猪小肠内容物中苯甲酸和马尿酸高效液相色谱检测方法的建立

Establishment of HPLC method for determinationof benzoic acid and hippuric acid in pig small intestine contents

试验为建立猪小肠内容物中苯甲酸及其代谢物马尿酸的高效液相色谱(HPLC)测定方法,采用乙酸乙酯为提取剂,亚铁氰化钾-乙酸锌为蛋白沉淀剂,提取样品中的苯甲酸,流动相为甲醇∶0.02%磷酸水;水、亚铁氰化钾-乙酸锌共同提取样品中的马尿酸,流动相为甲醇∶20 mmol/L乙酸铵溶液,紫外检测波长230 nm。结果显示:在猪小肠内容物中苯甲酸添加浓度为0.10~10.00μg/g内线性关系良好(R^(2)=0.9994);检测限为0.05μg/g,定量限为0.10μg/g;添加浓度在0.10、0.50、1.00、5.00、10.00μg/g时,回收率为(101.62±4.49)%,批内和批间变异系数分别低于4.80%和9.96%。猪小肠内容物中马尿酸添加浓度在0.30~10.00μg/g内线性关系良好(R^(2)=1.0000);检测限和定量限分别为0.10μg/g和0.30μg/g;马尿酸添加浓度在0.50、1.00、5.00、20.00μg/g时,回收率为(90.3±4.00)%,批内和批间变异系数分别低于5.93%和5.26%。研究表明,此方法苯甲酸及马尿酸预处理方法简单省时,无须固相萃取及使用有毒有害溶剂,节约成本,为猪小肠内容物中苯甲酸及马尿酸的检测提供参考。

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(R^(2)=0.9994).The limit of detection was 0.05μg/g,and the limit of quantification was 0.10μg/g.At 0.10,0.50,1.00,5.00,10.00μg/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 10.00μg/g(R^(2)=1.0000).The limits of detection and quantification were 0.1μg/g and 0.3μg/g,respectively.The recovery of hippuric acid was(90.3±4.00)%at 0.50,1.00,5.00,20.00μg/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.