Development of two ultra-sensitive UHPLC–QqQ-MS/MS methods for the simultaneous determination of hydroxyzine and its active metabolite (cetirizine) in human blood: applications to real cases of forensic toxicology

Both postmortem toxicological and medical-forensic examinations are very important in the case of analyzing various types of chemical substances. Hydroxyzine (HZ) is a first-generation antihistamine drug with a sedative effect that disrupts cognitive function and affects the ability to drive motor vehicles. Enzymatic oxidation of the hydroxy-methyl group to the carboxyl group leads to the formation of its main metabolite—cetirizine (CZ). CZ is the active substance of antiallergic drugs. Because it does not cross the BBB (blood–brain barrier) easily, it is less likely to cause drowsiness or affect memory and impair cognitive function. Therefore, in criminal studies, it is often important what medication had been taken by a person involved, e.g., in a car accident, HZ or CZ. The analysis of both antihistamine drugs is challenging, as usually very low concentrations of the compound of interest need to be determined. Thus, an ultra-sensitive UHPLC–QqQ-MS/MS method was developed for simultaneous determination of HZ and CZ in biological fluid samples. The lower limit of quantification (LOQ) for HZ and CZ was calculated as 0.345 and 0.3696 ng/mL, respectively. Together with a reduced sample volume to 200 μL, it makes the developed method suitable for a sensitive multidrug forensic toxicological analysis. Samples were extracted with simple and fast liquid–liquid extraction (ethyl acetate, pH 9). The present method for the determination of HZ and CZ in human blood proved to be simple, fast, selective, and sensitive. The quantification by LC–MS/MS was successfully applied to the samples coming from 28 authentic biological fluids (blood, urine, vitreous humor, bile and stomach content), both antemortem and postmortem. The performed studies confirm that the developed method is characterized by a high extraction efficiency. Its accuracy, reproducibility, simplicity, and selectivity suggest its application in clinical, toxicological, and forensic laboratories.