On-line incubation and real-time detection by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry for rapidly analyzing metabolites of anthraquinones in rat liver microsomes

The traditional studies on metabolism in liver microsomes were carried out in off-line form. In this paper, a rapid and convenient method for the study of metabolism of substrates in liver microsomes was established by means of ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS): on-line incubation and real-time detection of substrates in liver microsomes. The liver microsomal incubation system was placed in a sample chamber at 37 °C. On-line solid phase extraction (SPE) column was used for on-line sample treatment, its function was to enrich the drug prototype and its metabolites with weak polarity, and elute the phosphate in the samples. The incubation samples were analyzed by setting appropriate injection time, liquid phase elution procedure and mass spectrometry acquisition time. The phase I metabolites of anthraquinone compounds, aloe-emodin (A), rhein (R), emodin (E), chrysophanol (CP), physcion (PS) and their glucosides, were analyzed through this method. The results showed that 8 anthraquinone compounds underwent metabolic reactions such as hydrolysis, oxidation, reduction and hydroxylation in liver microsomal incubation system. In addition, a certain degree of mutual transformation of anthraquinones in liver microsomal incubation system was found. The results provide a reference for in vivo metabolism of anthraquinones in rhubarb. On-line incubation and real-time detection is a feasible, convenient and rapid method for the analysis of drug metabolism in vitro.