Hydrophobic deep eutectic solvent-based microextraction method for the simultaneous extraction of two benzodiazepines from saliva samples before determination by 96-well microplates-based spectrophotometer with the aid of chemometrics

In this study, a 96-well microplates-based spectrophotometer with the aid of the partial least squares (PLS) approach was used for the simultaneous determination of more common benzodiazepines, clonazepam, and nitrazepam, in human saliva samples. For this purpose, a hydrophobic deep eutectic solvent (DES) based on choline chloride and 1-phenylethanol in the mole ratio of 1:4 was prepared and utilized in the ultrasound-assisted emulsification-microextraction (USAEME) method followed by back-extraction to retrieve the drugs from the DES phase to 200 of 0.1 M NaOH solution. Central composite design (CCD) in combination with the desirability function was used for multi-response optimization of different parameters and the optimum values were found to be: pH 2.06; DES volume, 247.1 mu L; NaCl, 1.55% (w/v); sonication time, 5.33 min. The DES-based USAEME method showed wide linear ranges of 0.1-6 and 0.2-8 mu g/mL and good limits of detection of 0.02 and 0.05 mu g/mL for clonazepam and nitrazepam, respectively. Furthermore, the method was successfully used for determining the drugs in human saliva samples.