A semi-automated 96-well plate method for the simultaneous determination of oral contraceptives concentrations in human plasma using ultra performance liquid chromatography coupled with tandem mass spectrometry

Abstract

Two semi-automated, relatively high throughput methods using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) were developed for the simultaneous determination of ethinyl estradiol (EE) in combination with either 19-norethindrone (NE) or levonorgestrel (LN) in human plasma. Using 300 microL plasma, the methods were validated over the concentration ranges of 0.01-2 ng/mL and 0.1-20 ng/mL for EE and NE (or LN), respectively. The existing methods for the determination of the oral contraceptives in human plasma require large volumes of plasma (> or =500 microL), and sample extraction is labor-intensive. The LC run time is at least 6 min, enabling analysis of only about 100 samples a day. In the present work the throughput was greatly improved by employing a semi-automated sample preparation process involving liquid-liquid extraction and derivatization with dansyl chloride followed by UPLC separation on a small particle size column achieving a run time of 2.7 min. The validation and actual sample analysis results show that both methods are rugged, precise, accurate, and well suitable to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in a day.