Development and Validation of a Simple HPLC–UV‐Based Bioanalytical Method for Estimation of Acalabrutinib in Rat Plasma and Its Application in Evaluation of Drug‐Loaded Nanocrystal Formulation

Abstract

ABSTRACTA robust and reproducible HPLC–UV‐based bioanalytical method for acalabrutinib (ACP) was developed and validated in rat plasma using prednisone as internal standard. The samples were prepared by single‐step protein precipitation method using methanol with 1% v/v formic acid. A polar C18 stationary phase was used with a combination of 10 mM ammonium acetate (pH 3.5) and methanol (60:40 ratio), flow rate 1.1 mL/min, and 50 µL injection volume. ACP and internal standard were retained at ∼5.96 and 8.29 min, respectively. The method was validated according to the United States Food and Drug Administration and ICH M10 bioanalytical method validation guidelines. It was linear in the calibration range (0.08–5 µg/mL) with as 0.9989; accurate (% bias ≤ 0.50 ± 0.67), and precise (%RSD ≤ 0.986) for any quality control standard. Further, the method was applied in estimating ACP in rat plasma samples collected during in vivo pharmacokinetic study of the bulk drug as well as a nanocrystal (NC) suspension formulation. ACP‐NCs were formulated by bottom‐up solvent–antisolvent precipitation with mean particle size 266.32 ± 29.90 nm, 0.25 ± 0.05 PDI, with 92.7% ± 2.3% yield. The NC formulation improved both in vitro dissolution and in vivo absorption, supporting a further development of ACP‐loaded nanodrug delivery systems.