Quantification of lactoferrin in human milk using monolithic cation exchange HPLC

Human lactoferrin (hLF) is one of the most important whey proteins in human milk, known for its ability to modulate innate host immunity and multifunctional activities for neonatal growth. The objective of this study was to validate an efficient method for the detection and quantification of hLF using a unique technology of cation-exchange high-performance liquid chromatography (HPLC) on CIM® monolithic columns. Human milk samples were collected using manual expression or a breast pump, at different weeks of lactation. After sample preparation, hLF was detected and measured by HPLC method and further confirmed by SDS-PAGE. Selected fractions were analysed also by LC-MS/MS. Presumably, due to the high density of positive charge on the surface of the N-terminal domain, hLF binds strongly to the column and elutes last, enabling the high specificity of this method. The LC-MS/MS analysis indicated that hLF eluted in two clearly separated peaks, presumably representing two different molecular species of hLF. hLF concentration in the human milk samples ranged from 2.03 mg/mL to 5.79 mg/mL and was not significantly affected by the sample collection method whereas it was negatively correlated with the stage of lactation. These results suggest that cation exchange chromatography is an accurate, efficient, and robust method for the detection and quantification of hLF.