Determination of Melamine, Ammeline, Ammelide and Cyanuric Acid in Infant Milk-Based Formula and Other Food and Feed Products Using the 300-MS Triple Quadrupole GC/MS/MS

Importance of the Topic

Contamination of infant milk formula and food products with melamine and its analogues has led to severe health incidents worldwide. Accurate ultra-trace analysis of these compounds is essential to ensure consumer safety and regulatory compliance.

Objectives and Study Overview

This study aimed to develop and validate a sensitive and selective GC/MS/MS method using the Bruker 300-MS triple quadrupole system for simultaneous determination of melamine, ammeline, ammelide and cyanuric acid in infant formula, pet food and related matrices.

Methodology and Instrumentation

Sample Preparation:

• Weighed 0.5 g of homogenized sample and extracted with DEA/water/acetonitrile (10:40:50).
• Sonication for 30 min, centrifugation at 15 000 rpm, and filtration through 0.45 µm membranes.
• Evaporation to dryness and derivatization with BSTFA+1% TMCS in pyridine at 70 °C for 45 min.

GC/MS/MS Conditions:

• Instrument: Bruker 300-MS triple quadrupole mass spectrometer with 450-GC and CTC Combi PAL autosampler.
• Column: VF-5ms, 30 m × 0.25 mm × 0.25 µm; inlet at 220 °C; splitless injection of 1 µL.
• Oven program: 75 °C (4 min) to 300 °C at 15 °C/min (hold 4 min), then to 320 °C at 20 °C/min (total 24 min).
• MS/MS in MRM mode with optimized transitions for each analyte.

Main Results and Discussion

The method achieved excellent chromatographic separation and linearity (r > 0.99) over wide concentration ranges. Limits of quantitation for melamine reached 0.1 ppb (equivalent to 0.002 µg/g). Calibration curves demonstrated consistent response from 0.1 to 1000 ppb. Recovery studies in infant formula and pet food at 2.5–5 µg/g yielded 90–120% recoveries. Ion ratio precision met Chinese National Standard criteria, confirming robust qualitative confirmation and quantitation.

Benefits and Practical Applications of the Method

By coupling triple quadrupole MS with GC separation, the protocol offers:

• Ultra-trace sensitivity well below current FDA thresholds.
• High selectivity via MRM transitions, reducing matrix interferences.
• Capability to monitor multiple analogues in a single run.

This enables routine compliance testing in food safety laboratories and rapid screening of complex samples.

Future Trends and Possibilities for Application

Advances may include integration with automated high-throughput workflows, expansion to other nitrogen-rich contaminants, miniaturized sample preparation and coupling with emerging ionization techniques to further lower detection limits and broaden analyte scope.

Conclusion

The presented GC/MS/MS approach using the Bruker 300-MS system delivers sensitive, accurate and robust quantitation of melamine and related compounds at ultra-trace levels. It significantly surpasses regulatory requirements, offering a reliable tool for food safety and quality control.

Reference

• Interim melamine and analogues safety/risk assessment, Department of Health and Human Services, US Food and Drug Administration.
• Jonathan J. Litzau, Gregory E. Mercer, Kevin J. Mulligan, GC-MS Screen for the presence of Melamine, Ammeline, Ammelide and Cyanuric Acid, US FDA Laboratory Information Bulletin No.4423, Oct 2008.
• Determination of melamine in raw milk and dairy products, China National Standard.