[A reverse phase high performance liquid chromatography-UV spectrometry method for the analysis of several intrinsic...

Taking an advantage of the property of delta 4-steroid that have a maximum absorbance around 250 nm wave-length of ultraviolet, we devised an assay method for the determination of serum delta 4-steroids concentration using a reverse phase high performance liquid chromatography (HPLC)-UV spectrometry. The assay procedure was as follows: 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 1.5 ml/min. The main column and precolumn used were ERC-ODS-1161 (phi 6 mm X 10 cm) and ERC-ODS-1652 (phi 6mm X 3 cm), respectively. 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 ethyl acetate. 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), 17 alpha-hydroxyprogesterone (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.(ABSTRACT TRUNCATED AT 250 WORDS)