Iwata J, Suga T
Department of Clinical Biochemistry, Tokyo College of Pharmacy, Japan.
Clin Chem. 1989 May;35(5):794-9.
We describe a direct method for determining four sulfates and seven glucuronides of 17-oxosteroids (17OS) in urine without hydrolysis, by use of "high-performance" liquid chromatography (HPLC) with fluorometric detection. After pretreatment of urine samples with a Sep-Pak C18 cartridge, four 17OS sulfates and seven 17OS glucuronides in the pretreated urine samples were reacted with tetrapentylammonium ions to form ion pairs. Ion-paired 17OS sulfates were extracted with benzene. By adding sodium sulfate to the remaining sample, we could then extract ion-paired 17OS glucuronides with dichloromethane. Each extract was labeled with dansyl-hydrazine in an acetic acid-acetonitrile solution. The labeled steroids were separated by HPLC on a reversed-phase Capcell-Pak C8 (silicon-polymer-coated silica gel modified with octyl groups). We monitored each effluent with a fluorometric detector (330 nmexcitation, 535 nmemission).
我们描述了一种直接测定尿液中17-氧代类固醇（17OS）的四种硫酸盐和七种葡萄糖醛酸苷的方法，无需水解，采用带荧光检测的“高效”液相色谱（HPLC）。用Sep-Pak C18柱对尿液样本进行预处理后，预处理尿液样本中的四种17OS硫酸盐和七种17OS葡萄糖醛酸苷与四戊基铵离子反应形成离子对。离子对的17OS硫酸盐用苯萃取。通过向剩余样品中加入硫酸钠，然后我们可以用二氯甲烷萃取离子对的17OS葡萄糖醛酸苷。每种提取物在乙酸-乙腈溶液中用丹磺酰肼标记。标记的类固醇通过HPLC在反相Capcell-Pak C8（用辛基改性的硅聚合物涂层硅胶）上分离。我们用荧光检测器（激发波长330nm，发射波长535nm）监测每种流出物。
