Background: Infant formulas, pediatric and adult nutritional products are being fortified with bovine lactoferrin (bLF) due to its beneficial impacts on immune development and gut health. Lactoferrin supplementation into these products requires an analytical method to accurately quantify the concentrations of bLF to meet global regulatory and quality standards.
Objective: To develop and validate a lactoferrin method capable of meeting the AOAC Standard Method Performance Requirements (SMPR requirements 2020.005).
Method: Powder formula samples are extracted using warm dibasic phosphate buffer, pH 8, then centrifuged at 4 °C to remove insoluble proteins, fat, and other solids. The soluble fraction is further purified on a HiTrap heparin solid-phase extraction column to isolate bLF from interferences. Samples are filtered, then analyzed by LC-UV using a protein BEH C4 analytical column and quantitated via external calibrant.
Results: The limit of quantitation (2 mg/100g), repeatability (2.0-4.8% RSD), recovery (92.1-97.7%) and analytical range (∼4-193 mg/100g) all meet the method requirements as stated in SMPR 2020.005 for lactoferrin.
Conclusions: The reported single lab validation results demonstrate the ability of this lactoferrin method to meet or exceed the method performance requirements to measure soluble, intact, non-denatured bLF in infant and adult nutritional powder formulas.
Highlights: The use of a heparin affinity column to isolate lactoferrin from bovine milk products combined with a selective analytical chromatographic column provides suitable analyte specificity without requiring proprietary equipment or reagents.
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