A Validated Chiral HPLC Method for the Enantiomeric Separation Of Levosimendan in Bulk Drug Substances

The present paper describes the development and validation of novel chiral HPLC method for the enantiomeric separation of S-Levosimendan from R-Levosimendan and quantitative determination of S-Levosimendan enantiomer in Levosimendan bulk drugs. The enantiomers of levosimendan were baseline resolved on normal phase chromatographic separation on Amylose tris (3,5-dimethylphenylcarbamate) immobilized on 5μm silica-gel-Based Chiral Stationary Phase, Chiral pak IA column (250mm ×4.6mm i.d.; particle size,5μ) at a temperature of 30°C using a mobile phase consisting of MTBE:Ethanol:TFA (650 : 350 : 1.0, v/v/v) at a flow rate of 1.0mLmin-1 with an injection volume of 10μL. Quantitation was achieved with UV detection at 383nm based on peak area with linear calibration curves. The elution times of S-Levosimendan and R-Levosimendan were 6.8 min and 11.0 minutes respectively. In this proposed chiral HPLC method, the resolution between S-Levosimendan and R-Levosimendan was found to be greater than 8.0. The developed method was validated with respect to linearity, accuracy, precision, solution stability, ruggedness, robustness, limit of detection and limit of quantification. The recovery obtained for S-isomer was in between 99.1 % and 107.9%. The detection limit obtained for S-isomer was 0.025μg.mL-1 and the quantification limit was 0.075μg.mL-1 respectively. Linearity was performed for the S-isomer from LOQ to 150%. The correlation coefficient obtained for S-isomer was more than 0.999. The solution stability of Levosimendan bulk drug was determined and the compound was found to be stable up to 48 hrs. As the method has less run time (20 min), it can be useful in quality control laboratories for routine analysis.