Determination of Human Milk Oligosaccharides in Human Breast Milk by HPAE-PAD with On-Line Sample Cleanup

Importance of the Topic

Human milk oligosaccharides (HMOs) represent the third most abundant solid component of breast milk after lactose and lipids. Their structural diversity and biofunctional roles—including immunomodulation, anti-infective activity, and prebiotic effects—make them critical targets for nutritional research, infant health monitoring, and quality control in human milk–based products.

Study Objectives and Overview

This work aimed to establish and validate a robust, high-throughput method for quantifying six major HMOs (2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose) in human breast milk. Key goals included minimizing sample preparation time, avoiding derivatization, achieving low detection limits, and ensuring precision and accuracy suitable for both colostrum and mature milk analyses.

Methodology and Instrumentation

The analytical approach combined high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and an on-line cleanup using a polymeric reversed-phase trap column. A ‘‘dilute-and-shoot’’ protocol reduced manual handling:

• Milk was diluted 50-fold with deionized water.
• An on-line trap (Dionex IonPac NG1) removed hydrophobic contaminants.
• Separation was achieved on a Dionex CarboPac PA1 column at 20 °C with a NaOH/sodium acetate gradient.
• Pulsed amperometric detection employed a standard carbohydrate waveform on a gold electrode.

Used Instrumentation

• Thermo Scientific Dionex ICS-5000 HPAE-PAD system with dual pumps
• Dionex IonPac NG1 trap column (4 × 35 mm) and CarboPac PA1 analytical column (4 × 250 mm) plus guard column (4 × 50 mm)
• Autosampler with 500 µL loop; gold electrode, carbohydrate triple potential waveform

Main Results and Discussion

Linearity was demonstrated from 1 to 100 µg/mL with r2 > 0.999. Method detection limits ranged 4–21 µg/mL in solution (13–68 µg/mL in milk). Spike-recovery studies in reconstituted milk powder and pooled breast milk showed recoveries between 89% and 112%, with RSDs below 6%. The NG1 trap sustained over 300 injections without loss of performance and could be regenerated with 200 mM HCl in 80% acetonitrile when necessary.

Benefits and Practical Applications

The presented method offers:

• Substantial labor and time savings from minimal sample preparation
• High throughput suitable for large clinical and nutritional studies
• No need for derivatization, preserving analyte integrity
• Robustness across diverse milk matrices (colostrum and mature milk)

Future Trends and Applications

Advances may include coupling HPAE-PAD with mass spectrometry for expanded HMO profiling, integration of automated sample-preparation robotics, and applying similar workflows to other complex biological fluids or fortified dairy products. There is also scope for developing rapid screening kits for on-site monitoring of HMO content in donor milk banks or commercial formula.

Conclusion

This validated HPAE-PAD method with on-line cleanup enables accurate, precise quantification of six key HMOs in human milk with minimal sample handling and high throughput. It meets performance criteria for nutritional research and quality control in lactation and dairy applications.

Reference

J. Tan, F. Chai, C. Zhang. Determination of Human Milk Oligosaccharides in Human Breast Milk by HPAE-PAD with On-Line Sample Cleanup. Abbott Nutrition Custom Application Note 119, 2015.