Quantitative determination of zopiclone and zolpidem in whole blood by liquid-liquid extraction and UHPLC-MS/MS

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 0.1 mL whole blood. Deuterated analogues were used as internal standards (IS) for both compounds. The compounds were separated using a reversed phase C18-column (2.1 mm × 100 mm, 1.7 μm), with a flow rate of 0.5 mL/min, 1 μL injected and gradient elution with 5 mM 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 4.5 min including equilibration time. The calibration curves of extracted whole blood standards were fitted by linear-order calibration curves weighted 1/x, with R2 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 0.50 nmol/L (0.19 ng/mL) or 0.05 pg injected on column, and 3.5 nmol/mL (1.10 ng/mL) for zolpidem, or 0.27 pg injected on column. The limit of detection (LOD) was 0.2 nmol/L (0.08 ng/mL) for zopiclone and 0.3 nmol/L (0.09 ng/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.