CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China.
Anal Chem. 2012 Dec 4;84(23):10245-51. doi: 10.1021/ac301984t. Epub 2012 Nov 13.
Steroid hormones are crucial substances that mediate a wide range of vital physiological functions of the body. Determination of the levels of steroid hormones plays an important role in understanding the mechanism of the steroid hormone-related diseases. In this study, we present a novel targeted metabolic profiling method based on the introduction of an easily protonated stable isotope tag to a hydroxyl-containing steroid hormone with a synthesized derivatization reagent, deuterium 4-(dimethylamino)-benzoic acid (d(4)-DMBA), and liquid chromatography-mass spectrometry (LC-MS). Different from other reported derivatization reagents that have been used to enhance the sensitivities for estrogens or androgens, our method is comprehensive with the capability of covering hydroxyl-containing androgens, estrogens, corticoids, and progestogens. Furthermore, the nonderivatized steroid hormones (e.g., 17α-hydroxyprogesterone, progesterone, and androstenedione) were not destroyed during the derivatization process, and their levels could still be obtained in one LC-MS run. We were able to detect 24 steroid hormones at subng/mL levels (the lower limit of detection could reach 5 pg/mL for estrone and 16α-hydroxy estrone, which is equivalent to 0.1 pg on column) with maximum sensitivity enhancement factors of more than 10(3)- to 10(4)-fold after derivatization. The method was successfully applied to the measurement of free (unconjugated) steroid hormones in urine samples of males, females, and pregnant women. Because the significant role the steroid hormone pathway plays in humans, a comprehensive, sensitive, specific, and accurate method for profiling the steroid hormone metabolome shall offer new insights into hormone-related diseases.
甾体激素是调节人体广泛重要生理功能的关键物质。测定甾体激素的水平对于了解甾体激素相关疾病的机制起着重要作用。在本研究中，我们提出了一种基于在含有羟基的甾体激素上引入易于质子化的稳定同位素标记物的新型靶向代谢组学方法，该甾体激素与合成的衍生化试剂氘代 4-(二甲氨基）-苯甲酸（d(4)-DMBA）一起使用，并通过液相色谱-质谱联用（LC-MS）进行分析。与其他已报道的用于增强雌激素或雄激素灵敏度的衍生化试剂不同，我们的方法具有全面性，能够涵盖含羟基的雄激素、雌激素、皮质激素和孕激素。此外，在衍生化过程中未破坏非衍生化的甾体激素（例如 17α-羟孕酮、孕酮和雄烯二酮），并且仍然可以在一次 LC-MS 运行中获得它们的水平。我们能够以亚纳克/毫升级别的水平（检测下限可低至雌酮和 16α-羟雌酮的 5 皮克/毫升，相当于柱上的 0.1 皮克）检测到 24 种甾体激素，衍生化后的最大灵敏度增强因子超过 10(3)-到 10(4)-倍。该方法成功地应用于男性、女性和孕妇尿液样品中游离（未结合）甾体激素的测量。由于甾体激素途径在人类中的重要作用，全面、敏感、特异和准确的方法来分析甾体激素代谢组学将为激素相关疾病提供新的见解。
