Development and validation of a sensitive and fast bioanalytical LC-MS/MS assay for the quantitation of venetoclax and azacitidine in Rat Plasma: Application to pharmacokinetic study.

Abstract

The combination of venetoclax plus azacitidine (VTX-AZA) is FDA-approved to treat patients with acute myeloid leukemia (AML) aged ≥75 years and has become the standard of care for AML patients. However, the literature has not reported an analytical method for determining VTX-AZA in plasma samples. Therefore, developing an accurate and sensitive bioanalytical assay to quantify VTX-AZA in plasma is important. For the first time, this study describes the development of a new liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for the simultaneous determination of VTX and AZC in plasma samples with its application to pharmacokinetic study in rats. The assay employs repaglinide (RPG) as an internal standard. The chromatographic separations of VTX, AZC, and RPG are achieved within 2.5 min at 25 °C on an Eclipse plus C18 column (100 mm × 2.1 mm, 1.8 μm) and an isocratic mobile phase consisted of water with 0.1 % formic acid and acetonitrile (50:50, v/v, pH 3.2) at a flow rate of 0.30 mL/min. VTX and AZC have been extracted from rat plasma using the solid-phase extraction (SPE) procedure without interference from plasma endogenous. The FDA guidelines were followed in the validation of the developed assay, and linearity in rat plasma was observed for AZC and VTX, respectively, ranging from 5 to 3000 and 5-1000 ng/mL, with r ≥ 0.998. The lower limits of detection (LLOD) were 2 ng/mL for both drugs. In addition, the inter-day and intra-day accuracy were 0.8-6.6 % and 2.2-5.7 %; the inter-day and intra-day precision were 3-6.6 % and 1.5-7.1 %, respectively. The validated assay was effectively used in a pharmacokinetic investigation including the simultaneous oral administration of 40 mg/kg of AZA and 100 mg/kg of VTX to rats. The maximum plasma concentration (C_max) for AZC and VTX was 794 ± 99.6 ng/mL and 641 ± 96.9 ng/mL achieved at 0.5 ± 0.03 h and 6 ± 0.05 h, respectively. The AUC_0-∞ for AZC and VTX was 1253 ± 252.6 and 4881 ± 745.4 ng/mL.h; respectively.