Fluorescence high performance liquid chromatographic determination of 3 alpha-hydroxysteroids in urine of 21-hydroxylase deficiency.
A fluorescence high performance liquid chromatographic method using an immobilized 3 alpha-hydroxysteroid dehydrogenase column as a post-column enzymatic reactor was developed for the determination of corticosteroid metabolites in the urine of subjects with congenital adrenal hyperplasia. 3 alpha-Hydroxysteroids, such as pregnanetriol, pregnanediol and pregnanetriolone, in the eluate from mu-Bondapak phenyl column (300 x 3.9 mm I.D.) using 0.05% ammonium phosphate buffer (pH 7.1)-acetonitrile-methanol (100:55:15) as the mobile phase was mixed with NAD+ solution in the enzyme column at 30 degrees C to generate NADH, which was monitored by a fluorophotometric detector. Each steroid was measured at the 2.5 micrograms/dl at the highest sensitivity of the detector. The mean recoveries and reproducibilities were 91.5-108.2% with 0.9-6.5% (CV%).
D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000172:deficiency* / Q000652:urine; D011276:Pregnanediol / Q000652:urine*; D011279:Pregnanetriol / Q000031:analogs & derivatives* / Q000652:urine*; D013050:Spectrometry, Fluorescence
D011279:Pregnanetriol; C009030:pregnanetriolone; D006899:Mixed Function Oxygenases; D011276:Pregnanediol