1982 Volume 31 Issue 5 Pages 265-270
A simple and practical method for the determination of nitrite and nitrate in human milk and blood is described. The method is based on the nitration of 2-s-butylphenol, followed by the pentafluorobenzoylation to form pentafluorobenzoyl ester for nitrate determination and the formation of tetrazolophthalazine as reported in the previous paper for nitrite determination. Both products were extracted with benzene and simultaneously determined by the gas chromatography. The procedure was as follows : To 3 ml of blood sample, 1 ml of 1 N NaOH and 1 ml of 12 w/v % ZnSO4 were added. The solution was well mixed for 1 min and centrifuged at 3000 rpm for 5 min. To 5 ml of human milk sample, 1 ml of deproteinization solution {a mixture of 8 w/v% HgCl2, 9 w/v% NH4-CN and 25 w/v% Zn (CH3COO)2} and 3 ml of distilled water were added, and the mixture was shaken for 1 min. To 1 ml of the filtrate, 1 ml of 5 w/v% Ag2SO4 and 3 ml of distilled water were added. Then, in a ice-bath 7 ml of sulfuric acid was added gently, and finally 0.1 ml of 2-s-butylphenol solution was added. After reaction for 15 min at room temperature with occasional shaking, the reaction mixture was extracted with 10 ml of toluene. The organic phase was washed twice with 10 ml of distilled water, then re-extracted with 10 ml of 5 w/v% Na2CO3. The alkaline solution was transferred into another separating funnel and 10 μl of pentafluorobenzoyl chloride was added. After reaction with shaking for 1 min, the content was extracted with 20 ml of benzene and dried with Na2SO4 (anhydrous). The supernatant phase (5 ml for human milk or 1 ml for blood) was transferred into another test tube for nitrite determination, and reacted as described peviously, and then extracted twice with 10 ml of ethyl ether and dried with Na2SO4 (anhydrous) and evaporated at 40 °C in a water-bath. To the dried residue was added 2.5 ml of benzene extract, and concentrated to 1 ml. An adequate volume of the final solution was injected into gas chromatograph. The average recoveries of nitrite and nitrate added to human milk and human blood plasma range from 95.4% to 97.8 % for nitrite and 91.1% to 98.5 % for nitrate. The mean concentration was 0.04 ppm for nitrite and 0.42 ppm for nitrate in fifty human milk samples, and was 0.04 ppm for nitrite and 0.76 ppm for nitrate in fifty human blood samples. The method allowed the determination of N-NO2 in the concentration range (0.010.15)μg/ml and N-NO3 in the concentration range (0.010.1) μg/ml and the detection limit was 0.01 ppm for nitrite and 0.05 ppm for nitrate in human milk, 0.004 ppm for nitrite and 0.02 ppm for nitrate in human blood.
