High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

Significance of the topic

The addition of melamine to dairy products to inflate apparent protein content led to serious health crises in 2007–2008, highlighting the need for reliable screening methods. Dicyandiamide (DCD), used as a pasture growth promoter, has since been detected as a trace contaminant in milk powders. Accurate simultaneous quantification of melamine and DCD at low levels is essential for ensuring food safety and protecting public health.

Objectives and Study Overview

This study aimed to develop and validate a sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of melamine and dicyandiamide in infant milk powder. Key goals included achieving low limits of quantitation in a complex milk matrix and demonstrating robust performance characteristics.

Methodology

Sample preparation followed a modified FDA protocol:

• Weigh 2.0 g of milk powder and add 14 mL of 2.5% formic acid.
• Sonicate for 1 hour, then centrifuge at 6 000 rpm for 10 minutes.
• Transfer 4 mL supernatant into a 5 kDa MWCO centrifugal filter and spin at 7 500 rpm for 10 minutes.
• Collect the filtrate, dilute 1:20 with acetonitrile, and filter through a 0.2 µm PTFE syringe filter.
• Inject 5 µL of the final solution into the LC-MS/MS system.

Instrumentation Used

• Shimadzu Nexera UHPLC system coupled to an LCMS-8040 triple quadrupole mass spectrometer.
• Alltima HP HILIC column (3 µm, 150 × 2.1 mm).
• Electrospray ionization in positive mode with multiple reaction monitoring (MRM).

Key Results and Discussion

Calibration curves for both analytes were linear across 0.5–10 ng/mL (R2 > 0.997) in neat and matrix-matched standards. Retention times were 2.55 min for DCD and 6.29 min for melamine. Method precision (%RSD) at 0.5 and 1.0 ng/mL levels remained below 9.2%. Limits of quantitation in matrix were 0.16 ng/mL for DCD and 0.15 ng/mL for melamine. Matrix effects ranged from 62% to 70%, and recovery studies yielded 103% for DCD and 105% for melamine in spiked samples.

Benefits and Practical Applications

This LC-MS/MS method provides:

• Simultaneous quantification of high-level melamine adulteration and trace dicyandiamide residues.
• High throughput with a total run time under 9 minutes.
• Low detection limits suitable for regulatory monitoring of infant food products.

Future Trends and Potential Applications

Emerging directions include integrating stable-isotope internal standards, expanding to other food matrices, coupling with high-resolution MS for non-target screening, and developing portable MS platforms for on-site testing to further enhance food safety surveillance.

Conclusion

The validated LC-MS/MS approach on the Shimadzu LCMS-8040 platform achieves high sensitivity, precision, and accuracy for detecting melamine and dicyandiamide in milk powders. Its robust performance supports routine QA/QC workflows and regulatory compliance in the dairy industry.

References

1. Turnipseed S., Casey C., Nochetto C., Heller D. N., FDA Food, LIB No. 4421, Vol. 24, Oct. 2008.
2. MacMahon S., Begley T. H., Diachenko G. W., Stromgren S. A., Journal of Chromatography A, 1220, 101–107 (2012).
3. Natural News, Fonterra milk powder dicyandiamide contamination report, 2014.