Chromatographic determination of benzodiazepines in vitreous humor after microwave-assisted extraction

Abstract

An analytical method was developed for the simultaneous determination of bromazepam, alprazolam, lorazepam, lormetazepam, diazepam and tetrazepam in vitreous humor, using microwave-assisted extraction (MAE) optimized by a central composite design followed by desirability functions, and high performance liquid chromatography coupled to a photodiode array detector (HPLC-PDA). The optimal conditions were 98 °C for the extraction temperature, 10 min for the extraction time, and ethyl acetate (10 mL) as the solvent. The extract was evaporated to dryness, redissolved in 100 μL of mobile phase, and injected (30 μL) into a XBridge™ Shield RP18 column (250 × 4.6 mm id, 5 μm particle size). The mobile phase, composed of acetonitrile and 0.02 M phosphate buffer pH 6, was eluted at a flow rate of 0.8 mL min⁻¹ in gradient mode. The response of the detector was linear in the range of 0.05 to 5 μg mL⁻¹ vitreous humor, the limits of detection ranged between 10 and 13 ng mL⁻¹, and the coefficients of variation were always lower than 10%. The mean recoveries oscillated between 87.6% for diazepam and 101.2% for lormetazepam. Finally, the MAE/HPLC-PDA method was successfully applied to 16 vitreous humor samples from individuals intoxicated with benzodiazepines, with alprazolam, lormetazepam and diazepam being the most frequently found of these substances.