逆相系高速液体クロマトグラフィー-紫外吸光分析による内因性Δ⁴-ステロイドの同時測定法

Cortisone, Hydrocortisone, 21-deoxycortisol, 17α-hydroxyprogesteroneを中心として

抄録

Taking an advantage of the property of Δ⁴ -steroid that have a maximum absorbance around 250nm wave-length of ultraviolet, we devised an assay method for the determination of serum Δ⁴ -steroids concentration using a reverse phase high performance liquid chromatography (HPLC) -UV spectrometry.
The assay procedure was as follows : (1) A mixed solvent containing methanol, acetonitrile and water in 55/3/42 by volume was used as a mobile phase, and which was pumped at a constant flow rate of 1.5ml/min. (2) The main column and precolumn used were ERC-ODS-1161 (φ6mm × 10cm) and ERC-ODS-1652 (φ6mm × 3cm), respectively. (3) Two liquid-liquid extraction methods were used. One was a conventional method using dichloromethane for an extraction solvent, and the other was a simplified method using Extrelut column and ethylacetate. (4) Before a practical assay we examined the retention time of each steroid determined and its ratio of peak height to that of the internal standard (dexamethasone).
We found good correlations between the concentrations of cortisol (F), 17α-hydroxy-progesterone (17-OHP) and 21-deoxycortisol (21-DOF) estimated by this HPLC method and those by highly specific radioimmunoassay method.
The concentrations of cortisone (E) and F of eight umbilical venous blood specimens were 159.7 ± 26.3 (Mean SD) ng/ml and 93.3 ± 58.9 ng/ml, respectively, and 17-OHP was detected 7 of them and its concentration was 17.4 ± 12.4 ng/ml. On the other hand, 17-OHP and 21-DOF peaks could not be detected in 1 month old normal infants. In untreated patients with adrenal 21-hydroxylase deficiency (21-OHD), the concentrations of E and F were very low and those of 17-OHP and 21-DOF were both extremely high, which were 26.1 ± 18.5 ng/ml, 5.9 ± 4.3 ng/ml, 157.0 ± 122.6 ng/ml, 26.1 ± 10.0 ng/ml, respectively.
Our method is more simplified and has higher sensitivity than the methods previously reported, and several Δ⁴-steroids which are clinically important can be analysed in a brief time. It was concluded that we could make an easy and accurate diagnosis of disorders of steroid biosynthesis and metabolism including congenital adrenal hyperplasia by this method.