A method for separation and quantification of phospholipid classes in human milk.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Nutritional Science

Background:

• Human milk phospholipids are crucial for infant development.
• Accurate quantification of phospholipid classes is essential for nutritional and clinical studies.
• Existing methods for phospholipid analysis can be time-consuming or lack comprehensive resolution.

Purpose of the Study:

• To develop and validate a simple, isocratic High-Performance Liquid Chromatography (HPLC) method.
• To achieve efficient separation of major human milk phospholipid classes.
• To provide a rapid and reliable analytical technique for human milk phospholipid profiling.

Main Methods:

• Isocratic HPLC separation on a silica column.
• Elution using a solvent mixture of acetonitrile:methanol:sulfuric acid.
• Quantification of phospholipid classes via inorganic phosphorus analysis after acid digestion.

Main Results:

• Successful resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine, and sphingomyelin within 30 minutes.
• High repeatability with a coefficient of variation of 5.3% across 19 samples.
• Average analytical recovery of phospholipid standards was 98%, with minor variations among classes.

Conclusions:

• The developed HPLC method is simple, rapid, and effective for separating major human milk phospholipid classes.
• This method provides a reliable and repeatable approach for the quantitative analysis of human milk phospholipids.
• The technique is suitable for routine analysis in research and clinical settings involving human milk composition.