Czech J. Anim. Sci., 2025, 70(11):467-479 | DOI: 10.17221/114/2025-CJAS

Lactoferrin content determined in bovine milk by HPLC and mid-infrared spectrometry – Relation to udder health and potential for detection of milk adulterationOriginal Paper

Hana Nejeschlebová ORCID...^1,4, Oto Hanuš ORCID...¹, Klára Bartáková ORCID...², Craig Parsons³, Eva Samková ORCID...⁴, Lenka Vorlová ORCID...², Gavin Thompson ORCID...⁵, Lucie Hasoňová ORCID...⁴, Roman Konečný ORCID...⁶, Pavlína Navrátilová ORCID...², Marcela Klimešová ORCID...¹

¹ Dairy Research Institute Ltd., Prague, Czech Republic

² Department of Animal Origin Food and Gastronomic Sciences, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, Brno, Czech Republic

³ Bentley Instruments, Inc., Chaska, Minnesota, USA

⁴ Department of Food Biotechnologies and Agricultural Products’ Quality, Faculty of Agriculture and Technology, University of South Bohemia in České Budějovice, České Budějovice, Czech Republic

⁵ Bentley Czech Ltd., Prague, Czech Republic

⁶ Department of Animal Husbandry Sciences, Faculty of Agriculture and Technology, University of South Bohemia in České Budějovice, České Budějovice, Czech Republic

Lactoferrin (LF) is a multifunctional glycoprotein linked to udder health in dairy cows. This study aimed to develop a calibration model for LF quantification using mid-infrared spectrometry (MIR-FT), with ion-pair reversed-phase high-performance liquid chromatography (HPLC) as the reference method. Two sets of individual milk samples (A: n = 120; B: n = 91) were collected from five dairy farms in the Czech Republic. Set A included a higher proportion of samples with somatic cell count (SCC) above 300 000 cells/ml to ensure broad LF variability. After merging both sets and removing six outliers, a final calibration set C (n = 205) was created. The developed model achieved a cross-validated coefficient of determination of 0.588 7 and a standard error of cross-validation of 67.33 mg/l. Correlation analysis showed that several milk parameters correlated with LF determined by MIR-FT in patterns typical of mastitis (SCC: r = 0.450; lactose: r = –0.364; free fatty acids: r = 0.621; electrical conductivity: r = 0.442), indicating potential for MIR-FT in evaluating mammary gland health. The feasibility of using LF as an indicator of milk adulteration by artificial SCC reduction through centrifugation was also assessed. Two sample sets (n = 20 and n = 68) were analysed, each containing normal bulk tank milk and bulk tank milk supplemented with abnormal milk. Centrifugation caused minimal changes in LF determined by both HPLC and MIR-FT (maximum 3.27%) while SCC decreased by nearly 50%, suggesting that LF may serve as a marker for detecting artificial SCC reduction. However, practical application of MIR-FT for accurate LF determination is limited by the achieved validation parameters and the high expanded uncertainty (114.7 mg/l). The method is therefore more suitable for monitoring relative LF changes in milk than for determining exact LF content.

Keywords: calibration model; dairy cow; mastitis; milk centrifugation; milk composition; somatic cell count

Received: July 26, 2025; Accepted: October 15, 2025; Published: November 28, 2025  Show citation