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

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.