BIO-ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ETHINYL ESTRADIOL WITH ETHINYL ESTRADIOL-D4 AS INTERNAL STANDARD IN HUMAN K2-EDTA PLASMA BY LC-MS/MS

A sensitive method was developed to determine Ethinyl Estradiol in human plasma by liquid chromatography-tandem mass spectrometric (LC–MS/MS). Separation of analyte and the Internal Standard (IS) Ethinyl Estradiol-d4 using SPE followed by LLE in plasma with TBME, was performed on a LC/MS/MS-API-5500 instrument with SB C18 HT (50mm×3.0mm) 1.8 µm columns at a flow rate of 0.300 ml/min with mobile phase consisting of Acetonitrile: 2 mM Ammonium Formate Buffer (80:20 v/v). The interface used with the API 5500 LC/MS/MS was used a turbo ion spray. The positive ions were measured in MRM mode for the analyte and IS. The method was linear over range of 5.000 – 308.560 pg/mL. The correlation coefficient is ≥ 0.9942 for Ethinyl Estradiol. The mean % recovery of extraction across QC of Ethinyl Estradiol in plasma was 68.48%. The reproducibility of Ethinyl Estradiol QC sample at room temperature and at 5 ºC was between 91.80 to 101.20% and 90.63 to 101.44% respectively. The % CV of intra and inter day precision were less than 19.74%. In the present study, a novel, fast, sensitive and robust method to quantify at very low concentration (pg/mL) Ethinyl Estradiol in human plasma using Ethinyl Estradiol-d4 as the IS was described.