Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10–1000 nmol L⁻¹ showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD < 6.5% and RSD < 7.1%, respectively. Excellent repeatability (RSD < 6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method was successfully validated and applied to determination of bovine lactoferrin in dairy products including infant formulas.