Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Simultaneous Determination of 26 Benzodiazepines and Metabolites, Zolpidem and Zopiclone, in Blood, Urine, and Hair

Abstract

A liquid chromatography-tandem mass spectrometry method was developed for the simultaneous quantification of 26 benzodiazepines and metabolites, zolpidem and zopiclone, in blood, urine, and hair. Drugs were extracted from all matrices by liquid-liquid extraction with 1-chlorobutane. Chromatography was achieved using a XTerra MS C18 column eluted with a mixture of methanol and formate buffer. Data were acquired using positive electrospray ionization and multiple reaction monitoring using one precursor ion/product ion transition per compound. Quantification was performed using 13 deuterated analogues. Further confirmation of the identity of the compounds was achieved through a second injection of positive samples, monitoring two transitions per compound. The limits of quantification for all benzodiazepines ranged from 1 to 2 ng/mL in blood, 10 to 25 ng/mL in urine, and 0.5 to 10 pg/mg in hair. Linearity was observed from the limit of quantification of each compound to 200 ng/mL, 1000 ng/mL, and 1000 pg/mg for blood, urine, and hair, respectively (r2 > 0.99). Precision for quality control samples, spiked at three concentrations, was calculated (CV < 20% in most cases). Extraction recoveries for the three matrices ranged from 25.1 to 103.8%, except for one compound (cloxazolam in urine). Ion suppression was studied for all matrices. The validated assay was applied to authentic blood, urine, and hair samples from forensic cases.

The Clinical and Forensic Toxicology of Z-drugs

“…It can potentially complement tests done on blood and urine, though in some scenarios hair may be the only matrix available. Hair as a biological matrix has several advantages including ease of sampling, storage, and transportation [64]. In general, detection of Z-drugs in hair is difficult due to the low level of uptake into hair.…”

Zopiclone degradation in biological samples : Characteristics and consequences in forensic toxicology

“…In some analytical methods for ZOP the procedure included an enzymatic hydrolyzing step with β-glucuronidase [127,138], mainly because the methods also included benzodiazepines. Another study showed that ZOP metabolites are excreted mainly unconjugated [142].…”

“…Several analytical methods, including high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS), have been developed for the identification and quantification of ZOP in whole blood, serum and/or plasma [118,[120][121][122][123][124][125][126][127][128][129][130][131][132][133][134][135][136]. Some assays (HPLC, LC-MS, LC-MS/MS) have also been developed to separate and determine ZOP [127,[137][138][139] and/or its metabolites (NDZOP and ZOPNO) in urine [140][141][142].…”

“…Some assays (HPLC, LC-MS, LC-MS/MS) have also been developed to separate and determine ZOP [127,[137][138][139] and/or its metabolites (NDZOP and ZOPNO) in urine [140][141][142]. However, because of analytical detection or validation problems when ZOP was analysed in urine, the ZOP analyte was excluded from two of these methods [139,141].…”

DEVELOPMENT AND VALIDATION OF A GC-NPD/micro-ECD METHOD USING DUAL COLUMN FOR THE DETERMINATION OF BENZODIAZEPINE IN HUMAN WHOLE BLOOD AND PLASMA

“…Several analytical methods for the detection of multiple benzodiazepine drugs in human blood and urine samples have been reported using GC/MS, 7-10 LC/MS, 11,12 and LC/MS/ MS. [13][14][15][16][17][18] Some of these drugs and their metabolites possess polar groups in their molecules that require derivatization prior to GC/MS analysis.…”