HPLC Determination of Enantiomeric Purity of PF-04136309 Based on a Chiral Stationary Phase

Abstract

A precise and sensitive LC method was developed and further validated for the determination of enantiomeric purity of (S)-N-[2-(3-{trans-4-hydroxy-4-[5-(pyrimidin-2-yl)pyridin-2-yl] cyclohexylamino} pyrrolidin-1-yl)-2-oxoethyl]-3-(trifluoromethyl) benzamide (PF-04136309). Baseline separation with a resolution higher than 1.8 was accomplished within 40 min using a CHIRALPAK AD (250 × 4.6 mm; particle size 5 μm) column, with n-hexane:2-propanol (70:30 v/v) as mobile phase at a flow rate of 1 mL min−1. The eluted analytes were subsequently detected with a UV detector at 260 nm. The effects of mobile phase components and temperature on enantiomeric selectivity as well as the resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within a concentration range between 0.01 and 1 mg mL−1 (n = 9), and recoveries between 98.17 and 101.28 % were obtained, with relative standard deviation (RSD) lower than 1.44 %. The LOD and LOQ for PF-04136309 were 3.59 and 11.54 μg mL−1 and for its enantiomer were 3.39 and 11.28 μg mL−1, respectively. The developed method was demonstrated to be accurate, robust and sensitive for the determination of enantiomeric purity of PF-04136309, especially for the analysis of bulk samples.