二硫苏糖醇在奶粉含硫氨基酸检测中的应用

Application of dithiothreitol in the detection of sulfur amino acids in milk powder

目的建立一种在前处理过程中使用二硫苏糖醇(DTT)为还原剂,反相高效液相色谱法检测奶粉中含硫氨基酸的方法。方法奶粉经酸法水解后,加入二硫苏糖醇对水解液进行还原处理,用反相高效液相色谱法检测奶粉中含硫氨基酸,对还原条件、色谱分离条件进行优化,确定最佳检测条件。结果蛋氨酸(Met)、胱氨酸[(Cys)2]、半胱氨酸(Cys)检测限分别为0.7μmol/L、0.3μmol/L、0.5μmol/L,定量检测范围均为10～750μmol/L,相关系数分别为0.9998、0.9999、0.9991。最佳DTT作用条件为向1mL水解液中加入100μL0.1mol/LDTT溶液,室温反应1h。测得奶粉中Met、(Cys)2含量分别为0.37g/100g、0.14g/100g,加标回收率均在82%～87%之间,相对标准偏差(RSD)≤1.5%。结论在前处理过程中使用适量DTT做还原剂,用反向高效液相色谱法可准确检测奶粉中含硫氨基酸的含量。

Objective To develop a new method to detect sulfur amino acids by reverse-phase high performance liquid chromatography(HPLC) in milk powder reacted with dithiothreitol in pretreatment.Methods Milk powder was hydrolyzed by acid,further reduced by dithiothreitol,and then detected by HPLC.The optimum reduction conditions and chromatographic separation conditions were investigated.Results Detection limits of methionine,cystine and cysteine were 0.7 μmol/L,0.3 μmol/L and 0.5 μmol/L,respectively,and all of the quantitation range were 10～750 μmol/L with the correlation coefficients of 0.9998,0.9999 and 0.9991,respectively.The optimum operating conditions were as follows:100 μL dithiothreitol at the concentration of 0.1 mol/L was added in 1 mL hydrolyzate and reacted for 1h at room temperature.The contents of methionine and cystine in milk powder were 0.37 g/100 g and 0.14 g/100 g,respectively.The recoveries ranged from 82% to 87% with the relative standard deviation(RSD) of less than 1.5%.Conclusion Applying optimum dithiothreitol in pretreatment and using the reverse phase HPLC method could detect the content of sulfur amino acids in milk powder accurately.