A simplified small-scale workflow for determination of complete protein-bound amino acids using pre-column derivatization HPLC method

Abstract

Existing quantification methods of complete protein-bound amino acids profile of a food or feed often involve complicated hydrolysis setup and multiple hydrolysate treatment steps. Herein, we present a simplified workflow requiring only two steps: hydrolysis (in milligram scale) and direct sample dilution (in microliter scale), well adapted for automated pre-column O-phthaldialdehyde (OPA)/ 9-fluorenylmethyl chloroformate (FMOC) derivatized reverse phase high performance liquid chromatography (HPLC) method (the concentration range was established between 0.005 and 0.50 mM). This new workflow significantly reduces the analysis time (by up to 12 h for every 36 samples) while achieving over 90 % recovery for total protein-bound amino acids, including tryptophan, cysteine, and methionine. The easy hydrolysis setup eliminates the need for special hydrolysis tube or explosive reagents and substitutes laborious sample preparation steps like dry-down, reconstitution, neutralization with voluminous diluents and pH adjustment, with straightforward dilution using 0.1 N hydrochloric acid and deionized water. This method accommodates a small sample load, addressing concerns of limited sample availability. Robustness of this established protocol was validated by bovine serum albumin, and further confirmed by several plant proteins and casein standard. Good correlation of our results with an ISO accredited laboratory was verified (r = 0.992).