Abstract

A sensitive and rapid high performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of ten steroid hormones, including estrogens, androgens, progesterones, and corticosteroids four classes of steroids. The following ten steroid hormones were analyzed: progesterone, 21-deoxycortisol, estrone, 4-androstenedione, testosterone, dihydro-testosterone, androstenone, dehydroepiandrosterone, corticosterone and cortisone. Stable deuterated isotopes were used as internal standards for quantification. Sample preparation with and without derivatization were performed after liquid–liquid extraction, and the corresponding results were compared according to sensitivity and selectivity. Hydroxylamine derivatization was found to improve the ionization efficiency of the analytes for electrospray ionization MS analysis. The gradient of mobile phase and experimental parameters for HPLC separation were optimized. The lower limits of quantification were in the range of 0.05–5 ng mL⁻¹ with wide linear range for the ten steroid hormones. The intra-day precision < 11.1% and recovery of 84.5–120% with negligible matrix effect were achieved, where within the acceptance limits of the FDA guideline. Total HPLC–MS analysis time was 6 min. This method enables simultaneous quantification of steroids in human serum. It will be helpful for the serum steroid profiling in order to understand various endocrinology diseases.

Graphical Abstract

中文翻译：

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羟胺衍生化HPLC-MS法测定血清类固醇激素含量

摘要

建立了灵敏快速的高效液相色谱-质谱（HPLC-MS）方法，并同时定量了十种类固醇激素，包括雌激素，雄激素，孕酮和皮质类固醇四类类固醇。分析了以下十种类固醇激素：孕酮，21-脱氧皮质醇，雌酮，4-雄烯二酮，睾丸激素，二氢睾丸激素，雄烯酮，脱氢表雄甾酮，皮质酮和可的松。稳定的氘化同位素用作定量的内标。液-液萃取后，进行有无衍生化的样品制备，并根据灵敏度和选择性比较相应的结果。发现羟胺衍生化可提高用于电喷雾电离质谱分析的分析物的电离效率。优化了流动相的梯度和HPLC分离的实验参数。定量下限在0.05-5 ng mL范围内^-1，具有十种甾体激素的线性范围。在FDA准则的可接受范围内，日内精度<11.1％，回收率84.5–120％，基质效应可忽略不计。HPLC-MS的总分析时间为6分钟。这种方法可以同时定量人类血清中的类固醇。对于了解各种内分泌疾病，有助于血清类固醇分析。

图形概要