Kawasaki T, Maeda M, Tsuji A
J Chromatogr. 1981 Nov 13;226(1):1-12.
A fluorescence high-performance liquid chromatographic method is described for the determination of 17-oxosteroids in biological fluids. 17-Oxosteroids in urine samples are extracted with dichloromethane after enzymatic hydrolysis (beta-glucuronidase-sulfatase), and dehydroepiandrosterone sulfate in serum samples is solvolysed with sulfuric acid in ethyl acetate. 17-Oxosteroids are labeled with dansyl hydrazine in trichloroacetic acid-benzene solution, and then chromatographed on the microparticulate silica gel column using dichloromethane-ethanol-water (400 : 1 : 2) as the mobile phase. The eluate is monitored by a fluorophotometer at 365 nm (excitation) and 505 nm (emission). Linearities of the fluorescence intensities (peak heights) with the amounts of various 17-oxosteroids were obtained between 60 and 1000 pg. The assay proved satisfactory with respect to sensitivity, precision and accuracy. The results obtained by a radioimmunoassay and this method were in good agreement (r = 0.964, n = 81) for serum dehydroepiandrosterone sulfate. This method is also use for the simultaneous determination of individual 17-oxosteroids in serum and urine.
本文描述了一种用于测定生物体液中17-氧代类固醇的荧光高效液相色谱法。尿样中的17-氧代类固醇在酶促水解（β-葡萄糖醛酸酶-硫酸酯酶）后用二氯甲烷萃取，血清样品中的硫酸脱氢表雄酮用乙酸乙酯中的硫酸进行溶剂解。17-氧代类固醇在三氯乙酸-苯溶液中用丹磺酰肼标记，然后在微粒硅胶柱上进行色谱分析，以二氯甲烷-乙醇-水（400:1:2）为流动相。用荧光光度计在365nm（激发）和505nm（发射）处监测洗脱液。各种17-氧代类固醇的荧光强度（峰高）与含量在60至1000pg之间呈线性关系。该测定法在灵敏度、精密度和准确度方面均令人满意。放射免疫分析法和本法对血清硫酸脱氢表雄酮的测定结果吻合良好（r = 0.964，n = 81）。该方法还可用于同时测定血清和尿液中的单个17-氧代类固醇。
