Development and validation of a novel LC-MS/MS method for simultaneous quantification of alvocidib and its glucuronide: Application to pharmacokinetic and tissue distribution study in rats.

Abstract

Alvocidib, an approved drug for the treatment of myeloid leukemia, undergoes glucuronidation metabolism. The purpose of this study is to develop a robust and sensitive LC-MS/MS method to simultaneously quantify alvocidib and its metabolite, alvocidib-glucuronide (Alvo-G), and apply this method in a pharmacokinetic and tissue distribution study in rats. A UHPLC system coupled to an AB Sciex QTrap 4000 triple quadrupole mass spectrometer was used for the analysis. The analytes were separated using a Biphenyl column with 0.1% formic acid in water and acetonitrile as the mobile phases and were quantified under positive electrospray ionization (ESI) mode using a multiple reaction monitoring (MRM) approach. Protein precipitation with acetonitrile was used in sample processing. The method showed a good linearity within the range of 2.4nM - 5000.00 nM for both analytes. The method was reproducible, with intra and inter-day accuracy and precision ranging from 84-98% and <13% respectively. The extraction recovery and matrix effect achieved acceptable ranges (82-105%). Stability studies demonstrated that they were stable under bench-top (91-99%), freeze-thaw (83-110%), and long-term storage conditions (80-106%). The PK results showed that after oral administration of alvocidib, both alvocidib and Alv-G were detected in plasma with the parent drug showing highest distribution in the colon, and lowest in the small intestine. In contrast, Alv-G showed the highest distribution in the small intestine and the lowest amount in the colon compared to the other tissues. This method is applicable for PK and tissue distribution studies to quantify both alvocidib and its glucuronide.