Optimization of ultrasound-assisted dispersive liquid–liquid microextraction for ultra performance liquid chromatography determination of benzodiazepines in urine and hospital wastewater

Abstract

Ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME) combined with ultra-performance liquid chromatography (UPLC) was developed for the determination of seven benzodiazepines (alprazolam, bromazepam, clonazepam, diazepam, lorazepam, lormetazepam and tetrazepam) in urine samples and hospital wastewater. Several parameters affecting UA-DLLME efficiency such as type and dispersive solvent volume and ionic strength were investigated. The effects of pH, ultrasonic time and extraction solvent volume on the extraction efficiency were studied simultaneously using a Doehlert design and desirability functions. The optimal conditions were: 0.5 mL of sample; 1.6 mL acetone, as a dispersive solvent; 160 μL of chloroform, as an extraction solvent; pH 9; 4.5 min of ultrasound application; 0.2 g of NaCl and 8 min of centrifugation at 3500 rpm. The chromatographic separation of benzodiazepines was carried out by a UPLC system using a binary mobile phase composed of aqueous phosphate buffer (0.001 M, pH 6) and acetonitrile, ACN in gradient elution mode (4 min total analysis). Under the optimized conditions, the calibration curves showed good linearity in the studied ranges with determination coefficients higher than 0.994. The limits of quantification (LOQs) ranged from 1.8 to 7.4 ng mL−1 and 0.05 to 0.42 ng mL−1 in urine and wastewater, respectively. Intra- and inter-day precision ranged from 3.1 to 14.1% and the recoveries ranged from 81 to 124%. The application of the proposed analytical procedure in human urine samples and wastewater from a hospital and municipal treatment plants revealed the presence of benzodiazepines.