Screening, library‐assisted identification and validated quantification of 23 benzodiazepines, flumazenil, zaleplone, zolpidem and zopiclone in plasma by liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization

Abstract

A liquid chromatographic/mass spectrometric assay with atmospheric pressure chemical ionization (LC/APCI-MS) is presented for fast and reliable screening and identification and also for precise and sensitive quantification in plasma of the 23 benzodiazepines alprazolam, bromazepam, brotizolam, camazepam, chlordiazepoxide, clobazam, clonazepam, diazepam, flunitrazepam, flurazepam, desalkylflurazepam, lorazepam, lormetazepam, medazepam, metaclazepam, midazolam, nitrazepam, nordazepam, oxazepam, prazepam, temazepam and tetrazepam, triazolam, their antagonist flumazenil and the benzodiazepine BZ1 (omega 1) receptor agonists zaleplone, zolpidem and zopiclone. It allows confirmation of the diagnosis of an overdose situation and monitoring of psychiatric patients' compliance. The analytes were isolated from plasma using liquid-liquid extraction and were separated on a Merck LiChroCART column with Superspher 60 RP Select B as the stationary phase. Gradient elution was performed using aqueous ammonium formate and acetonitrile. After screening and identification in the scan mode using the authors' LC/MS library, the analytes were quantified in the selected-ion monitoring mode. The quantification assay was fully validated. It was found to be selective proved to be linear from sub-therapeutic to over therapeutic concentrations for all analytes, except bromazepam. The corresponding reference levels the assay's accuracy and precision data for all studied substances are listed. The accuracy and precision data were within the required limits with the exception of those for bromazepam. The analytes were stable in frozen plasma for at least 1 month. The validated assay was successfully applied to several authentic plasma samples from patients treated or intoxicated with various benzodiazepines or with zaleplone, zolpidem or zopiclone. It has proven to be appropriate for the isolation, separation, screening, identification and quantification of the drugs mentioned above in plasma for clinical toxicology, e.g. in cases of poisoning, and forensic toxicology, e.g. in cases of driving under the influence of drugs.

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

“…ethanol, acetonitrile, dichloromethane) [92,102]. Analysis of ZOP in biological specimens is complicated by its instability in certain solvents such as methanol, and under acid or basic pH conditions [101,117,118]. Standard solution should be prepared in acetonitrile and extraction must be done at neutral pH to improve stability [101,119].…”

“…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].…”

“…Studies of the stability of ZOP have also been carried out from stability experiments in connection with method development and validation. Long-term stability tests with spiked human plasma showed that ZOP was stable for one month [118,129], for 74 days [131] and for six months [119] when stored at -20°C or lower. Freeze-thaw stability tests verified that ZOP was stable in spiked human plasma for three freeze-thaw cycles [118,129] but stability for five freezethaw cycles has also been reported [131].…”

“…Long-term stability tests with spiked human plasma showed that ZOP was stable for one month [118,129], for 74 days [131] and for six months [119] when stored at -20°C or lower. Freeze-thaw stability tests verified that ZOP was stable in spiked human plasma for three freeze-thaw cycles [118,129] but stability for five freezethaw cycles has also been reported [131]. Short-term or in process stability tests give no evidence of degradation in plasma when quality control samples were stored at room temperature for 12 h [131] or 24 h [129].…”

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.…”