Determination of Ethinyl Estradiol and Levonorgestrel in Human Plasma with Prednisone as Internal Standard Using Ultra-performance Liquid Chromatography-Tandem Mass Spectrometry.

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Objective:Ethinyl estradiol and levonorgestrel as a combination of oral contraceptive drugs have very low dosage levels; hence, a highly sensitive and selective method of using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) is needed.Materials and Methods:This method was developed using prednisone as an internal standard, thus the purpose of this research was to get the optimum condition. The analytical method had been fully validated according to the European Medicines Agency guidelines, 2011. A reverse-phase chromatography separation was performed on ACQUITY UPLC ethylene bridged hybrid C18 column (1.7 μm, 2.1 × 50mm), eluted at a 0.3 mL/min flow rate under a gradient of mobile phase of 0.1% formic acid in water and acetonitrile within 5 min. Sample preparation used protein precipitation followed by liquid–liquid extraction. Quantification analysis was performed by a triple quadrupole mass spectrometry with electrospray ionization in positive-ion mode. The multiple reaction monitoring was set at m/z: 530.16 → 171.08 for ethinyl estradiol derivatized by dansyl chloride; m/z: 313.16 → 245.10 for levonorgestrel; and m/z: 359.10 → 147.04 for prednisone.Results:The validated method was accurate, precise, and sensitive with a lower limit of quantification at 5 and 100 pg/mL for ethinyl estradiol and levonorgestrel, respectively.