A rapid ultrasound-assisted dispersive liquid–liquid microextraction followed by ultra-performance liquid chromatography for the simultaneous determination of seven benzodiazepines in human plasma samples

A simple and efficient ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME) method has been developed for the determination of seven benzodiazepines (alprazolam, bromazepam, clonazepam, diazepam, lorazepam, lormetazepam and tetrazepam) in human plasma samples. Chloroform and methanol were used as extractant and disperser solvents, respectively. The influence of several variables (e.g., type and volume of dispersant and extraction solvents, pH, ultrasonic time and ionic strength) was carefully evaluated and optimized, using an asymmetric screening design 3²4²//16. Analysis of extracts was performed by ultra-performance liquid chromatography coupled with photodiode array detection (UPLC-PDA). Under the optimum conditions, two reversed-phases, Shield RP18 and C18 columns were successfully tested, obtaining good linearity in a range of 0.01–5 μg mL⁻¹, with correlation coefficients r > 0.996. Quantification limits ranged between 4.3–13.2 ng mL⁻¹ and 4.0–14.8 ng mL⁻¹, were obtained for C18 and Shield RP18 columns, respectively. The optimized method exhibited a good precision level, with relative standard deviation values lower than 8%. The recoveries studied at two spiked levels, ranged from 71 to 102% for all considered compounds. The proposed method was successfully applied to the analysis of seven benzodiazepines in real human plasma samples.