Development and validation of a LC-MS/MS method for the simultaneous determination of cycloicaritin and its carbamate prodrug in rat plasma: application to pharmacokinetic study

A novel leucine carbamate prodrug (3-L) has been synthesized to improve the low oral bioavailability of cycloicaritin. To assess the pharmacokinetic properties of the prodrug, a selective, sensitive and reliable liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of cycloicaritin and its carbamate prodrug 3-L in rat plasma with daidzein as internal standard was developed and validated. Ethanoic acid was added to plasma samples to maintain the stability of the prodrug. The samples were prepared employing a liquid–liquid extraction (LLE) method using tert-butyl methyl ether in a low plasma sample volume of 25 μL, and then separated on an ACE Excel 2 C18-Amide column (50 mm × 2.1 mm, 2 μm) by gradient elution at a flow rate of 0.4 mL min−1. A triple quadrupole tandem mass spectrometer system with a positive ESI interface was used for quantification in multiple reaction monitoring (MRM) mode. Cycloicaritin and 3-L both showed excellent linearity over the concentration range of 1–2000 ng mL−1 (r ≥ 0.995), with a lower limit of quantification of 1 ng mL−1. The accuracy varied from −5.2% to 14% for all analytes, and the intra- and inter-day precision was less than 14% across quality control levels. The method described herein was successfully applied to the pharmacokinetic study of orally administered 3-L in rats.