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      Improved RP-HPLC method for determination of bovine lactoferrin and its proteolytic degradation in simulated gastrointestinal fluids.

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          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 µg/mL, and the equation of linearity was y = 86.10x + 178.31 with the correlation coefficient (r(2)) 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 log(10) [A(t)]/[A(0)] = -0.03x (r(2) = 0.85), and log(10) [A(t)]/[A(0)] = -0.01x (r(2) = 0.81) for trypsin and chymotrypsin combination. Pepsinolysis of bLf in simulated gastric fluid was relatively fast with the half-life t(1/2) 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.

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          Author and article information

          Journal
          Biomed. Chromatogr.
          Biomedical chromatography : BMC
          Wiley-Blackwell
          1099-0801
          0269-3879
          Feb 2013
          : 27
          : 2
          Affiliations
          [1 ] School of Pharmacy, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand.
          Article
          10.1002/bmc.2771
          22684820

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