Reverse Micelle Hollow Fiber Liquid-Phase Microextraction Coupled with HPLC for the Determination of Q-Markers of Anthraquinones in Rhubarb and Their Plasma Protein Binding Rates

Abstract

A three-phase hollow fiber liquid-phase microextraction based on reversed micellar supramolecule was developed and employed for the determination of quality markers of three anthraquinones in Rhubarb and their plasma protein binding rates. Moreover, the extraction mechanism of reversed micellar supramolecule and determination mechanism of plasma protein binding rate by the proposed method are both explored. In this procedure, n-nonanol was impregnated in the wall pores and reverse micelle of tetrabutylammonium chloride/n-nonanol filled in lumen of hollow fibers, which were covered with a thin salt film and used for the microextraction of target compounds prior to HPLC analysis. Important extraction parameters including extraction solvent type, reversed micellar supramolecule type and concentration, sample phase pH, salt concentration, stirring rate, extraction time, and binding time between active compound and plasma protein were investigated. Under the optimized conditions, limits of detection were 0.1–0.4 ng/mL with enrichment factors in the range of 54.2–100.7. The linearities were 0.5–500 ng/mL with r2 ≥ 0.9838. Satisfactory accuracies (relative recoveries 95.7–98.3%) were also obtained. The average plasma protein binding rates of rhein, chrysophenol, and physcion were 96.7%, 41.8%, and 46.7%, respectively. The results showed that the proposed procedure can not only extract and concentrate anthraquinones in Rhubarb, but also determine their plasma protein binding rates.