UPLC–MS/MS assay for the simultaneous determination of ethinyl estradiol, norgestimate and 17-Desacetyl norgestimate at low pg/mL in human plasma

•A UPLC–MS/MS assay has been validated for the simultaneous quantitation of EE2, NGM and DNGM in human plasma at low pg/mL range using dansyl chloride as the derivatization reagent.•EE2, NGM and DNGM were separated from endogenous interference by careful optimization of chromatographic conditions and selection of MRM transitions.•The validated 3-in-1 method has been applied to a NGM/EE2 BE study for the simultaneous determination of EE2, NGM and DNGM concentrations in human plasma.

Previously, because of the difficulty of measuring very low levels (pg/mL) of norgestimate (NGM) due to rapid metabolism to its active and major metabolite, 17-Desacetyl norgestimate (DNGM), only DNGM and the co-administered ethinyl estradiol (EE2) were required to be analyzed in bioequivalence (BE) studies for oral contraceptive NGM/EE2 tablets. Recently, with more sensitive assays available, health authorities have requested that bioequivalence of NGM be also demonstrated in addition to DNGM and EE2. Therefore, it was important to establish a 3-in-1 method for the quantitation of EE2, NGM and DNGM in human plasma. Here a UPLC–MS/MS method for the simultaneous quantitation of EE2, NGM and DNGM in human plasma at low pg/mL range is described. EE2, NGM, DNGM and their isotopic labeled internal standards (IS) EE2-d4, NGM-d6 and DNGM-d6 in 0.4 mL of human plasma were extracted with hexane/ethyl acetate. The extracts were evaporated to dryness and derivatized with dansyl chloride, to enhance the mass spec response. The derivatives were reconstituted with methanol and analyzed by UPLC–MS/MS. In order to minimize the ex-vivo conversion of NGM to DNGM, sodium fluoride/potassium oxalate was used as anti-coagulant. To achieve desirable throughput for sample analysis, UPLC–MS/MS with a run time of 4.4 min was utilized. The calibration curve ranges were 5–500 pg/mL for EE2 and NGM, and 25–2500 pg/mL for DNGM, respectively. The chromatographic separation was achieved on a Waters Acquity UPLC HSS T3 (100 × 2.1 mm, 1.8 μm) column with a gradient elution. Assay accuracy, precision, linearity, selectivity, sensitivity and analyte stability covering sample storage and analysis were established. This validated UPLC–MS/MS method has been applied to a BE study for the determination of EE2, NGM and DNGM concentrations in human plasma.