Calibration of pre-equilibrium HF-LPME and its application to the rapid determination of free analytes in biological fluids.

Abstract

This study establishes a novel calibration method for pre-equilibrium hollow-fiber liquid-phase microextraction (PE-HF-LPME), where the time constant of the extraction of the analyte from sample matrix to the extraction phase (organic solvent) is obtained from a simple concentration curve. Comparing to the traditional kinetic calibration method, where the time constant was obtained from the extraction time profile, the new calibration approach shows improved accuracy and precision. More importantly, deuterated standards are not required in the new method, thus significantly improving its cost-effectiveness and extending its applicability to a wide range of analytes lack of deuterated analogs serving as internal standards. In addition, mass spectrometry is not necessary for the quantification of analytes with the new calibration method, which may further extend the applicability of PE-HF-LPME to some laboratories without mass spectrometers. This study has been substantiated with both theoretical and experimental evidences. Further, the feasibility of the method for real biological samples was demonstrated by measuring the free concentration of flunitrazepam in urine and plasma samples and its drug-protein binding ratio in plasma. The results showed that the method had a short analysis time and was easily implemented with high accuracy and good reproducibility.