Abstract
Most present colorimetric methods of assay 1-4 for the estrogenic hormones have two serious defects: a lack of specificity and the production of interfering colors with inactive impurities. Bachman, 5 however, has recently reported a modification of the Kober reaction which appears to be specific for estriol.
This report deals with an investigation of guaiacolsulfonic acid: (CH3+O) (HO) (C6H3+SO3H), substituted for phenolsulfonic acid as the color reagent in the Kober 1 reaction. The reagent was prepared by dissolving the potassium salt, thiocol, in concentrated sulfuric acid.
Conditions for the reaction with crystalline estrone∗ in both 1.0 cc and 7.5 cc final volumes have been studied.
Guaiacolsulfonic acid gave a pink color with estrone which was similar in intensity to that given by phenolsulfonic acid. The resultant color of the reaction in the 1 cc final volume was more stable than that given by the Cartland 1 phenolsulfonic acid procedure. The color in the 7.5 cc final volume was at least as stable as that given by the Venning 1 phenolsulfonic acid procedure modified to that volume.
Beer's law applied to the reaction with the new reagent over at least 25-fold variation in hormone concentration.
A 10% solution of thiocol and concentrated sulfuric acid was found to be most satisfactory for the 1 cc procedure. This procedure involves the same series of steps as the Kober 1 reaction except that the optimum times for each step were as follows:
1. Six minutes initial heating in the boiling water bath.
2. Cooling in ice water 5 minutes.
3. After dilution, heating in a boiling water bath for 7 minutes.
4. Cooling again in ice water for 5 minutes before the final dilution.
Get full access to this article
View all access options for this article.