Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
41, Number 5, May/June 2003, pp 251-254
Reversed-Phase High-Performance Liquid Chromatography Separation
of Adrenal Steroids Prior to Radioimmunoassay: Application in Congenital Adrenal
Hyperplasia
Vânia Tonetto Fernandes[1],[2], Luciane M. Ribeiro-Neto[1],
Samira Barbosa Lima[3], José G.H. Vieira[1], Ieda T.N. Verreschi[1],
and Claudio E. Kater[1]
[1]Division of Endocrinology, Department of Medicine, Universidade Federal
de São Paulo (UNIFESP), São Paulo, Brazil;
[2]Pediatric Endocrinology
Service, Hospital Infantil Darcy Vargas, São Paulo, SP, Brazil; and
[3]Faculdade de Medicina Veterinária e Zootecnia, Universidade de São
Paulo–USP, Brazil
21-Hydroxylase deficiency (21-OHD) is the most common form of
congenital adrenal hyperplasia (CAH), followed by 11b-hydroxylase deficiency
(11b-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 11b-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 11b-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.
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