Development of enantioselective assay and evaluation of pharmacokinetic properties of lenalidomide by HPLC & LC-MS/MS

Abstract

Immobilised polysaccharide based chiral stationary phase (CSPs) are a new generation of chromatographic materials combining the remarkable enantioselective performance of the polysaccharide derivatives and solvent versatility for enantiomeric resolution. Based on extensive experimental work, this work will focus on the approach to efficient method development with these Immobilised CSPs (Chiralpak IA, Chiralpak IB and Chiralpak IC) by applying a limited number of mobile phases. The experiments were performed under normal phase conditions. The influence of polar modifier, mobile phase composition, flow rate and column temperature on the etention time and separation factor behavior of Thalidomide (TLM) and Lenalidomide (LLM) was evaluated. Apparent thermodynamic parameters were also deduced from Van t Hoff plots, and some aspects of chiral recognition mechanism were studied. Lenalidomide, 3-(4-amino-1-oxo-3H-isoindol-2-yl) piperidine-2,6-dione, currently on the market and in therapeutic use as a racmate. In order to investigate single enantiomers of lenalidomide in pharmaceutical formulations both were chromatographically purified and characterized. High Performance Liquid Chromatography (HPLC) has enantiomerically separated the two enantiomers of lenalidomide (LLM) at semi-preparative scale on a polysaccharide based chiral stationary phase (Chiralpak IC, 250x10 mm, and 5 µm). The effect of organic modifier (2-propanol) was studied, and different injection volumes and concentrations of the lenalidomide racemic mixture were evaluated in order to obtain high enantiomeric purities. Better results were achieved using concentration overloading instead of volume overloading. The recoveries decreased when the requirements of enantiomeric purity or the load increased, but it was possible to recover gt99.1% of both enantiomers at an enantiomeric purity higher than gt99.60% under some loading conditions, like injecting 1 ml of a solution of 6 g/lit.