一种灵敏而稳健的 UPLC-MS/MS 方法，用于定量检测人血清中的雌激素和孕激素。
A sensitive and robust UPLC-MS/MS method for quantitation of estrogens and progestogens in human serum.
机构信息
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA.
Department of Obstetrics, Gynecology, and Reproductive Sciences and Center for Family Planning Research, School of Medicine, University of Pittsburgh, Pittsburgh, PA; Magee-Womens Research Institute, Pittsburgh, PA.
出版信息
Contraception. 2019 Apr;99(4):244-250. doi: 10.1016/j.contraception.2018.12.010. Epub 2019 Jan 24.
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.
目的
随着甾体激素避孕药和激素替代疗法的广泛应用，人们对可靠的分析方法的需求日益增加。我们报告了一种灵敏而强大的超高效液相色谱-串联质谱（UPLC-MS/MS）方法的开发，用于定量人血清中的内源性和合成甾体激素。
研究设计
我们开发并验证了一种 UPLC-MS/MS 方法，用于定量孕激素（依托孕烯、左炔诺孕酮、醋酸甲羟孕酮、去氧孕烯、孕酮）和雌激素（雌二醇和乙炔雌二醇），具有良好的准确性、高灵敏度和出色的稳健性。然后，我们将该方法应用于 451 名临床研究参与者的血清中甾体激素的分析。
结果
每个 UPLC-MS/MS 分析用时 6.5 分钟。孕激素的定量下限（LLOQ）为 25pg/ml，雌二醇和乙炔雌二醇的 LLOQ 分别为 2.5 和 5.0pg/ml。当不评估孕激素而仅分析雌二醇时，LLOQ 降低至 1pg/ml。校准曲线的线性范围分别为 25-50,000、2.5-2000（仅雌二醇分析为 1-2000）和 5-2000pg/ml。不仅在常规验证（日内和日间）中，而且在长期（>2 年）外部对照的分析稳健性中，准确性和精密度均低于±15%，从而证明了该方法在孕激素和雌激素多年临床研究评估中的实用性。我们应用该方法对 1804 个临床样本进行了定量分析。
结论
我们成功开发了一种 UPLC-MS/MS 方法，克服了基质抑制，能够灵敏地定量人血清中的合成和内源性甾体激素。
意义
我们开发了一种灵敏而强大的 UPLC-MS/MS 方法，可准确测量血清中甾体激素的水平。该方法克服了基质干扰的障碍，实现了出色的长期稳定性和重现性（≥96.9%的准确性；在 2 年内使用外部对照测量，相对变异性≤13.0%），证明了其在临床样本分析中的实用性。
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