Determination of Difenidol and Diphenhydramine in Blood Samples by Ultrasonic-assisted Ionic Liquid-based Dispersive Liquid-liquid Microextraction Coupled with LC-MS/MS

A method for the determination of difenidol and diphenhydramine in blood samples utilizing ultrasonic-assisted ionic liquid dispersive liquid-liquid microextraction coupled with LC-MS/MS was established. 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]) was used as the extractant, and acetone was used as the dispersant. The samples were separated by a ZORBAX Eclipse Plus C18 RRHD column. Aqueous solution containing 0.1% formic acid and methanol was used as mobile phases. A gradient elution mode was employed with a flow rate of 0.2 mL/min, followed by electrospray ion source mode (ESI⁺) analysis in scheduled multiple reaction monitoring mode (MRM). The detection limits of difenidol and diphenhydramine in blood samples were 0.01 ng/mL, and the quantification limits were 0.02 ng/mL for both. The intraday and interday precisions (relative standard deviation, RSD) were both less than 20%, and the relative recovery rates ranged from 98.38% to 115.10%. The concentrations of difenidol and diphenhydramine in the blood of a fatal case were 4.02 µg/mL and 3.78 µg/mL, respectively. This study demonstrated the feasibility of using ultrasonic-assisted ionic liquid dispersive liquid-liquid microextraction combined with LC-MS/MS for the determination of difenidol and diphenhydramine in blood samples. The method has the advantages of environmentally friendly, high enrichment efficiency, and high sensitivity, making it suitable for forensic identification of difenidol and diphenhydramine.