Simultaneous Detection of Suvorexant, Daridorexant and Lemborexant in Human Blood by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Suvorexant, daridorexant, and lemborexant, as a class of novel hypnotic agents, have been increasingly reported in cases of drug-facilitated crimes (DFCs), such as sexual assault and poisoning. However, highly selective and sensitive methods for simultaneous detection of these drugs in human blood remain scarce. This study aimed to develop and validate a method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for simultaneous qualitative and quantitative analysis of these drugs in blood. The sample preparation procedure was systematically investigated and optimized. Chromatographic separation was achieved using UPLC, and MS detection was performed under positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The results demonstrated excellent chromatographic performance, with well-resolved peaks and satisfactory separation for all three target analytes. The method exhibited acceptable precision and post-preparative stability. It proved to be highly sensitive, with limits of detection (LOD) of 0.05, 0.06 and 0.07 ng/mL, and limits of quantification (LOQ) of 0.17, 0.21 and 0.24 ng/mL, for suvorexant, lemborexant, and daridorexant, respectively. The matrix effect, recovery, and accuracy for all analytes met the predefined criteria for bioanalytical method validation. Excellent linearity was observed for the three drugs over a concentration range of 0.25 to 100 ng/mL, with correlation coefficients (r2) greater than 0.998. In conclusion, the developed UPLC-MS/MS method was simple, rapid, sensitive, and reliable, and suitable for routine application in forensic toxicological analysis, showing potential in pharmacokinetic studies of these drugs.