UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets

Suvorexant is a novel sedative/hypnotic drug approved for treatment of insomnia. It has significant forensic importance due to its hypnotic and depressant effects on central nervous system. In this study, a highly sensitive UPLC-MS/MS assay was developed and validated for the determination of suvorexant in urine sample. A simplified dispersive liquid-liquid microextraction followed by ultrasound assisted back extraction from solidified floating organic droplets was employed for sample preparation. The 20 μL of 1-undecanol and 200 μL of acetonitrile were used as extraction solvent and dispersive solvent, respectively. An ultrasound assisted back extraction step was employed to enable the cleanup procedure compatible with mass spectrometric detection. Acquity CSH ^™ C ₁₈ column with mobile phase composition of 15 mM ammonium acetate: acetonitrile: formic acid (15:85:0.1%; v/v/v) were used for chromatographic separation. The multiple reaction monitoring transition of 451.12 →104.01 and 451.12→186.04 were used for identification and quantification of suvorexant, respectively, whereas 237.06→194.1 was used for IS in positive mode. The assay demonstrated good linearity in the range of 0.27–1000 ng mL ⁻¹ with limit of detection (LOD) and quantification (LOQ) of 0.10 and 0.27 ng mL ^-1 , respectively. Assay validation was performed by following SWGTOX guidelines and all validation results were found to be within acceptable limits. This is the first report of dispersive liquid-liquid microextraction based on solidification of floating organic droplets employed to UPLC-MS/MS for application in biological fluids.