Determination of Anions in Breast Milk

Importance of the Topic

Monitoring anionic species in breast milk is critical for ensuring optimal infant nutrition and health. Inorganic ions such as chloride, nitrate, phosphate, and organic anions like citrate play vital roles in maintaining electrolyte balance, supporting renal function, and promoting cardiovascular development. Excess or deficiency of these ions in early life nutrition can lead to physiological imbalances or organ stress.

Objectives and Overview of the Study

This study aimed to develop and validate a robust ion chromatography (IC) method for the simultaneous sensitive determination of multiple anions in human breast milk. Key focuses included achieving low detection limits, high reproducibility, and accurate quantitation in a complex biological matrix, enabling dietary adjustments through maternal nutrition monitoring.

Methodology and Instrumentation

The analytical workflow consisted of:

• Sample Preparation: Dilution of breast milk with deionized water, liquid–liquid extraction using trichloromethane, centrifugation, and cleanup via reversed-phase OnGuard cartridge followed by 0.45 µm syringe filtration.
• Calibration: Preparation of mixed standard solutions for nine target anions with concentrations ranging from 0.02 to 5.0 mg/L using gravimetric dilution of 1000 mg/L stock solutions.
• Chromatographic Separation: Gradient elution with potassium hydroxide generated in-line, using a quaternary-ammonium IonPac AS19 analytical column at 30 °C and 1.2 mL/min flow rate.
• Detection: Suppressed conductivity detection in recycle mode for quantitation, complemented by negative-mode ESI mass spectrometry with selected ion monitoring (SIM) at characteristic m/z values (35 for chloride, 62 for nitrate, 97 for phosphate, 191 for citrate) to confirm peak identities.

Used Instrumentation

• Thermo Scientific Dionex ICS-5000+ HPIC system with DP Dual Pump, EG Eluent Generator, and DC Detector module
• Dionex AS-AP autosampler (25 µL loop)
• Dionex AERS-500 electrolytically regenerated suppressor
• MSQ Plus single-quadrupole mass spectrometer with ESI source
• Thermo Scientific Sorvall ST16 centrifuge
• OnGuard II RP cartridges and 0.45 µm nylon syringe filters
• Chromeleon CDS software version 7.2

Main Results and Discussion

Separation of nine anions was achieved with baseline resolution using the AS19 column. Short-term precision tests (seven injections at 1 mg/L) yielded retention time RSDs ≤ 0.01% and peak area RSDs ≤ 0.64%. Calibration curves for chloride and nitrate were linear over 0.02–5.0 mg/L with r2 ≥ 0.9995. Method detection limits (S/N=3) were 8 µg/L for chloride and 10 µg/L for nitrate. In real samples diluted 100-fold, SIM traces confirmed the presence and identity of chloride (35 m/z), nitrate (62 m/z), phosphate (97 m/z), and citrate (191 m/z). Spike recovery experiments in breast milk demonstrated recoveries of 97% for chloride and 98% for nitrate, indicating high accuracy and minimal matrix effects.

Benefits and Practical Applications of the Method

This IC method provides a rapid, sensitive, and selective approach for multi-anion profiling in breast milk. It supports routine quality control in clinical and nutritional research laboratories, guiding maternal dietary recommendations and ensuring infant electrolyte balance. The reagent-free eluent generation simplifies operation and reduces contamination risks.

Future Trends and Possibilities for Use

• Integration with high-resolution mass spectrometry for expanded analyte coverage and structural confirmation.
• Automation of sample preparation workflows to increase throughput for large-scale epidemiological studies.
• Application to other biofluids (e.g., serum, urine) for comprehensive electrolyte monitoring in neonatology and pediatric care.
• Development of miniaturized or portable IC systems for point-of-care nutritional assessment.

Conclusion

A reagent-free high-pressure ion chromatography method with suppressed conductivity and MS confirmation has been established for sensitive determination of key anions in breast milk. The approach offers excellent reproducibility, low detection limits, and accurate quantitation in complex matrices, making it a valuable tool for nutritional monitoring and research.

References

• Gaucheron F, Le Graet Y, Piot M, Boyaval E. Determination of Anions of Milk by Ion Chromatography. Lait 1996, 76(5):433–443.
• Thermo Scientific. Data Sheet: IonPac AS19 Anion-Exchange Column. Sunnyvale, CA, 2010.