Analysis Of Lynestrenol In Human Plasma In Vitro By High-Performance Liquid Chromatography Uv-Vis: European Medicines Agency Guideline

ABSTRACT

Objective: Development and validation of reverse phase high performance liquid chromatographic (RP-HPLC) method with UV-Vis detector for in vitro determination of lynestrenol with levonorgestrel as an internal standard in human plasma. Methods: The RP-HPLC method was developed using a C18 Sunfire© waters column with a mobile phase of acetonitrile containing 0.1% formic acid in water (6040), respectively, at a flow rate of 1.0 ml/min and was detected at a wavelength of 204 nm. Lynestrenol and levonorgestrel were extracted from human plasma using pentane with protein precipitation method. Results: The RP-HPLC method was able to selectively quantify lynestrenol in blood plasma on 40 ng/ml. The assay exhibited a linear dynamic range 40-1000 ng/ml for lynestrenol with retention time 4.0 second, and the coefficient correlation (r) was 0.9994. Accuracy (% diff) of this method was-10.81% to 8.72% with precision (CV) being 3.84% to 8.12%, and complete recovery was established to be 98.27% to 106.49%. The method was sensitive, selective, and has simple sample preparation extraction lynestrenol in plasma with pentane was successfully developed. Conclusion: The method can be used to analyze lynestrenol in blood plasma, with a simple pretreatment procedure using pentane.