Quantitative Stability Indicating Bio-analytical Method Development and Validation of Apalutamide - Apalutamide D3 By Using Ultra Performance Liquid Chromatography in Human Plasma

Abstract

A simple, convenient, specific, precise and highly conventional stability-indicating ultra-performance liquid chromatographic‑ diode array method was developed for the quantification of Apalutamide in human plasma. The Phenomenex Luna (100x4.6x5µ) column was used for apalutamide separation. The mobile phase was composed with 5 mM ammonium fumarate and acetonitrile in the ratio of 15:85 v/v, and buffer pH 3.5 was adjusted with glacial acetic acid and detected at 345 nm. The Apalutamide‑D3 used as internal standard and K2‑EDTA used as a coagulant. The liquid-liquid extraction process used for extraction of drug from human plasma with tert butyl methyl ether. The retention times of Apalutamide and Apalutamide D3 (ISTD) was 1.48 min & 1.97 min, respectively. The assay of the method was validated in human plasma in the concentration range from 307.26-200013.87 pg/ml with the accuracy and precision ranging from 3.86 to 4.87. Recovery studies were found to be 103.79%, 90.93% & 96.83% for HQC, MQC and LQC respectively. The stability of the drug was evaluated in human plasma with different conditions of the auto-sampler, freeze-thaw, bench top, short term and long term stability studies were performed. The method was proved as highly sensitive and selective for the quantification of Apalutamide and determined at the picogram level. There was no matrix effect observed and proved as a stability-indicating method.