Development and validation of a liquid chromatography–atmospheric pressure photoionization–mass spectrometry method for the quantification of alprazolam, flunitrazepam, and their main metabolites in haemolysed blood

A LC–APPI–MS method was developed and validated for the detection of alprazolam, flunitrazepam and their major metabolites in haemolysed blood. Samples were diluted with water (2:1, v:v) and extracted with a hydrophobic–lipophilic balanced copolymer. The method was fully validated according to ICH guidelines and SFSTP protocols. Deuterated internal standards of both parent drugs were used and good quantitative performance was achieved in terms of trueness and precision (repeatability and intermediate precision) since accuracy profiles were achieved within the acceptance limits (±30% for biological samples). The LC–APPI–MS method was linear over the concentration range of 1–1000 and 3–1000 ng/mL, for alprazolam and flunitrazepam, respectively. Lower limits of quantification as low as 1 ng/mL in haemolysed blood were reached and the method was successfully applied to the quantification of alprazolam, flunitrazepam and their major metabolites in real toxicological samples.