Mitamura Kuniko, Satoh née Okihara Rika, Kamibayashi Mami, Sato Kanta, Iida Takashi, Ikegawa Shigeo
Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan.
Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan; Department of Chemistry, College of Humanities & Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan.
Steroids. 2014 Jul;85:18-29. doi: 10.1016/j.steroids.2014.03.015. Epub 2014 Apr 8.
A liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of eighteen tetrahydrocorticosteroid sulfates in human urine has been developed. The analytes were 3- and 21-monosulfates and 3,21-disulfates of tetrahydrocortisol (THF), tetrahydrocortisone (THE), tetrahydro-11-deoxycortisol (THS), and their corresponding 5α-H stereoisomers. The mass spectrometric behavior of these sulfates in negative-ion ESI-MS/MS revealed the production of intense structure specific product ions within the same group of sulfates and permitted distinction between regioisomeric sulfates by collision-induced fragmentation with the MS/MS technique using a linear ion-trap instrument. For the quantitative analysis, selected reaction monitoring analysis in the negative-ion detection mode using triple-stage quadrupole mass spectrometer was performed by monitoring transitions from M-H to the most abundant product ion of each tetrahydrocorticosteroid sulfate. After addition of 3- and 21-monosulfates of [2,2,3β,4,4-d5]-THF, -THE, and -THS as internal standards, urine sample was applied to a solid phase extraction using a lipophilic-weak anion exchange cartridge column, and then analyzed by LC/ESI-MS/MS. The method had satisfactory performance in terms of intra- and inter-assay precision (less than 9.7% and 9.6%, respectively), and accuracy (91.2-108.2%). The limit of quantification was lower than 2.5 ng/mL for all sulfates examined. We applied this method to determine the concentration of eighteen tetrahydrocorticosteroid sulfates in the urine of healthy subjects. Thus, we have developed a sensitive, precise and accurate assay for urinary tetrahydrocorticosteroid sulfates that should be useful for clinical and biological studies.
已开发出一种液相色谱（LC）/电喷雾电离（ESI）-质谱（MS）方法，用于直接测定人尿中18种四氢皮质类固醇硫酸盐。分析物为四氢皮质醇（THF）、四氢可的松（THE）、四氢-11-脱氧皮质醇（THS）的3-和21-单硫酸盐以及3,21-二硫酸盐，及其相应的5α-H立体异构体。这些硫酸盐在负离子ESI-MS/MS中的质谱行为显示，在同一组硫酸盐中会产生强烈的结构特异性产物离子，并且通过使用线性离子阱仪器的MS/MS技术进行碰撞诱导裂解，可以区分区域异构体硫酸盐。对于定量分析，使用三级四极杆质谱仪在负离子检测模式下进行选择反应监测分析，通过监测从M-H到每种四氢皮质类固醇硫酸盐最丰富的产物离子的跃迁来实现。加入[2,2,3β,4,4-d5]-THF、-THE和-THS的3-和21-单硫酸盐作为内标后，将尿样应用于亲脂性弱阴离子交换柱进行固相萃取，然后通过LC/ESI-MS/MS进行分析。该方法在批内和批间精密度（分别小于9.7%和9.6%）以及准确度（91.2-108.2%）方面具有令人满意的性能。所有检测的硫酸盐的定量限均低于2.5 ng/mL。我们应用该方法测定了健康受试者尿液中18种四氢皮质类固醇硫酸盐的浓度。因此，我们开发了一种灵敏、精确且准确的尿四氢皮质类固醇硫酸盐测定方法，该方法应有助于临床和生物学研究。
