Quantitative determination of fifteen basic pharmaceuticals in ante- and post-mortem whole blood by high pH mobile phase reversed phase ultra high performance liquid chromatography–tandem mass spectrometry

An ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for the determination of fifteen basic pharmaceuticals, for analysis of post- and ante-mortem whole blood samples. The following compounds were included: amitriptyline and its metabolite nortriptyline, trimipramine, mianserin, mirtazapine, citalopram, paroxetine, sertraline, and venlafaxine (all antidepressants), levomepromazine and quetiapine (antipsychotics), ketobemidone and tramadol (analgesics), alimemazine (sedative antihistamine), and metoprolol (beta-blocker). The sample pretreatment consisted of liquid–liquid extraction (LLE) using ethylacetate:n-heptane (80:20, v/v). Six deuterated analogues were used as internal standards (IS). The compounds were separated using a reversed phase C18-column (2.1 mm × 100 mm, 1.7 μm), a flow rate of 0.5 mL/min, 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, using two transitions for the compounds and one transition for the IS. The run time of the method was 8 min including equilibration time. The calibration curves had R2 values above 0.995 for all the compounds. The intermediate precision had a relative standard deviation (RSD, %) ranging between 2.0 and 16%. Recoveries of the compounds were ≥81%. The lower limits of quantifications (LLOQs) for the compounds varied from 5.0 nmol/L to 0.10 μmol/L (1.3–26 ng/mL) and the limits of detections (LODs) from 1.0 to 20 nmol/L (0.24–5.3 ng/mL). LLOQ corresponds to 0.28–5.5 pg injected on column. Matrix effects (ME) were between 91 and 113% when calculated against an IS. A comparison with former confirmation LC–MS methods at the Norwegian Institute of Public Health, Division of Forensic Medicine and Drug Abuse Research (NIPH) was performed during method validation. Good correlation was seen for all compounds except sertraline, where the old LC–MS method was showing 33% higher results. The method has been running on a routine basis for more than a year, and has proven to be very robust and reliable with results for external quality samples, including sertaline, corresponding well to consensus mean or median.