Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

New Method for the Monitoring of Antidepressants in Oral Fluid Using Dried Spot Sampling

The increase in the consumption of antidepressants is a public health problem worldwide, as these are a class of compounds widely used in the treatment of several illnesses, such as depression and anxiety. This work aimed to develop and optimize a method for the quantification of a number of antidepressants and their metabolites (fluoxetine, venlafaxine, O-desmethylvenlafaxine, citalopram, sertraline, and paroxetine) in 100 µL of oral fluid using the dried saliva spots (DSS) sampling approach and gas chromatography coupled with tandem mass spectrometry (GC–MS/MS). The method was validated, presenting linearity within the studied range, with detection and quantification limits ranging between 10 and 100 ng/mL, and coefficients of determination (R2) of at least 0.99 for all analytes. Recoveries were between approximately 13 and 46%. The analysis of precision and accuracy presented acceptable coefficients of variation and relative errors, considering the criteria usually accepted in the validation of bioanalytical procedures. The method herein described is the first to be reported using DSS for the extraction of antidepressants, proving to be a sensitive, simple, and fast alternative to conventional techniques, and capable of being routinely applied in clinical and forensic toxicology scenarios.

Low Temperature Dynamic Chromatography for the Separation of the Interconverting Conformational Enantiomers of the Benzodiazepines Clonazolam, Flubromazolam, Diclazepam and Flurazepam

Benzodiazepines (BZDs) are an important class of psychoactive drugs with hypnotic-sedative, myorelaxant, anxiolytic and anticonvulsant properties due to interaction with the GABAa receptor in the central nervous system of mammals. BZDs are interesting both in clinical and forensic toxicology for their pharmacological characteristics and potential of abuse. The presence of a non-planar diazepine ring generates chiral conformational stereoisomers, even in the absence of stereogenic centers. A conformational enrichment of BZD at the binding sites has been reported in the literature, thus making interesting a stereodynamic screening of a wide range of BZDs. Herein, we report the investigation of three stereolabile 1,4-benzodiazepine included in the class of “designer benzodiazepines” (e.g., diclazepam, a chloro-derivative of diazepam, and two triazolo-benzodiazepines, flubromazolam and clonazolam) and a commercially available BZD known as flurazepam, in order to study the kinetic of the “ring-flip” process that allows two conformational enantiomers to interconvert at high rate at room temperature. A combination of low temperature enantioselective dynamic chromatography on chiral stationary phase and computer simulations of the experimental chromatograms allowed us to measure activation energies of enantiomerization (ΔG‡) lower than 18.5 kcal/mol. The differences between compounds have been correlated to the pattern of substitutions on the 1,4-benzodiazepinic core.

Hyphenated high-resolution mass spectrometry—the “all-in-one” device in analytical toxicology?

This trend article reviews papers with hyphenated high-resolution mass spectrometry (HRMS) approaches applied in analytical toxicology, particularly in clinical and forensic toxicology published since 2016 and referenced in PubMed. The article focuses on the question of whether HRMS has or will become the all-in-one device in these fields as supposed by the increasing number of HRMS presentations at scientific meetings, corresponding original papers, and review articles. Typical examples for the different application fields are discussed such as targeted or untargeted drug screening, quantification, drug metabolism studies, and metabolomics approaches. Considering the reviewed papers, HRMS is currently the only technique that fulfills the criteria of an all-in-one device for the various applications needed in analytical toxicology.Graphical abstract

Ultrasound-assisted liquid-liquid extraction for the determination of methadone in urine samples using gas chromatography-mass spectrometry

Background: Methadone abuse and dependence has been reported as a growing concern in some countries. In present study, a rapid, simple, easy and sensitive method for determination of methadone in human urine samples for use in clinical and forensic toxicology and drug screening laboratories was developed and validated. Methods: We determined methadone in urine sampled by gas chromatography/mass spectrometry (GC/MS) and used ultrasound assisted liquid-liquid extraction (UALLE) method for the extraction and preconcentration of methadone before analysis. Results: The limit of detection was 2.1 ng/mL and the limit of quantification 7 ng/mL. Correlation coefficient was 0.9984 for the methadone calibration curve in linear range from 7 to 10,000 ng/mL. The method is accurate and precise. Recovery was in the range of 81.3 % to 97.4 % and enrichment factor was 8.7. The method was successfully applied for determining methadone in clinical and postmortem urine samples. Conclusions: The present method is a rapid, simple, easy and sensitive procedure and can be used in clinical and forensic toxicology laboratories as routine method for qualitative and quantitative analysis of methadone.