Dong Zhen, Wang Caihong, Zhang Jinlan, Wang Zhe
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, 100050, China.
Anal Bioanal Chem. 2017 Aug;409(20):4751-4769. doi: 10.1007/s00216-017-0419-2. Epub 2017 Jun 20.
It is important to profile steroids in many physiological and pathological processes. Recently, hair has been used for the long-term measurement of endogenous steroid hormones. Analyzing hair has advantages of being noninvasive and time sequential compared with other bio-specimens. Liquid chromatography-mass spectrometry (LC-MS) techniques have been widely used over the past decades; however, it is challenging to profile estrogens in hair by LC-MS, and more comprehensive steroid profiling is required. In this paper, an ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to simultaneously profile 28 multifunctional steroids, including corticosteroids (n = 6), estrogens (n = 13), androgens (n = 5) and progestogens (n = 4), in human scalp hair in a single run. To optimize the sample preparation procedure, we evaluated extraction time, post-incubation purification and hair fragment length; 30 mg hair samples were washed with hexane, cut into 5 mm pieces and incubated in methanol for 18 h at 25 °C. Methanol extraction derivatized using Girard P and dansyl chloride reagent was analyzed within 25 min using an automated injection program combined with a diverter valve switch and step analysis (AIDSA). The method was well validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect and recovery, and was successfully applied to a steroid profile from male and female hairs. Significant differences were observed between genders. In addition, steroids showed a declining trend from the proximal to more distal hair segments; thus, care should be taken when obtaining hair samples for analysis to account for this difference in steroid levels along the length of hair. Graphical Abstract The workflow of the estabished UHPLC-MS/MS method.
在许多生理和病理过程中对类固醇进行分析具有重要意义。近年来，毛发已被用于内源性类固醇激素的长期检测。与其他生物样本相比，分析毛发具有非侵入性和时间连续性的优点。液相色谱-质谱（LC-MS）技术在过去几十年中得到了广泛应用；然而，通过LC-MS对毛发中的雌激素进行分析具有挑战性，需要更全面的类固醇分析。本文开发了一种超高效液相色谱-串联质谱（UHPLC-MS/MS）方法，可在一次运行中同时分析人头皮毛发中的28种多功能类固醇，包括皮质类固醇（n = 6）、雌激素（n = 13）、雄激素（n = 5）和孕激素（n = 4）。为了优化样品制备程序，我们评估了提取时间、孵育后纯化和毛发片段长度；将30 mg毛发样品用己烷洗涤，切成5 mm片段，并在25°C下于甲醇中孵育18 h。使用Girard P和丹磺酰氯试剂衍生化的甲醇提取物，通过自动进样程序结合分流阀切换和阶梯分析（AIDSA）在25分钟内进行分析。该方法在线性、检测限（LOD）、定量限（LOQ）、精密度、准确度、基质效应和回收率方面得到了很好的验证，并成功应用于男性和女性毛发的类固醇分析。观察到性别之间存在显著差异。此外，类固醇从毛发近端到远端呈现下降趋势；因此，在获取毛发样本进行分析时应注意毛发长度上类固醇水平的这种差异。图形摘要：所建立的UHPLC-MS/MS方法的工作流程。
