AN AUTOMATED METHOD FOR THE DETERMINATION OF URINARY 17-HYDROXYCORTICOSTEROIDS

Abstract

SUMMARY An automated method for measuring urinary 17-hydroxycorticosteroids is described. The preliminary borohydride reduction of pre-existing 17-oxo-steroids is performed manually and the reduced urine transferred to the sample cups of an Auto-Analyzer. The urine is submitted to periodate oxidation, mild alkaline hydrolysis and then extracted with dichloromethane. The dichloromethane extract is mixed with an aqueous solution of m-dinitrobenzene in hyamine and the mixture passed through a 'solvent exchanger' at 75° to evaporate the dichloromethane. Alkali is added and the Zimmermann reaction performed at 55°. After extraction of the reaction products into n-hexanol their light absorption is measured at 510 mμ. The method has been evaluated for accuracy, precision, sensitivity and validity. Urine samples can be assayed in duplicate at the rate of 5/hr. with adequate accuracy. Passage time through the automated procedure is 36 min.; thus 30 to 40 samples can easily be assayed in a normal working day. Details are given of the recovery of cortisone from urine and of comparison between the results obtained with the automated method and with a manual method. The choice of cortisone as the standard, and the role of the temperature and the pH maintained during periodate oxidation are discussed.