Validation of a simple and quick method for determination of psychoactive substances, drugs and their metabolites from human blood by LC-MS/MS

• Liquid-liquid extraction enables the rapid isolation of multiple analytes in one process. • LC-MS enables the simultaneous and unequivocal identification of a large number of analytes. • Rapid analysis of blood samples across a wide range of analytes allows the identification of toxic substances and rapid treatment initiation. • A quick, sensitive and specific method of analyzing a large number of psychoactive substances allows the detection of illegal substances. The development of the black market, and thus the increase in the number of available psychoactive substances, forces laboratories to take measures to prevent poisoning and deaths caused by the use of, inter alia, new psychoactive substances (NPS). It is important to selectively determine psychoactive substances and their metabolites in blood samples in the shortest possible time, which will allow medical services to act timely. Bearing in mind the above, the aim of our study was to develop an analytical method that would enable the unequivocal identification of over 500 psychoactive substances in blood samples with one analysis. The techniques selected for this purpose were liquid–liquid extraction (LLE) and liquid chromatography coupled with mass spectrometry (LC-MS/MS). Acetonitrile with ammonium formate and formic acid was used for the extraction. The best column was found to be the Kinetex C18 column (Phenomenex, 3.0 × 100 mm; 2.6 µm). MRM pair monitoring mode was used (two pairs for each analyte). 522 psychoactive substances and their metabolites have been validated, and only 2 out of 522 failed to meet the validation criteria recommended by SWGTOX (CV: ± 20 % and BIAS ± 20 %). The developed method enables the quantitative analysis of 520 psychoactive substances during a 30-minute run. Limits of quantification (LOQ) range from 1 to 200 ng/mL. The developed method was introduced into the routine toxicological analysis of blood samples. It also offers opportunities for further development in line with the needs of toxicology.