title: Reversed-phase high-performance liquid chromatography separation of adrenal steroids prior to radioimmunoassay: application in congenital adrenal hyperplasia.
abstract: 21-Hydroxylase deficiency (21-OHD) is the most common form of congenital adrenal hyperplasia (CAH), followed by 11beta-hydroxylase deficiency (11beta-OHD). Diagnostic serum markers for these conditions are 17-hydroxyprogesterone (17-OHP) and 11-desoxycortisol (S), respectively. In 21-OHD, the large amounts of 17-OHP are further 11beta-hydroxylated to form 21-deoxycortisol (21-DF), making it also an excellent marker of this disease. These steroids can be measured in blood by radioimmunoassay (RIA). In this paper, we report the use of high-performance liquid chromatography (HPLC) for steroid purification, prior to RIA determinations of 21-DF, S, 17-OHP, and testosterone (T) in ether-extracted serum. The chromatographic separation is developed in a BDS-Hypersil column using water-methanol (53:47, v/v) as the mobile phase. The method is applied to 35 patients with the classic form of 21-OHD (18 females, 17 males, 5.1-14.2 years old) and 2 with 11beta-OHD (1 female, 1 male, 9.5 and 12.6 years old). Thirteen control children (5 females and 8 males, 5.2-15.2 years) are also studied. The results obtained for all measured steroids are compatible with those reported in the literature. The method is precise, and recovery is adequate. The HPLC technique proved to be of value for the purification of several steroids from single serum samples prior to RIA in patients with CAH.
authors.affiliation: Division of Endocrinology, Department of Medicine, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Vânia Tonetto
authors.initials: VT
authors.lastname: Fernandes
authors.forename: Luciane M
authors.initials: LM
authors.lastname: Ribeiro-Neto
authors.forename: Samira Barbosa
authors.initials: SB
authors.lastname: Lima
authors.forename: José G H
authors.initials: JG
authors.lastname: Vieira
authors.forename: Ieda T N
authors.initials: IT
authors.lastname: Verreschi
authors.forename: Claudio E
authors.initials: CE
authors.lastname: Kater
mesh_terms: D019326:17-alpha-Hydroxyprogesterone / Q000097:blood*; D000293:Adolescent; D000312:Adrenal Hyperplasia, Congenital / Q000097:blood*; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003350:Cortodoxone / Q000097:blood*; D005260:Female; D006801:Humans; D008297:Male; D011863:Radioimmunoassay / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D019326:17-alpha-Hydroxyprogesterone; D003350:Cortodoxone