A simple method to quantify lactoferrin in milk by Micro-Batch Resin Extraction (MBRE) before RP-HPLC

The quantification of lactoferrin in dairy products is mainly performed using immunological methods (ELISA, radio-immunodiffusion and optical biosensing). These methods are species-specific and considered to be sensitive (ng on a diluted sample basis) but are expensive and reproducibility is average. Separative techniques such as electrophoresis and HPLC can interestingly discriminate different forms of lactoferrin but appeared disappointing for quantification. Indeed, caseins and fat should be removed before analysis, which results in the dilution and loss of lactoferrin, and critical resolution from whey proteins was often observed. In the proposed method, skimmed cow’s milk sample (1 mL) is deposited as is in a microtube containing 100 mg of macroporous cation-exchange sulfonated resin and stirred rotationally for 90 minutes at 20-25 °C. Lactoferrin which is positively charged at the pH of native milk (6.8) is quantitatively adsorbed while the other non-adsorbed negatively-charged whey proteins and caseins are removed by centrifugation. After washing 4 times the resin with 1 ml of water, lactoferrin is quantitatively desorbed after 30 minutes stirring with 1 mL 2M NaCl containing phenylalanine as a dilution marker. Lactoferrin is then completely separated from the co-desorbed cationic peptides and lactoperoxidase using RP-HPLC at 220 nm. This cheap, universal and flexible method improves selectivity (no protein interference) and sensitivity (no dilution) over previous separative methods. Its intra-laboratory validation (six concentrations in triplicate over five days) demonstrated linearity up to 500 μg.mL-1, accuracy (98-110% recovery), sensitivity (LOQ=25 μg.mL-1 milk) and precision (< 4%) which challenge most immunochemical methods. MBRE-HPLC and monoclonal ELISA-sandwich gave similar results for individual raw cow’s milks. Nevertheless different evolutions were observed with milk heated at increasing temperature (60-87° C, 30 s) indicating that the epitope sensitive in ELISA and cationic part implicated in MBREHPLC are situated in different portions of the molecule that are affected differently by heating.