Determination of hippuric acid in biological fluids using single drop liquid-liquid-liquid microextraction

A simple, sensitive, and inexpensive single drop liquid liquid-liquid microextraction (LLLME) followed by isocratic reverse phase high performance liquid chromatography (RP-HPLC) and UV detection was developed for determination of hippuric acid in human urine and serum samples. The analyte was extracted from an acidic aqueous sample solution (pH 3) through a thin layer of organic solvent membrane and back-extracted to a basic acceptor drop (pH 11) suspended on the tip of a 10-mu L HPLC syringe in the organic layer. The influence of several important parameters on extraction efficiency of hippuric acid was evaluated. Under optimized experimental conditions, the calibration graph was linear in the concentration range of 1-400 mu g L-1 with coefficient of determination 0.998. The limits of detection and quantification were 0.3 and 1.0 mu g L-1, respectively. Intra-day and inter-day precision were in the range of 1.1-2.7% and 1.1-3.1%, respectively. This procedure was successfully applied to the determination of hippuric acid in spiked urine and serum samples with satisfactory results. The relative recoveries of urine and serum samples ranged from 91.4 to 99.3%, with relative standard deviations varying from 1.6 to 4.2%.