Simultaneous assay of segesterone acetate (Nestorone®), estradiol, progesterone, and estrone in human serum by LC-MS/MS

Abstract

Objective: To develop a method to simultaneously quantify the synthetic contraceptive progestin segesterone acetate (Nestorone®, NES) and the endogenous steroid hormones estradiol (E2), progesterone (P4), and estrone (E1) in human serum samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Study design: We analyzed 615 serum samples collected from 67 reproductive-age women actively using a contraceptive vaginal ring (CVR) designed to release NES (200 mcg/d) and E2 (75-200 mcg/d). Samples were taken prior to and up to 30 days after CVR insertion and analyzed for concentrations of NES, E2, P4, and E1 in human serum using a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. Precision, accuracy, and sensitivity for all analytes were determined across multiple assays.

Results: The assay ranges for NES, E2, P4, and E1 in this analytical method were 10 pg/mL to 10 ng/mL with a lower limit of quantification of 10 pg/mL for all targets. Assay precisions were less than or equal to 14.5% and accuracies ranged from 87.0% to 110.8%. When applied to the 615 clinical samples, 550 samples had quantifiable concentrations of NES (value range 0.014-1471 ng/mL). Similarly, 595 samples had quantifiable concentrations of E2 (0.010-0.312 ng/mL), 596 samples had quantifiable concentrations of P4 (0.010-5.791 ng/mL), and 609 samples had quantifiable concentrations of E1 (0.010-0.416 ng/mL).

Conclusions: The LC-MS/MS platform results in a robust, accurate, and sensitive method for the simultaneous quantification of NES and endogenous steroid hormones in human serum.

Implications: The analytical method described allows for the simultaneous quantification of NES and endogenous steroids and can be used to monitor NES concentrations during clinical trials and subject adherence to treatment with NES.

Keywords: Contraception; Estradiol; LC–MS/MS; Nestorone®; Segesterone acetate.

Figure 1.

Representative multiple reaction monitoring (MRM) chromatograms of unlabeled and stable isotope-labeled endogenous steroid hormones and Nestorone. The chromatogram in the top panel is representative off the simultaneous analysis of E2, E1, NES, and P4 (0.150 ng/mL each). Below are representative chromatograms for each individual analyte from a single injection of a mixed standard at the lower limit of quantification (LLOQ) for each hormone, with the LLOQ indicated in each respective panel. Peaks are colored to represent the different ions used in analysis of a particular target. MRMs used for quantification and other MS parameters can be found in Table 2. E2, estradiol; E1, estrone; NES, Nestorone; P4, progesterone; d, deuterium; CPS, counts per second; min, minutes.

Figure 2.

Representative multiple reaction monitoring (MRM) chromatograms of unlabeled and stable isotope-labeled endogenous steroid hormones and Nestorone in a charcoal-stripped serum (CSS) blank and a subject sample. The chromatogram in the top panel is a representative CSS blank, demonstrating absence of appreciable endogenous steroids. The chromatogram in the bottom panel is a representative simultaneous analysis of E2 (180 pg/mL), E1 (102 pg/mL), NES (178 pg/mL), and P4 (45 pg/mL) in a subject sample. E2, estradiol; E2, estrone; NES, Nestorone; P4, progesterone; d, deuterium; CPS, counts per second; min, minutes; CSS, charcoal-stripped serum.