[A method for the quantitative analysis of steroid hormones by HPLC/RIA]

To solve the problem of cross-reaction in immunoassay and determine various steroid hormones simultaneously in a small amount of sample, a method for the quantitative analysis of steroid hormones was developed. This method is a combination of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The purpose of the study is the comprehensive analysis of steroid hormones profiles in normal subjects and adreno-cortical diseases. One hundred microliters of plasma was extracted by ether and the ether layer was evaporated. The residue was redissolved and separated by HPLC. Then fractions of steroid hormones were taken and determined by RIA. In this study, cortisol (F), androstenedione (A), 17 alpha-hydroxyprogesterone (17-OHP), testosterone (T), progesterone (P), estrone (E1) and estradiol (E2) were analyzed in normal adults, congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and Cushing's syndrome. Results in normal adults were similar with those had been previously reported. In CAH, F was remarkably low and 17-OHP, A, T and P were remarkably high before treatment. During treatment some cases showed that 17-OHP, A, T and P were high, and 17-OHP and P tended to be within normal range, if F had been kept higher than about 20 micrograms/dl. In the analysis of Cushing's syndrome before treatment, there were definite differences between adenoma and hyperplasia. A, 17-OHP, T, E1 and E2 were higher in hyperplasia than those in adenoma. It is suggested that it is possible to diagnose the type of Cushing's syndrome with a small amount of plasma using this method. As a mass screening method of CAH at present, 17-OHP in dried blood on filter paper is determined, therefore the quantitative analysis of 17-OHP in dried blood on filter paper (9 mm disc) was attempted. The quantitative analysis proved to be possible, and it was considered to be applied as the secondary screening method of CAH by the use of dried blood on filter paper.