Simultaneous determination of dorzolomide and timolol in aqueous humor: A novel salting out liquid–liquid microextraction combined with HPLC

A Snovel method for the simultaneous separation and determination of two antiglaucoma drugs namely, dorzolamide hydrochloride (DOR) and timolol maleate (TIM) in aqueous humor samples (AH) was developed by using salting-out assisted liquid–liquid microextraction (SALLME) combined with HPLC–UV method. Box–Behnken experimental design and response surface methodology were employed to assist the optimization of SALLME conditions, including salt concentration, the pH of sample solution and vortex time as variable factors. The optimal extraction conditions were as follows: to 50µL of AH sample, 100µL of phosphate buffer (100mmolL−1, pH 11.9), 90µL of acetonitrile (ACN) and 0.11g of (NH4)2SO4 salt were added into an Eppendorf vial (1mL) then vortexed for 1.1min. As an effort to miniaturize SALLE system, a 1mL syringe adapted with a capillary tube was employed as the phase separation device. Once the phase separation occurred, the upper layer could be narrowed into the capillary tube by pushing the plunger; thus, the collection of the upper layer solvent was simple and convenient. By miniaturization, the consumption of the organic solvent was decreased as low as possible. The chromatographic separation was achieved on Gemini C18 column using a mobile phase of ACN: 30mmolL−1 potassium dihydrogen phosphate buffer containing 0.1% triethylamine, pH 3.5 (20:80, v/v) at a flow rate of 1mLmin−1 and UV detection at 254 and 295nm for DOR and TIM, respectively. Mepivacaine hydrochloride was used as an internal standard. The described method showed better separation with enhanced sensitivities than the previously reported methods with limits of quantitation of 8.75 and 10.32ngmL−1 in aqueous solution and 15.97 and 23.53ngmL−1 in AH for DOR and TIM, respectively. The simple, rapid and eco-friendly SALLME–HPLC method has been successfully applied for the simultaneous pharmacokinetic studies of DOR and TIM in rabbit AH. [Display omitted] •Dorzolomide and timolol have been analyzed in aqueous humor by SALLME–HPLC.•Box–Behnken design and response surface were used for SALLME optimization.•The method was simple, rapid, economic, eco-friendly and with high sensitivity.•The method was applied to the pharmacokinetic studies of the studied drugs in AH.