Estrogens are among the most important hormones in the female body, which pass through distant organs, interact with multiple organ systems, and play a pivotal role in the physiologic events that occur during a woman’s life.  Presence of endogenous hormones in the plasma poses challenges for the pharmacokinetic and bioanalytical considerations in terms of endogenous baseline levels, low plasma concentration, analytical range, and possible effect of circulating hormones. In order to quantify plasma concentrations of estrogens in clinical trials, it was necessary to develop and validate an assay with appropriate sensitivity, selectivity, accuracy and precision. 
The objective of the present study is to develop the more sensitive, rapid and simultaneous quantification of estrogen i.e. Estradiol (E2) and Estrone (E1) in human plasma.
1. Chemicals: The pure substances of Estradiol, Estrone were procured from the council of Europe, (Strasbourg, Cedex). Amlodipine besilyate (internal standard) procured from Sigma-Aldrich. Dansyl chloride was procured from Acros organics (New Jersey, USA). Bovine serum albumin was used as control. HPLC grade methanol used for the chromatography. Analytical grade sodium hydroxide, sodium hydrogen carbonate was used (Sigma-Aldrich). Water was deionized using a Milli-Q system from Millipore (Bedford, MA, USA).
2. LC-MS/MS apparatus and conditions: The HPLC Alliance HT 2795 series (Waters, USA) is equipped with binary pump, degasser and autosampler with thermostat, thermostated column compartment and control module. The chromatography was on Genesis C18 (5-μm, 50 mm X 4.6 mm i.d.) at 40 ˚C. The mobile phase composition was a mixture of 0.1 % formic acid buffer and methanol in gradient mode, with a flow rate of 0.30 mL/min. Mass spectrometric detection was performed on ESI triple quadrupole instrument Quattro Premier (Micromass MS technologies, Waters, USA)...