DETERMINATION OF MICRO-AMOUNTS OF 5β-PREGNAN-3α-OL-20-ONE IN URINE USING INFRARED-SPECTROMETRY

Abstract

A procedure for the quantitative determination of 5β-pregnan-3α-ol-20-one in urine is described. After acid hydrolysis of the pregnanolone-conjugates in urine, the free steroids are extracted with toluene. Pregnanolone is isolated in a pure form as its acetate; after chromatographic separation of the free steroids on alumina, the fraction containing pregnanolone is acetylated and rechromatographed on alumina. Quantitative determination of the isolated pregnanolone-acetate is carried out with the aid of the infrared spectrum recorded by a micro KBr-wafermethod. The reliability of the method under various conditions is discussed under the headings, specificity, accuracy, precision and sensitivity. It is possible to determine 30–40 μg pregnanolone in a 24-hours urine portion with a precision of 25%.