Abstract
An LC-MS/MS assay based on a signature peptide was developed and fully validated for the quantitation of bovine lactoferrin in infant formulas. Three unreported signature peptides were derived and identified from the tryptic peptides of bovine lactoferrin. The peptide ETTVFENLPEK was used for quantification based on assay performance. The blank matrix camel milk powder and bovine lactoferrin protein standards were mixed and spiked with stable isotope-labeled internal standard to establish a calibration curve. The established method was extensively validated by determining the linearity (R2 > 0.999), sensitivity (limit of quantitation, 0.16 mg/100 g), recovery (83.1–91.6%), precision (RSD < 5.4%) and repeatability (RSD < 7.7%). To validate the applicability of the method, four different brands of infant formulas in China were analysed. The acquired contents of bovine lactoferrin were 52.60–150.56 mg/100 g.
Highlights
Lactoferrin is an 80 kDa iron-binding glycoprotein of the transferrin family that is widely distributed in colostrum and milk [1,2]
The endogenous tryptic peptides derived from digested bovine lactoferrin standard were separated by LC and confirmed by MS in multiple reaction monitoring (MRM) mode
Tryptic fragment ETTVFENLPEK with highest MS abundance, intensity and sensitivity was chosen as the signature peptide of bovine lactoferrin
Summary
Lactoferrin is an 80 kDa iron-binding glycoprotein of the transferrin family that is widely distributed in colostrum and milk [1,2]. As part of the innate immune system, lactoferrin possesses a wide spectrum of important biological functions, including ion metabolism, antimicrobial, antiviral, antioxidation, anti-inflammatory, and anticancer functions, wound healing, and immune modulation [3,4,5,6,7]. The lactoferrin content in milk varies between different mammalian species and, within a given species, between lactation periods [3]. High concentrations of lactoferrin are found in human breast milk [9], while the content of lactoferrin in cow’s milk is relatively low. It is worth noting that there is a very high (77%) structural homology between bovine lactoferrin (extracted and purified from cow milk) and human lactoferrin
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