Determination of native lactoferrin in milk by HPLC on HiTrap Heparin HP column

Foods supplemented with lactoferrin are an increasing trend owing to its multiple functions. Therefore, a reliable analytical method for the nutritional assessment and quality control of lactoferrin containing foods must be established. A method for the determination of native lactoferrin in milk using a HiTrap Heparin HP column coupled with HPLC was developed and validated. The conditions for lactoferrin enrichment in milk using the HiTrap(TM) Heparin HP column were optimized. A linear range of 2 to 100 mg L-1 was obtained for lactoferrin standards, with a R-2 value of 0.9989. The detection limit (LOD) and quantification limit (LOQ) of the lactoferrin standards were 2.00 and 6.67 mg L-1, respectively. Considering the enrichment factor involved in the pre-treatment procedures, LOD and LOQ for detection of milk sample were 0.57 and 1.90 mg L-1, respectively. In addition, milk samples spiked with lactoferrin at three concentration levels (2, 5, and 10 mg/L) showed overall mean recovery of 88.3%, 90.2% and 95.1%, respectively. The relative standard deviations of intra-day and inter-day precision were 1.3% similar to 4.8%, and 2.1% similar to 5.7%, respectively, indicating that the present method showed good performances. Furthermore, the successful quantification of lactoferrin in raw milk and processed milk demonstrated the usefulness and effectiveness of the proposed method.