Technical Note: Optimizing and Validating an RP-HPLC Method to Determine Lactoferrin in Porcine Colostrum and Milk

Abstract

This Technical Note provides a detailed description of a sample preparation procedure, along with the validation of a reversed-phase high-performance liquid chromatography (RP-HPLC) method for quantitatively determining lactoferrin (LF) in porcine colostrum and milk. The analysis of native milk samples is a challenging process due to the complex composition of the sample. Raw milk is an emulsion and colloid of fat globules in a water-based liquid containing dissolved carbohydrates and protein aggregates with minerals. This paper aims to optimize a method for preparing porcine colostrum and milk samples, which involves a new combination of homogenization, centrifugation, dilution, and filtration techniques for the subsequent analysis of LF via RP-HPLC. A validation process was implemented to ensure the reliability and repeatability of this analytical approach. The results showed that the quantification of LF content in porcine colostrum (1684.58 ± 466.68 µg/mL; n = 10) and milk (849.75 ± 85.82 µg/mL; n = 10) is feasible with successful validation. In addition, the improved sensitivity due to the novel combination of these preparation steps is shown by low limits of detection and quantification of 0.21 µg/mL each. The advantage of this optimized methodology is that the sample preparation can be carried out relatively simply and using standard laboratory equipment, thus enabling the accurate determination of LF in porcine colostrum and milk samples.