Abstract
The objective of this study was to qualitatively and quantitatively evaluate bovine lactoferrin (bLf) and its stability using a rapid RP-HPLC method. bLf could be rapidly detected within 20?min and quantitated at levels down to 5?mu g/mL, and the equation of linearity was y?=?86.10x?+?178.31 with the correlation coefficient (r2) 0.9997. Quantitative data obtained in the present study proved the improved RP-HPLC method to be a sensitive and accurate analytical tool for bLf determination. The proteolytic cleavage of bLf in simulated human gastrointestinal fluids was further analyzed by RP-HPLC, and found to follow pseudo-first-order kinetics. The typical equation obtained by pepsin was log10 [At]/[A0]?=?-0.03x (r2?=?0.85), and log10 [At]/[A0]?=?-0.01x (r2?=?0.81) for trypsin and chymotrypsin combination. Pepsinolysis of bLf in simulated gastric fluid was relatively fast with the half-life t1/2 23.1?min. The digestion of bLf in simulated intestinal fluid was slower with about a 3-fold increase in half-life (69.3?min). After the complete proteolysis of bLf, small cleaved peptide fragments were fully separated and identified by RP-HPLC. The proteolytic study indicated that this validated RP-HPLC was able to evaluate bLf stability though monitoring the derivatization products. Copyright (c) 2012 John Wiley & Sons, Ltd.