ANALYSIS OF LYNESTRENOL IN HUMAN PLASMA IN VITRO BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY UV-VIS: EUROPEAN MEDICINES AGENCY GUIDELINE
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 (60:40), 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.
2. Kaur S, Kaur T, Kaur G, Verma S. Development and validation of a UV-Spectrophotometric method for estimation of hydroquinone in bulk marketed cream and prepared NLC formulation. Int J Appl Pharm 2017;9:102-8.
3. Korhonen T, Miia T, Ari T, Kari L, Olavi P. Identification of the human cytochrome P450 enzymes involved in the in vitro biotransformation of lynestrenol and norethindrone. J Steroid Biochem Mol Biol 2008;100:56-66.
4. Yasuda J, Hideo H, Hirosi O. Metabolism of lynestrenol: characterization of 3-hydroxylation using rabbit liver microsomes in vitro. J Steroid Biochem 1984;2:777-80.
5. Lalitha KV, Raveendra RJ, Devanna N. Validated stability-indicating high-performance liquid chromatography method for the estimation of torsemide. Asian J Pharm Clin Res 2019;13:26-32.
6. Shi JYQ, Yao FL, Hu CQ, Yuan QM, Zhang S, Jin H. Establishment of an HPLC identification system for detection of counterfeit steroidal drugs. J Pharm Biomed Anal 2008;46:663–9.
7. Meyer FR. Practical high-performance liquid chromatography. 4th Ed. John Wiley and Sons, New York; 2004.
8. Blister DM. Validating chromatographic methods, a Practical Guide, John Wiley and Sons, Inc., Hoboken; 2006.
9. Harahap Y, Nufus, Firda Z, Sunarsih. Effect of various anticoagulants on ethinyl estradiol and levonorgestrel analysis in human plasma in vitro by ultra-performance liquid chromatography-tandem mass spectrometry. J Global Pharma Tech 2018;10:42-9.
10. European Medicines Agency. Guideline on Bioanalytical Method Validation Vol. 44. London: An agency of the European Union; 2011.
11. Wenkui L, Ying HL, Austin C, Shaolin Z, Weng N. Simultaneous determination of norethindrone and Ethinyl estradiol in human plasma by high-performance liquid chromatography with tandem mass spectrometry-experience on developing a highly selective method using derivatization reagent for enhancing sensitivity. J Chromatography B 2005;825:223-32.
12. Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. United States of America: Department of Health and Human Service; 2013.
This work is licensed under a Creative Commons Attribution 4.0 International License.