Development and validation of UPLC-MS/MS method for simultaneous determination of gestodene and ethinyl estradiol in rat plasma.

A selective and sensitive ultra-performance liquid chromatography method with tandem mass spectrometric detection for simultaneous determination of gestodene (GES) and ethinyl estradiol (EE) in rat plasma was developed and validated. GES, EE and the internal standard, norgestrel, were extracted with ethyl acetate, derivatized (EE only) with dansyl chloride and then back-extracted into diethyl ether-hexane (2:1, v/v). The separation was performed on an ACQUITY UPLC BEH C(18) column with gradient elution using mobile phase consisting of acetonitrile and water (both containing 0.1% formic acid). The detection was carried out by means of electrospray ionization (ESI) mass spectrometry in positive ion mode with multiple-reaction monitoring. Calibration curves of GES and EE were linear (r(2) >or= 0.99) over the concentration ranges 1.59-159 and 0.196-78.4 ng/mL, respectively. The intra- and inter-day precisions were not more than 6.9 and 12.9% for GES and 10.6 and 9.0% for EE, and the accuracies were -2.5-8.0% for GES, and -7.2-0.19% for EE, respectively. The method herein described was superior to previous methods and was applicable to the pharmacokinetic study of GES and EE in rats.