Optimization of Extraction Buffer Conditions for Improved Heparin Affinity-Based Quantification of Bovine Lactoferrin in Various Dairy Products

BACKGROUND: Bovine lactoferrin (bLF) has been supplemented to a variety of foods, particularly dairy products, to promote human health. Reliable analytical methods are required for accurate quantification of bLF in foods. Isolation of bLF from foods using a heparin column and quantification via HPLC have been reported (heparin method); however, its applicability remains limited. OBJECTIVE: To develop a broadly applicable method for determining bLF in dairy products by optimizing the extraction buffer conditions used in the heparin method. METHOD: bLF was spiked into different dairy products and extracted using buffers with or without additives, such as urea, sodium chloride, EDTA, and Tween 20. These additives reduced interactions between bLF and dairy components and improved the interaction of bLF to the heparin column, with the buffer pH adjusted to 6.0. The extracted bLF was isolated using a heparin column and quantified via C4 reversed-phase HPLC using an external standard calibration curve. RESULTS: The optimized extraction buffer improved bLF recovery rates across all tested dairy product matrixes. CONCLUSION: An improved extraction buffer was developed that enhanced the applicability of bLF quantification using the heparin method. This optimized extraction buffer enabled reliable recovery of bLF from diverse and complex dairy product matrixes. HIGHLIGHT: The optimized extraction buffer significantly improves the recovery and quantification of bLF in various dairy products, providing a versatile solution for accurate bLF analysis.