The Establishment of the Assay-system of Blood Adrenal Steroids in the Method of Gaschromatograph/Mass Spectrometry (GC/MS)

Abstract

In this study, we established a method for the quantitative measurement of native adrenal steroids with GC-MS equipped with capillary column (cross-linked methyl silicone 25 m X 0.2 mm I.D., 0.11 m thin film). 1 ml of serum sample containing 5 alpha-cholestane as internal standard (IS) was elicited by organic solvent using extrelunt column. These samples were derived by n-butylboronic acid, o-methylhydroxylamine and trimethyl-silylating agents, then were finally applied to GC-MS. The intensities of molecular ions were used for the measurement of the serum concentration of steroids. The molecular ion peaks of steroids were obtained at m/z460 (17 alpha-hydroxyprogesterone; 17OHP), m/z548 (corticosterone; B), m/z470 (11-deoxycortisol; S), m/z417 (pregnenolone; PL), m/z372 (progesterone; PT), m/z558 (cortisol; F), m/z389 (dehydroepiandrosterone; DHEA), m/z371 (estrone; E1), m/z416 (estradiol; E2), m/z504 (estriol; E3), m/z389 (testosterone; T), m/z344 (androstenedione; A) and m/z372 (IS). The curve of calibration for each steroid showed good linearity. The sensitivities of the GC/MS method were less than 5pg/one shot of each sample. The coefficients of variations of accuracies and precisions in this GC/MS method were less than 15% of each steroid. The samples from normal subjects after metyrapone and ACTH loading tests, and the patients of congenital adrenal hyperplasia showed a good correlationship between the data of GC/MS and the data of RIA after sephadex LH-20 column-chromatography. These results implied the usefulness of our system in clinical application. Moreover, this assay takes only 3 hrs. Thus it saves much time in comparison with the time-consuming radioimmunoassay system.