Simultaneous determination of daidzein, its prodrug and major conjugative metabolites in rat plasma and application in a pharmacokinetic study

Abstract

An isoleucine carbamate prodrug of daidzein was designed to address the challenge of its metabolic instability with the aim of improving oral bioavailability. To explore the process of biotransformation between daidzein and its prodrug, and establish whether the prodrug could protect daidzein from phase II metabolism, a simple HPLC-MS/MS method for the simultaneous determination of daidzein, its carbamate prodrug and main conjugative metabolites in rat plasma was developed and validated. Ethanoic acid was added to plasma samples to avoid transformation of the prodrug into daidzein. Analytes were extracted from 50 μL rat plasma via a one-step protein precipitation method using methanol. Chromatographic separation was performed on the Kinetex® C18 column (100 × 2.1 mm, 2.6 μm) under gradient elution in 6.2 min. Good linear correlation curves were achieved over concentration ranges of 0.8–1000 ng mL⁻¹ for daidzein and 4–5000 ng mL⁻¹ for the prodrug and conjugates (daidzein-7-4′-disulfate and daidzein-7-O-glucuronide). The intra- and inter-day trueness and precision results of all quality control levels were within ±15%. Finally, the newly developed sensitive and high-throughput method was successfully applied to conduct pharmacokinetic evaluation of daidzein and its prodrug. Our collective results indicate that the isoleucine carbamate prodrug improves the oral bioavailability of daidzein by reducing its metabolites.