Dataset Information

A Sensitive and Specific ESI-Positive LC-MS/MS Method for the Quantitation of Estrogens in Human Serum in Under Three Minutes.

ABSTRACT: Amplifex Diene reagent was employed to derivatize estradiol (E2) to enhance the analyte signal at low picogram concentrations. This derivatization enabled measurement of E2 (and other estrogens) in ESI+ mode, earlier retention times for analytes than other methods, avoidance of MS harmful ammonium fluoride in mobile phases, and an LLOQ below 1 pg/mL. The sample preparation workflow involved liquid-liquid extraction followed by Amplifex Diene derivatization for 10 min at ambient temperature. Samples were chromatographed using a standard C18 column and analyzed using a SCIEX 6500+ mass spectrometer. The assay calibrators were prepared in-house, traceable to certified reference materials, and ranged from 1.29 to 624 pg/mL. A method comparison to samples from the CDC HoSt program yielded a correlation coefficient of 0.9858 and bias of -1.37%. The LLOQ using certified reference material was 0.66 pg/mL. The intra-run precision was <9.00% for low- and high-level samples, whereas the inter-run precision was 15.2 and 5.43% for low- and high-level samples, respectively. No interference from other clinically relevant steroids was found. Amplifex Diene derivatized E2 and estrone (E1) was found to be stable for over 6 months, both refrigerated and frozen.

SUBMITTER: Stella A

PROVIDER: S-EPMC7882671 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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A Sensitive and Specific ESI-Positive LC-MS/MS Method for the Quantitation of Estrogens in Human Serum in Under Three Minutes.

Stella Aaron A Dey Subhakar S

Journal of chromatographic science 20210201 3

Amplifex Diene reagent was employed to derivatize estradiol (E2) to enhance the analyte signal at low picogram concentrations. This derivatization enabled measurement of E2 (and other estrogens) in ESI+ mode, earlier retention times for analytes than other methods, avoidance of MS harmful ammonium fluoride in mobile phases, and an LLOQ below 1 pg/mL. The sample preparation workflow involved liquid-liquid extraction followed by Amplifex Diene derivatization for 10 min at ambient temperature. Samp ...[more]

PMID: 33306780

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Project description: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, 2.5-2000 (1-2000 for estrogens-only analysis) and 5-2000 pg/ml, respectively. Both the accuracy and precision were below±15% not only for routine validation (intraday and interday), but for long-term (>2 years) assay robustness with external controls, thereby, demonstrating the utility of this method for multi-year clinical trial assessments of progestogens and estrogens. We applied the method to quantify sex-steroid levels in 1804 clinical samples. CONCLUSIONS:We successfully developed a UPLC-MS/MS method, and overcame the matrix suppression to allow sensitive quantitation of both synthetic and endogenous sex-steroid hormones in human serum. IMPLICATIONS:We developed a sensitive and robust UPLC-MS/MS method to accurately measure the levels of sex-steroid hormones in serum. The method overcame matrix interference barriers and achieved excellent long-term stability and reproducibility (≥96.9% accuracy; ≤13.0% relative variability measured with external controls over 2 years), demonstrating its utility in clinical sample analysis.

Dataset Information

A Sensitive and Specific ESI-Positive LC-MS/MS Method for the Quantitation of Estrogens in Human Serum in Under Three Minutes.

Publications

A Sensitive and Specific ESI-Positive LC-MS/MS Method for the Quantitation of Estrogens in Human Serum in Under Three Minutes.

Similar Datasets

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