Determination of Sulfur Amino Acids in Milk and Plant Proteins

A method for the determination of the sulfur amino acids methionine and cysteine in milk- and plant-derived protein powders and in protein-based nutritional products is described. Samples and calibration standards are prepared by acid hydrolysis (6-M HCl, 110 °C, 24 h) with 3,3’dithiopropionic acid, evaporation, and resuspension. The released methionine and cysteine mixed disulfide are quantified by direct LC/UV, without pre- or post-column chromophore/fluorophore derivatization. Since tyrosine is also released by the acid hydrolysis and elutes in the same chromatogram, it was included in all determinations. Method performance has been defined by assessments of linearity (R 2 averaged > 0.999, n = 10, for 5-point plots of each analyte), intermediate precision (within-day RSD < 2%, n = 3, and day-to-day RSD < 3%, n = 3 days, for each analyte), accuracy (cystine spike recovery = 101%; measured methionine, cystine, and tyrosine concentrations were 95–111% of published concentrations), selectivity (reagent blanks and peak purity found analyte bias ≤ 2%), and quantitation limit (25 mg of methionine and tyrosine, and 50 mg of cystine, per 100 g of protein powder). The method provides a relatively simple means for the accurate and precise determination of both sulfur amino acids in protein powders and in nutritional products, and may be performed by conventional LC/UV, without specialized amino acid analysis instrumentation. Graphical abstract