BIO-ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ETHINYL ESTRADIOL WITH ETHINYL ESTRADIOL-D4 AS INTERNAL STANDARD IN HUMAN K2-EDTA PLASMA BY LC-MS/MSAbstract
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.
S. Udhayavani *, V. Girija Sastry, R. Govinda Rajan, A. Srinivas and J. K. D. Tejaswi
Department of Pharmaceutical Sciences, College of Pharmacy, Andhra University, Visakhapatnam, Andhra Pradesh, India.
27 December, 2016
10 February, 2017
17 February, 2017
01 July, 2017