A novel and sensitive method for ethinylestradiol quantification in human plasma by high-performance liquid chromatography coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometry: Application to a comparative pharmacokinetics study

Abstract

In the present study, a novel, fast, sensitive and robust method to quantify ethinylestradiol in human plasma using 17α-ethinylestradiol-d4 as the internal standard (IS) is described. The analyte and the IS were extracted from acidified plasma by liquid–liquid extraction (LLE) using diethyl ether–hexane followed by online solid phase extraction (SPE) using online C18 cartridges. Extracted samples were analyzed by high-performance liquid chromatography coupled to atmospheric pressure photoionization tandem mass spectrometry (HPLC–APPI-MS/MS). Chromatography was performed isocratically on a C18, 5 μm analytical column. The method had a chromatographic run time of 2.50 min and a linear calibration curve over the range 5–500 pg ml −1 ( r 2 > 0.9992). The lowest concentration quantified was 5 pg ml −1, demonstrating acceptable accuracy and precision. The intra-assay precisions ranged from 2.1 to 14.6%, while inter-assay precisions ranged from 4.4 to 11.4%. The intra-assay accuracies ranged from 94.6 to 103.8%, while the inter-assay accuracies ranged from 98.9 to 101.6%. The recovery of ethinylestradiol was determined as part of the assay validation process and was 73.1 and 79.0% for the concentrations 15 and 375 pg ml −1, respectively. Short-term stability showed that ethinylestradiol was stable in plasma for at least 19 h at room temperature or for at least 385 days when stored at −20 °C. In the study of bioequivalence conducted in Brazil, healthy volunteers received two ethinylestradiol 0.035 mg tablet formulations using an open, randomized, two-period crossover design with a 2-week washout interval. Since the 90% confidence interval for C max and area under the curve ratios were all inside the 80–125% interval proposed by the US Food and Drug Administration, it was concluded that the two ethinylestradiol formulations are bioequivalent with respect to both the rate and the extent of absorption.