Rapid and simple multi-analyte LC-MS/MS method for the determination of benzodiazepines and Z-hypnotic drugs in blood samples: Development, validation and application based on three years of toxicological analyses.

Benzodiazepines (BZDs) and Z-drugs have been particularly important treatments for sleeping and anxiety disorders for many years. However, recently, a number of new benzodiazepines (named designer benzodiazepines, DBZDs) were synthesised, but some of them have never been used in the clinic; they reached the black drug market as new psychoactive substances and are used for recreational purposes. The abuse of these substances has led to many crimes and even deaths. Therefore, it is necessary to develop new methods for their quantification for forensic and clinical toxicology. A liquid chromatography-tandem mass spectrometry-based method was developed for the simultaneous determination of 20 classical BZDS, 4 DBZDs and 3 Z-hypnotic drugs in human whole blood. As a sample preparation step, liquid-liquid extraction requiring the use of only 0.5 mL of blood sample and 1 mL of extraction solvent was applied. The selectivity, linearity, carry-over effects, limits of detection (LOD) and quantification (LOQ), precision, accuracy (both intra- and inter-day assays) and recovery were evaluated for validation. Calibration curves were linear with r values > 0.98. The LODs ranged from 0.01 to 0.33, and the LOQs were assumed to be 1 ng/mL. Inter-day precisions and accuracies were in the ranges of 87.8% - 108.5% and 1.8% - 11.2%, respectively. The recovery values ranged from 81.0% to 106.7%. The developed method proved to be sensitive, specific, simple, and fast and can be quickly modified and expanded for new compounds by the optimization of MRM. The method was applied for analysis of blood samples in 145 toxicological cases over a three-year study (2017 - 2019), which allowed us to obtain information on the prevalence of the use of these substances. The most frequently determined compounds were nordazepam (87 cases; 60%), diazepam (81 cases; 55.9%), temazepam (72 cases; 49.7%), oxazepam (56 cases; 38.7%), and midazolam (36 cases; 24.8%). The ranges of concentrations were wide and are presented as box plots. The results were used for the preparation of medico-legal opinions, which proved the utility of the method for routine toxicology analyses.

Banaszkiewicz L ，Woźniak MK ，Kata M ，Domagalska E ，Wiergowski M ，Szpiech B ，Kot-Wasik A ... -  《-》

Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography-tandem mass spectrometry.

Benzodiazepines and zolpidem are generally prescribed as sedative, hypnotics, anxiolytics or anticonvulsants. These drugs, however, are frequently misused in drug-facilitated crime. Therefore, a rapid and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for identification and quantification of benzodiazepines, zolpidem and their metabolites in urine using deuterium labeled internal standards (IS). Urine samples (120 μL) mixed with 80 μL of the IS solution were centrifuged. An aliquot (5 μL) of the sample solution was directly injected into the LC-MS/MS system for analysis. The mobile phases consisted of water and acetonitrile containing 2mM ammonium trifluoroacetate and 0.2% acetic acid. The analytical column was a Zorbax SB-C18 (100 mm × 2.1 mm i.d., 3.5 μm, Agilent). The separation and detection of 18 analytes were achieved within 10 min. Calibration curves were linear over the concentration ranges of 0.5-20 ng/mL (zolpidem), 1.0-40 ng/mL (flurazepam and temazepam), 2.5-100 ng/mL (7-aminoclonazepam, 1-hydroxymidazolam, midazolam, flunitrazepam and alprazolam), 5.0-200 ng/mL (zolpidem phenyl-4-carboxylic acid, α-hydroxyalprazolam, oxazepam, nordiazepam, triazolam, diazepam and α-hydroxytriazolam), 10-400 ng/mL (lorazepam and desalkylflurazepam) and 10-100 ng/mL (N-desmethylflunitrazepam) with the coefficients of determination (r(2)) above 0.9971. The dilution integrity of the analytes was examined for supplementation of short linear range. Dilution precision and accuracy were tested using two, four and ten-folds dilutions and they ranged from 3.7 to 14.4% and -12.8 to 12.5%, respectively. The process efficiency for this method was 63.0-104.6%. Intra- and inter-day precisions were less than 11.8% and 9.1%, while intra- and inter-day accuracies were less than -10.0 to 8.2%, respectively. The lower limits of quantification were lower than 10 ng/mL for each analyte. The applicability of the developed method was successfully verified with human urine samples from drug users (n=21). Direct urine sample injection and optimized mobile phases were introduced for simple sample preparation and high-sensitivity with the desired separation.

Jeong YD ，Kim MK ，Suh SI ，In MK ，Kim JY ，Paeng KJ ... -  《-》

An analytical strategy for designer benzodiazepines and Z-hypnotics determination in plasma samples using ultra-high performance liquid chromatography/tandem mass spectrometry after microextraction by packed sorbent.

Ares-Fuentes AM ，Lorenzo RA ，Fernández P ，Carro AM ... -  《-》

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

Vårdal L ，Wong G ，Øiestad ÅML ，Pedersen-Bjergaard S ，Gjelstad A ，Øiestad EL ... -  《-》

Direct determination of diazepam and its glucuronide metabolites in human whole blood by μElution solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A μElution solid-phase extraction (SPE) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of diazepam, nordiazepam, oxazepam, oxazepam glucuronide, temazepam and temazepam glucuronide in human whole blood is presented. 200 μL of whole blood samples were loaded onto a Waters Oasis HLB 96-well μElution SPE plate using 75 μL of methanol as the elution solvent, and the eluents were injected into an Eclipse XDB C18 column. No hydrolysis, solvent transfer, evaporation or reconstitution was involved in the sample preparation procedures. Tandem mass spectrometric detection with Turbo Ion Spray was conducted via multiple reaction monitoring (MRM) under positive ionization mode. The method was validated and proved to be accurate (accuracy within 93-108%), precise (intra-day RSD<9.9% and inter-day RSD<7.2%) and sensitive with limits of detection (LOD) in the range of 0.05-0.25 ng/mL for all the compounds. Extraction recoveries were in the range of 31-80% for all the analytes. This method demonstrated to be reproducible and reliable. The applicability of the method was demonstrated by analysis of several forensic cases involving diazepam and its metabolites.

Wang R ，Wang X ，Liang C ，Ni C ，Xiong L ，Rao Y ，Zhang Y ... -  《-》