A sensitive and robust UPLC–MS/MS method for quantitation of estrogens and progestogens in human serum

Abstract

Objective: With the widespread use of sex-steroid hormones in contraceptives and hormone replacement therapy, there is an increasing need for reliable analytical methods. We report the development of a sensitive and robust UPLC-MS/MS method for quantitation of both endogenous and synthetic sex-steroid hormones in human serum. Study Design: We developed and validated a UPLC-MS/MS method to quantify progestogens (etonogestrel, levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone) and estrogens (estradiol and ethinyl estradiol) with good accuracy, high sensitivity, and excellent robustness. We then applied the method to the analysis of sex-steroid hormones in serum from 451 clinical research participants. Results: Each UPLC-MS/MS analysis was 6.5 min. The lower limits of quantitation (LLOQs) were 25 pg/ml for the progestogens, and 2.5 and 5.0 pg/ml for estradiol and ethinyl estradiol, respectively. When estradiol was analyzed without assessment of progestogens, the LLOQ was reduced to 1 pg/ml. The calibration curves were linear from 25-50,000, 2.5-2,000 (1-2,000 for estrogens-only analysis) and 5-2,000 pg/ml, respectively. Both the accuracy and precision were below ±15% not only for routine validation (intraday and interday), but for long-term (> two years) assay robustness with external controls, thereby, demonstrating the utility of this method for

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

“…Advantages of the LC-MS/MS method for NES analysis we describe here include requirement of a relatively small sample size (200 μL) and a sample preparation method that does not require derivatization for the simultaneous measurement of endogenous estrogens. While derivatization is common to reach an appropriate LLOQ for E2 measurement [19], we were able to create a method with an adequate LLOQ for E2 without the use of chemical derivatization during sample preparation. Of the 615 samples to which the method was applied, three samples were undetectable or below the 3 pg/mL MDL for E2; one was less than the MDL for P4, and no samples measured below the MDL for E1.…”

Spatiotemporal regulation of human IFN-ε and innate immunity in the female reproductive tract

“…The samples were then transferred to 70% ethanol solution at room temperature. Blood samples were also evaluated by UPLC/MS/MS for quantification of estrogens and progestogens as previously described at the Magee-Womens Research Institute, Pittsburgh, PA, USA (14).…”

Determination of estrogens in human serum using a novel chemical derivatization–assisted liquid chromatography‐electrospray ionization‐tandem mass spectrometry method

“…To date, a variety of analytical strategies have been developed for the quantitative detection of estrogens, such as enzyme-linked immunosorbent assay (ELISA), [9][10][11] radioimmunoassay (RIA), [12][13][14] gas chromatography-mass spectrometry (GC-MS), 15 and liquid chromatography-mass spectrometry (LC-MS). [16][17][18][19][20][21] Immunoassays such as RIA and enzyme-linked immunoassay have been widely used in clinical and environmental laboratories. 19 However, ELISA has poor selectivity and is likely to cross-bind to exogenous compounds rather than the target estrogens.…”

“…15 LC-MS/MS has advantages such as high sensitivity, high accuracy, and high throughput and thus has become the primary technology platform for the detection of estrogens in complex matrices. [18][19][20][21] However, many steroid hormones are present in vivo in low concentrations and a large dynamic range, such as androgens and estrogens, in children before puberty or postmenopausal women. A major obstacle in these analyses is that many estrogens are weakly ionized during electrospray ionization (ESI).…”