Search

An ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of zopiclone and zolpidem in whole blood, for use in cases with suspected driving under influence of drugs (DUID) and autopsy cases. Sample preparation was performed with liquid-liquid extraction (LLE) using ethyl acetate/n-heptane (80:20, v/v) and 0.1mL whole blood. Deuterated analogues were used as internal standards (IS) for both compounds. The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7μm), with a flow rate of 0.5mL/min, 1μL injected and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode. The run time of the method was 4.5min including equilibration time. The calibration curves of extracted whole blood standards were fitted by linear-order calibration curves weighted 1/x, with R(2) values above 0.999 for both compounds. Intermediate precision and accuracies (bias) were 2.4-12.9% RSD and from -5.9 to 6.8%, respectively. Recoveries of the compounds were ≥70%. The lower limit of quantification (LLOQ) for zopiclone was 0.50nmol/L (0.19ng/mL) or 0.05pg injected on column, and 3.5nmol/mL (1.10ng/mL) for zolpidem, or 0.27pg injected on column. The limit of detection (LOD) was 0.2nmol/L (0.08ng/mL) for zopiclone and 0.3nmol/L (0.09ng/mL) for zolpidem. Matrix effects (ME) were between 108 and 115% when calculated against IS. A comparison with former confirmation LC-MS method at the Norwegian Institute of Public Health, Division of Forensic Medicine (NIPH) was performed during method validation. Good correlation was seen for both compounds. The method has been running on a routine basis for two years, and has proven to be very robust and reliable with satisfactory long term precision and bias and with results for external quality samples corresponding well to consensus mean or median. Zopiclone and zolpidem concentrations in post mortem and ante mortem cases were reported. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs introduced in the Norwegian Road Traffic Act Law from 2012., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Benzodiazepines and their pharmacologically related drugs, zolpidem and zopiclone are widely prescribed as safe drugs, but these drugs are also abused in cases of crime, suicide and drug-facilitated sexual assault. We developed a rapid and quantitative screening method for 43 benzodiazepines, their metabolites, zolpidem and zopiclone in human plasma by liquid chromatography/mass spectrometry with a small particle column. All drugs were successfully separated within 12 min using combined scan and selected ion recording (SIR) mode. The calibration curves of most drugs were linear in the concentration range 0.5-250 ng/mL with correlation coefficients exceeding 0.99. Within-day precisions (RSD, %) of this method were 1.8-15.6% (10 ng/mL) and 0.6-10.1% (100 ng/mL) and between-day precisions (RSD, %) were 1.6-16.9% (10 ng/mL) and 0.6-16.7% (100 ng/mL). The average recoveries were 70.1% (10 ng/mL) and 87.1% (100 ng/mL). The limit of detection ranged from 0.2 to 8.0 ng/mL in 37 drugs and was below 0.2 ng/mL in 6 drugs. The established method is sensitive and rapid, thus it should be useful in forensic and clinical toxicological analyses.

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm x 4.6 mm i.d., 3.5 microm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5-150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1-150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5-109.1% and 3.0-14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were > or = 90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.