An Efficient Heparin Affinity Column Purification Method Coupled with Ultraperformance Liquid Chromatography for the Quantification of Native Lactoferrin in Breast Milk

Abstract

Lactoferrin (LF) is a bioactive multifunctional protein and found in the highest amounts in human milk. Several methods can be used to quantify LF. However, quantification of native LF has garnered relatively little interest to date. This study aimed to develop a novel efficient two-step method for quantifying native LF in breast milk. During the analysis, LF was first extracted with phosphate buffer (pH 5.0), purified using a heparin affinity column. Subsequently, LF was detected using ultraperformance liquid chromatography (UPLC) at a wavelength of 201 nm. A linear calibration curve was obtained in the range of 5-200 mg/L. The limit of detection and limit of quantitation were 1 mg/L and 5 mg/L, respectively, indicating that the validated method could be employed to quantify LF in breast milk. Compared with previous HPLC methods, this method demonstrated several remarkable advantages, including simple operation, low-cost detection, and high accuracy. Hence, the results demonstrate an efficient method that can be employed commercially to purify and analyze LF in human milk samples.