A facile method for simultaneously derivatizing cyst(e)ine with 3,3‘-dithiodipropionic acid (DTDPA) to the mixed disulfide (Cys-X) and hydrolyzing protein prior to amino acid analysis was applied to complex samples consisting of diets and digesta from an animal feeding study. Modified chromatographic conditions allowed adequate separation of all acid-stable amino acids in 90 min with Cys-X eluting between valine and methionine. Repeated chromatography of these hydrolysates led to loss of column performance, which could be reversed by back-flushing the column with a high pH/ion strength regenerant. Passing hydrolysates through a cation exchange cleanup column removed the interfering compound and allowed quantitative recovery of all amino acids. The conversion of cyst(e)ine to Cys-X increased as the molar ratio of DTDPA to cystine increased from 2.6 to 211. Hydrolysis times >75 min had no effect on production of Cys-X. Total amino acid yield from hydrolysates increased with time up to 540 min for most sampl...