An Entire Process Optimization Strategy for Comprehensive In Vivo Metabolite Profiling of Prucalopride in Rats Based on Ultra-Performance Liquid Chromatography With Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometry.

Lihua Zuo,Yantao Yang,Min Chen,Liwei Liu,Huafeng Zhang,Jiabo Wang,Zhi Sun,Jian Kang,Xiaojian Zhang,Jie Yang

doi:10.3389/fphar.2021.610226

Abstract

Prucalopride was widely used for chronic constipation, which is difficult to be adequately relieved by laxatives in adult patients in clinic. Due to the difficulty in metabolite identification, metabolic process of prucalopride had not been investigated in vivo. In this study, an efficient strategy was proposed for comprehensive metabolite profiling of prucalopride after oral administration in rat plasma, urine, and feces samples. This strategy was composed of five steps. First, the samples at multiple time points after oral administration were collected to increase the representativeness of the samples. Second, different sample preparation methods were investigated to obtain superior extraction efficiency. Third, the raw data of test sample and blank sample were acquired using ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution accurate mass spectrometry under the positive and negative full-scan/dd MS2 mode. Fourth, combined mass defect filter with background subtraction model in soft of compound discovery, all peaks were constructed to filter potential metabolites after retention time alignment and ion filtration, which could remove large amounts of interference ions. Besides, it can predict potential biotransformation, promoting to understand how to metabolize the drug. This provides multiple possibilities and prevents us conjecturing the potential metabolites blindly. Finally, the verification procedure was implemented through exporting the structure and MS2 spectrum to the analytical tool of Mass Frontier. The proposed strategy significantly improved the targeted detection and identification for metabolites in vivo. A total of 47 metabolites were tentatively characterized in the plasma, urine, and feces samples after oral administration of prucalopride. This study could provide a valuable reference for systematic metabolite profile of drug in vivo.

Highlights

The drug-related metabolites could provide abundant information related to the side effects and formation of active or other toxic metabolites (Balasaheb et al, 2018; Godinho et al, 2018; Radko and Olejnik, 2018; Shankar et al, 2018)
In order to extract and detect more metabolites of prucalopride from plasma samples, we systematically investigated the sample preparation method, including the commonly used protein precipitation (PP) with methanol and acetonitrile and liquid–liquid extraction (LLE) with ethyl acetate and n-butanol
An entire process optimization strategy was proposed for comprehensive in vivo metabolite profiling of prucalopride in rat plasma, urine, and feces samples after oral administration based on ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution mass spectrometry

Summary

INTRODUCTION

The drug-related metabolites could provide abundant information related to the side effects and formation of active or other toxic metabolites (Balasaheb et al, 2018; Godinho et al, 2018; Radko and Olejnik, 2018; Shankar et al, 2018). A comprehensive strategy was proposed for the rapid identification of the metabolites of prucalopride after oral administration based on the ultra-performance liquid chromatography with Q-Exactive hybrid quadrupole–orbitrap high-resolution accurate mass spectrometry (UHPLC-HRMS). This strategy could be divided into five steps. It can predict potential biotransformation, which could help us in inferring the metabolic process of the drug The structure and mass spectrometry were further exported to the Mass Frontier 7.0 (MF, Thermo Fisher Scientific) to observe whether the structure and fragments match

RESULTS AND DISCUSSION

CONCLUSION

ETHICS STATEMENT