Method development and validation for determination of entinostat in human plasma and its application in breast...

Entinostat is an oral histone deacetylase (HDAC) inhibitor used in HR+ breast cancer therapy, for which reliable plasma concentration monitoring is essential for dose optimization and toxicity management. This article described an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantitative analysis of entinostat in human plasma. Samples were concentrated under vacuum after methanol precipitation and separated on a Kinetex C18 column (100 mm × 2.1 mm, 2.6 µm). The mobile phase consisted of acetonitrile and an aqueous solution containing 10 mM ammonium acetate and 0.01% formic acid. The quantitative ion pair for entinostat was m/z 377.2 → 104.0, and for carbamazepine it was m/z 237.1 → 194.1, used as an internal standard (IS). The calibration curves showed good linearity (r ² > 0.99) across plasma concentrations ranging from 0.2 to 200 ng mL^-1. Precision (RSD%) was within the range of 1.22-6.36%, accuracy ranged from 97.58% to 110.67%, the matrix effect ranged from 85% to 115%, and analyte recoveries ranged from 85% to 115%, meeting acceptable standards. This validated UPLC-MS/MS method was successfully applied to determine plasma concentration in seven breast cancer patients receiving 3 mg weekly doses of entinostat, providing a powerful tool for clinical pharmacokinetic studies and therapeutic drug monitoring.