Abstract
Recently a method was described for the estimation of sulfanilamide (para-aminobenzenesulfonamide) in blood and urine. 1 In the rabbit and human subject this drug is partly excreted in the form of an acetylated derivative. 2 In developing a method for determining this conjugated derivative in blood, we have been able to improve the original procedure.
The present modification possesses the advantages of being more sensitive, of giving a more stable color, and of requiring fewer manipulations. Sulfanilamide can be determined very accurately by this method, and in addition one can obtain a fairly good estimate of the conjugated form present in blood. The present method consists of preparing a blood filtrate with toluenesulfonic acid, utilizing the acidity of the precipitant to perform diazotization and coupling with the amine. In determining the conjugated sulfanilamide in blood, the acidity of the blood filtrate is sufficient for hydrolysis on heating.
One volume of oxalated blood is measured into a flask, diluted and laked with 7 volumes of 0.05% saponin solution.∗ After laking is complete (1 or 2 minutes) 2 volumes of para-toluenesulfonic acid solution (20 gm. dissolved in water and diluted to 100 cc.) are added with shaking. After 5 minutes' standing, the mixture is filtered, and 10 cc. of the clear filtrate (a smaller amount can be used with proportionate reduction of the nitrite and dimethyl-a-naphthylamine reagents) is measured into a small flask and 1 cc. of 0.1% freshly prepared† sodium nitrite solution is added. After 3 minutes' standing, 5 cc. of a solution of dimethyl-α-naphthylamine (1 cc. in 250 cc. of 95% ethyl alcohol) is added from a burette.
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