Three phase liquid phase microextraction of phenylacetic acid and phenylpropionic acid from biological fluids

Three phase liquid phase microextraction (three phase LPME) technique coupled with HPLC-UV has been applied as a sensitive and efficient sample preparation method to determine phenylacetic acid (PAA) as a biomarker of depressive disorders and phenylpropionic acid (PPA) in biological fluids. The compounds were extracted from 3.0 ml aqueous solution with the adjustment of pH at a fixed value in the range of 2.0–3.5 (donor solution) into an organic phase (1-hexanol) layered on the surface of the donor solution and finally back-extracted into 4.0 μl of the acceptor microdrop (pH 11.1) located at the end of the microsyringe needle. After a prescribed back-extraction time, the acceptor microdrop was withdrawn into the microsyringe and then directly injected into the HPLC system. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. At the optimum conditions (donor solution: 2.3 M Na2SO4, pH 2.0–3.5; organic membrane: 95 μl of 1-hexanol; acceptor solution: 4.0 μl of 0.1 M NH3/NH4+ with pH 11.1; donor solution temperature: 45–50 °C; extraction time: 20 min and back-extraction time: 12 min), up to 110-fold enrichment factor was obtained. The calibration curve for these analytes was linear in the range of 1–5000 μg/l with r2 > 0.998. The intraday and interday RSD% were below 6.5% and the limits of detection (LODs) for both analytes were 0.2 μg/l (based on S/N = 3). The proposed technique is a low cost, simple and sensitive method with highly clean-up effect. Finally, this technique was successfully utilized for the detection of target analytes in human urine, serum and plasma.