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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 establish a method for the determination of sulfur-containing amino acids in milk powder using dithiothreitol (DTT) as reductant and RP-HPLC in the pretreatment. Methods After the milk powder was hydrolyzed by acid method, dithiothreitol was added to hydrolyze the hydrolyzate, and the content of sulfur amino acids in the milk powder was detected by RP-HPLC. The conditions of reduction and chromatographic separation were optimized to determine the best detection condition. Results The detection limits of methionine, cystine and cysteine were 0.7μmol / L, 0.3μmol / L and 0.5μmol / L, respectively. The detection limits were 10 ~ 750μmol / L, the correlation coefficients were 0.9998,0.9999,0.9991. The optimal conditions of DTT is to add 100μL 0.1mol / L LDT solution to 1mL hydrolyzate and react at room temperature for 1h. The contents of Met and (Cys) 2 in milk powder were 0.37g / 100g and 0.14g / 100g, respectively. The recoveries of spiked milk were between 82% ~ 87% and the relative standard deviations (RSDs) were less than or equal to 1.5%. Conclusions In the pretreatment process, an appropriate amount of DTT is used as the reducing agent, and the content of sulfur-containing amino acids in milk powder can be accurately detected by reverse-phase high performance liquid chromatography.