Establishment of HPLC method for determination of benzoic acid and hippuric acid in pig small intestine contents
In order to establish a high performance liquid chromatography(HPLC)method for the determination of benzoic acid and its metabolite hippuric acid in the contents of pig small intestine,ethyl acetate was used as extraction agent,potassium ferrocyanide zinc acetate was used as protein precipitator to extract benzoic acid from the sample,and the mobile phase was methanol:0.02％phosphoric acid water.Hippuric acid was extracted from samples with water,potassium ferrocyanide and zinc acetate,and the mobile phase was methanol:20 mmol/L ammonium acetate solution.The detection wavelength was 230 nm.The results showed that the linear relationship between the concentration of benzoic acid in the small intestine contents of pigs was good(R2=0.999 4).The limit of detection was 0.05 μg/g,and the limit of quantification was 0.10 μg/g.At 0.10,0.50,1.00,5.00,10.00 μg/g,the recovery was(101.62±4.49)％,and the coefficient of variation was lower than 4.80％and 9.96％,respectively.The addition concentration of hippuric acid in the contents of pig small intestine had a good linear relationship between 0.30 and 10.00 μg/g(R2=1.000 0).The limits of detection and quantification were 0.1 μg/g and 0.3 μg/g,respectively.The recovery of hippuric acid was(90.3±4.00)％at 0.50,1.00,5.00,20.00 μg/g,and the coefficient of variation within and between batches was lower than 5.93％and 5.26％,respectively.The results show that the pretreatment method of benzoic acid and hippuric acid is simple and time-saving,without solid phase extraction and use of toxic and harmful solvents,and saves cost.It can provide a reference for the detection of benzoic acid and its metabolite hippuric acid in the contents of pig small intestine.
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