Extraction of Melamine from Milk (Infant Formula) Using EVOLUTE® EXPRESS CX Solid Phase Extraction Columns with Analysis by LC-MS/MS

Significance of the Topic

Melamine is sometimes illegally added to milk and infant formula to inflate apparent protein levels, posing serious health risks including kidney stones and renal failure, especially in infants. Reliable detection and quantification of melamine at low levels in complex dairy matrices are therefore critical for food safety and regulatory compliance.

Objectives and Study Overview

This application note describes the development and validation of a solid‐phase extraction (SPE) protocol using EVOLUTE EXPRESS CX mixed‐mode strong cation exchange columns to isolate melamine from infant formula, followed by quantitation via liquid chromatography tandem mass spectrometry (LC‐MS/MS). The study evaluates recovery, precision, and robustness across powdered and liquid baby milk samples.

Methodology

• Sample Pre‐treatment: Reconstitute powdered formula per manufacturer instructions, cool, then dilute 1 mL sample with 1 mL of 50 mM ammonium acetate buffer (pH 5).
• Column Conditioning: Flush SPE column with 3 mL methanol.
• Column Equilibration: Equilibrate with 3 mL of 50 mM ammonium acetate buffer (pH 5).
• Sample Loading: Apply 2 mL of pre‐treated sample at 3 mL/min.
• Interference Washing: Elute polar/ionic interferences with 3 mL ammonium acetate buffer, followed by 3 mL methanol for non‐polar contaminants.
• Analyte Elution: Recover melamine with 3 mL methanol containing 5% ammonium hydroxide (v/v).
• Post‐Extraction: Evaporate eluate to dryness at 40 °C and reconstitute in 500 µL acetonitrile/water (90:10, v/v) before analysis.

Instrumentation

• SPE Columns: EVOLUTE EXPRESS CX 60 mg/3 mL (Biotage).
• Liquid Handling: Waters 2795 system for sample delivery.
• Analytical Column: Phenomenex Luna HILIC, 3 µm, 100×2.0 mm.
• Chromatography: Isocratic mobile phase of acetonitrile:20 mM ammonium formate pH 3.2 (75:25, v/v), flow rate 0.3 mL/min, ambient temperature.
• Detection: ESI‐MS/MS in positive mode, MRM transitions 127>85 (CE 12 eV) and 127>68 (CE 14 eV), collision gas pressure 2.4×10⁻³ mbar.

Results and Discussion

Recoveries of melamine from various infant formula matrices ranged from 84% to 98%, with relative standard deviations below 10% (n=6), demonstrating high extraction efficiency and method precision. The optimized SPE procedure effectively removed proteins, salts, phospholipids, and other matrix interferences, yielding clean extracts suitable for robust LC‐MS/MS quantitation.

Benefits and Practical Applications

• High recovery and precision enable reliable monitoring of melamine in routine QA/QC laboratories.
• Mixed‐mode SPE reduces matrix effects and extends instrument uptime.
• Method simplicity and reproducibility support implementation in food safety testing and regulatory compliance workflows.

Future Trends and Applications

Advancements may include automated SPE platforms for high‐throughput screening, miniaturized column formats to reduce solvent consumption, integration with high‐resolution mass spectrometry for broader contaminant profiling, and development of greener sorbent materials to enhance sustainability.

Conclusion

The described EVOLUTE EXPRESS CX SPE method coupled with LC‐MS/MS provides a robust, accurate, and reproducible approach for melamine detection in infant formula, meeting the needs of modern food safety laboratories.

References

• Williams L, et al. Extraction of melamine from various matrices using resin‐based mixed‐mode cation exchange SPE and analysis with LC‐MS/MS. Poster presented at ASMS, Philadelphia, May–June 2009.