Specific determination of nitrate and nitrite of chicken egg by gas-liquid chromatography with special reference to the turnover of these anions in laying hens.

Abstract

Nitrate and nitrite were successfully extracted from deproteinized chicken egg with aqueous solution, and analyzed by gasliquid chromatography with an electron capture detector without further cleaning. The distribution of these anions in 50 egg samples was the logarithmic normal distribution in each case, that is, N(2J96 (=0.063ppm), 0.295) and /?{0.052ppm<^< 0.076ppm}=0.95 for nitrate-TV, and N(2.470 (=0.030ppm), 0.227) and >{0.026ppm</*< 0.034ppm} =0.95 for nitrite-N.Whenthe chickens were fed with a commercial diet containing elevated levels (1,000 or 5,000ppm) of nitrate-or nitrite-N, the concentration of these anions in their eggs markedly increased and proceeded to the steady state within 2 or 3 days, where the level was proportional to that of anions added to the diet. After withdrawing the excess of anions from the diet, the concentrations of anions in the eggs decreased exponentially, where the rate constants for nitrate and nitrite were about 0.6 day"1 and 1.0 day"1, respectively. In the series of experiments, it was assumed that the reactions NO3~->NO2ãnd NO2-~>NO3p roceed simultaneously in the body of chickens.Chicken egg is one of the most popular and important foods, and is abundant in nitrogen. In the previous paper,1} we reported a sensitive and practical determination for trace amounts ofnitrite in egg ranging from 0.01 to 0.ll ppm.The toxicity of nitrite towards humans has been noticed,2'3) and therefore the turnover and behaviour of nitrate-and nitrite-N in egg due to the nitrogen source in a diet are of great interest. Because of the difficulty in determining the nitrate-and nitrite-N in egg, the distribution and behaviour of these anions in egg have been hardly reported. Wehave developed a specific method for nitrate determination besides nitrite by introducing a pentafluorobenzoyl ester of 4-nitro-2-^c-butylphenol, which is very sensitive in gas chromatographic analysis with an electron capture detector (GLC-ECD).4) Applying the proposed method, the distribution of nitrate-and nitrite-N in egg was statistically analyzed, and the turnover of these anions in eggs obtained from laying hens during and after feeding with various levels of these anions was studied. MATERIALS AND METHODSChemicals. NaNO2and KNO3were dried at 1 10°C for 1 hr under vacuum immediately before use. A stock nitrate or nitrite solution wasprepared to give a concentration of 1 /ig/ml of nitrate-or nitrite-N in distilled water. 2-secButylphenol solution (5%, w/v) and pentafluorobenzoyl' chloride (PFB-C1) for use in the determination of nitrate-N were prepared as described previously.4) The reagents used for the determination of nitrite-N were as described previously.5>6) The deproteinising solutions A and B (NH4SCN+HgCl2 and Zn(CH3COO)2) were also prepared as before.7) All other reagents and solvents were of special high purity and obtained from Wako Pure Chemical Industries.Equipment and GCconditions. A Shimadzu GC-7 AG gas chromatograph with an ECD(63Ni) was used for all analysis. The GCconditions for nitrate-N and nitrit...