Food Analytical Methods

, Volume 12, Issue 11, pp 2518–2526 | Cite as

Determination of native lactoferrin in milk by HPLC on HiTrapTM Heparin HP column

  • Meixia Chen
  • Fang Wen
  • Yangdong Zhang
  • Peng Li
  • Nan Zheng
  • Jiaqi WangEmail author


Foods supplemented with lactoferrin are an increasing trend owing to its multiple functions. Therefore, a reliable analytical method for the nutritional assessment and quality control of lactoferrin containing foods must be established. A method for the determination of native lactoferrin in milk using a HiTrapTM Heparin HP column coupled with HPLC was developed and validated. The conditions for lactoferrin enrichment in milk using the HiTrapTM Heparin HP column were optimized. A linear range of 2 to 100 mg L-1 was obtained for lactoferrin standards, with a R2 value of 0.9989. The detection limit (LOD) and quantification limit (LOQ) of the lactoferrin standards were 2.00 and 6.67 mg L-1, respectively. Considering the enrichment factor involved in the pre-treatment procedures, LOD and LOQ for detection of milk sample were 0.57 and 1.90 mg L-1, respectively. In addition, milk samples spiked with lactoferrin at three concentration levels (2, 5, and 10 mg/L) showed overall mean recovery of 88.3%, 90.2% and 95.1%, respectively. The relative standard deviations of intra-day and inter-day precision were 1.3% ~ 4.8%, and 2.1% ~ 5.7%, respectively, indicating that the present method showed good performances. Furthermore, the successful quantification of lactoferrin in raw milk and processed milk demonstrated the usefulness and effectiveness of the proposed method.


lactoferrin heparin HPLC milk enrichment 



This work was supported by a grant from the Agricultural Science and Technology Innovation Program (ASTIP-IAS12), Special Fund for Agro-scientific Research in the Public Interest (201403071) and Modern Agro-Industry Technology Research System of the PR China (CARS-36). We thank Simon Partridge, PhD, from Liwen Bianji, Edanz Editing China (, for editing the English text of a draft of this manuscript.

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Conflicts of Interest

The authors declare no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Informed consent

Not applicable.

Supplementary material

12161_2019_1572_MOESM1_ESM.docx (863 kb)
ESM 1 (DOCX 863 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Meixia Chen
    • 1
    • 2
    • 3
  • Fang Wen
    • 1
    • 2
    • 3
  • Yangdong Zhang
    • 1
    • 2
    • 3
  • Peng Li
    • 1
    • 2
    • 3
  • Nan Zheng
    • 1
    • 2
    • 3
  • Jiaqi Wang
    • 1
    • 2
    • 3
    Email author
  1. 1.Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal SciencesChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  2. 2.Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal SciencesChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  3. 3.State Key Laboratory of Animal Nutrition, Institute of Animal ScienceChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China

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