving performance. It is clinically available as a racemic formulation, with S-enantiomer being more active than R-enantiomer. In humans, it is mainly polimorphically metabolized by glucuronide conjugation at the 3-carbon hydroxyl group, yielding stable diastereomeric glucuronides (R- and S-oxazepam glucuronide). Relevant metabolic and stereoselective interspecies differences have been reported. In this work, the pharmacokinetics of oxazepam with particular focus on metabolic pathways is fully reviewed. Moreover, the metabolic profile of other prescribed benzodiazepines that produce oxazepam as a metabolite is also discussed. It is aimed that knowing the metabolism of oxazepam and related benzodiazepines may lead to the development of new analytical strategies for its early detection and help in further toxicological and clinical interpretations.

Characterization and identification of eight designer benzodiazepine metabolites by incubation with human liver microsomes and analysis by a triple quadrupole mass spectrometer.

Designer benzodiazepines (DBZDs) have become of particular importance in the past few years. The metabolite monitoring of DBZD in biological fluids could be of great interest in clinical and forensic toxicology. However, DBZD metabolites are not known or not commercially available. The identification of some DBZD metabolites has been mostly explored by self-administration studies or by in vitro studies followed by high-resolution mass spectrometry. The question arose whether a unit resolution instrument could be efficient enough to allow the identification of DBZD metabolites. In this study, we used an in vitro experiment where eight DBZDs (diclazepam, flubromazepam, etizolam, deschloroetizolam, flubromazolam, nifoxipam, meclonazepam and clonazolam) were incubated with human liver microsomes (HLMs) and metabolite identification was carried out by using a UHPLC coupled to a QTRAP triple quadrupole linear iontrap tandem mass spectrometer system. Post-mortem samples obtained from a real poisoning case, involving deschloroetizolam and diclazepam, were also analysed and discussed. Our study using HLM allowed the identification of 26 metabolites of the 8 DBZDs. These were denitro-, mono- or di-hydroxylated and desmethyl metabolites. In the forensic case, diclazepam was not detected whereas its metabolites (lormetazepam and lorazepam) were present at high concentrations in urine. We also identified hydroxy-deschloroetizolam in urine, while the parent compound was not detected in this matrix. This supports the approach that LC coupled to a simple QTRAP could be used by laboratories to identify other not-known/not-commercialized new psychoactive substance (NPS) metabolites.