Liquid Chromatographic Separation of a Novel Oxazolidinone Antibacterial Agent WCK 2996 Stereoisomers and Investigation of Thermodynamic Parameters on Separation

Abstract

WCK 2996 is a new molecular entity comprising of two stereoisomers. It possesses potent antibacterial activity active against gram-positive bacteria. This article describes liquid chromatographic method for the separation of stereoisomers of WCK 2996. The separation was achieved on Chiralpak AD-H (amylose based chiral stationary phase) using a mobile phase consisting of hexane: 2-propanol: methanol: acetic acid (70:10:20:0.2, v/v/v/v) at a flow rate 1.0 mL min-1. Chromatographic resolution (Rs) between two isomers was found to be more than 4. The developed method was extensively validated and proved to be robust. The detector response for stereoisomer-1 (SI-1) and stereoisomer-2 (SI-2) showed an excellent linear correlation over the concentration range 0.05 - 1.0 mg mL-1. The limit of detection (LOD) and limit of quantification (LOQ) were 0.15 and 0.45 μg mL-1 for SI-1 and 0.18 and 0.56 μg mL-1 for SI-2. Average recovery was in the range of 98.0 % to 101.0 % for SI-1 and 99.1 % to 102.0 % for SI-2. Analytical sample solutions were found to be stable up to 48 hrs. The method was found to be specific, sensitive, precise and accurate for quantitative determination of either of isomers. The method was scaled up for preparative separation and successfully applied to isolate the individual pure stereoisomers. One of the isolated stereoisomers was found to be 2 to 4 times more active than the other against various strains of bacteria. The thermodynamic parameters were studied to understand the effect of temperature on separation of stereoisomers. In addition, the effect of organic peak modifier on selectivity of stereoisomers was also studied.