Quantification of lactoferrin in human milk using monolithic cation exchange HPLC

Area of Science:

• Biochemistry
• Analytical Chemistry
• Neonatal Nutrition

Background:

• Human lactoferrin (hLF) is a key whey protein in human milk, vital for neonatal immunity and growth.
• Accurate quantification of hLF is essential for understanding its role in infant development.
• Existing methods may lack efficiency or specificity for hLF detection.

Purpose of the Study:

• To validate an efficient cation-exchange high-performance liquid chromatography (HPLC) method for human lactoferrin (hLF) detection and quantification.
• To assess the influence of sample collection methods and lactation stage on hLF concentration.

Main Methods:

• Cation-exchange HPLC using CIM® monolithic columns for hLF separation and quantification.
• Sample preparation followed by HPLC analysis.
• Confirmation of hLF detection using SDS-PAGE and LC-MS/MS.

Main Results:

• The cation-exchange HPLC method demonstrated high specificity for hLF due to its strong binding and late elution.
• LC-MS/MS analysis revealed two distinct peaks for hLF, suggesting different molecular forms.
• hLF concentration ranged from 2.03 to 5.79 mg/mL, unaffected by collection method but negatively correlated with lactation stage.

Conclusions:

• Cation-exchange chromatography is an accurate, efficient, and robust method for quantifying hLF in human milk.
• hLF levels decrease as lactation progresses.
• This validated method can support research on hLF's role in neonatal health.