Simultaneous Extraction of Melamine and Cyanuric Acid from Food Products Using Strata® Melamine SPE and Ultra-fast LC/MS/MS Analysis Using Kinetex™ HILIC, Rapid LC/MS/MS Analysis on Luna® HILIC, or Rapid GC/MS Analysis on Zebron™ ZB-XLB HT

Significance of the Topic

Rapid and reliable analysis of melamine and cyanuric acid in food is critical due to health risks such as renal failure from the poorly soluble melamine cyanurate complex and stringent global regulations. A simultaneous extraction and analysis approach reduces sample preparation time, limits solvent use, and enhances laboratory throughput.

Objectives and Study Overview

This application note presents a combined solid phase extraction method using Strata® Melamine SPE to isolate melamine and cyanuric acid from food matrices, coupled with ultra-fast LC/MS/MS or GC/MS analysis. The goals were to streamline workflow, achieve low detection limits, and offer flexible analytical platforms tailored to laboratory preferences.

Methodology and Instrumentation

• Sample Preparation: Baby formula spiked with standards, protein precipitation with acetonitrile and 0.2 N HCl, centrifugation, followed by Strata Melamine SPE cleanup.
• SPE Cartridge: 200 mg/3 mL Strata Melamine (Phenomenex) conditioned and equilibrated prior to sample loading; analytes eluted with methanol and ammonium hydroxide in methanol.
• LC/MS/MS: Agilent 1100 series HPLC with on-line degasser and column oven, Applied Biosystems API3000 tandem mass spectrometer, HILIC columns: Kinetex 2.6 µm (50×2.1 mm) for <1 min separation or Luna 3 µm (100×2.0 mm) for <3.5 min runs; MRM with polarity switching.
• GC/MS: Zebron ZB-XLB-HT Inferno column (15 m×0.25 mm×0.25 µm), on-column injection, derivatization with BSTFA/pyridine, run time <9 min.

Main Results and Discussion

Absolute recoveries averaged 67 % for melamine and 68 % for cyanuric acid, with relative recoveries of 67 % and 94.5 %, respectively; precision (CV) < 5 %. Calibration curves in baby formula (20–2000 ng/g) exhibited excellent linearity (R2 > 0.999). Limits of detection and quantitation were 20 ng/g and 200 ng/g, respectively, meeting FDA and WHO guidelines. Ultra-fast analysis with the Kinetex HILIC column reduced runtime to <0.4 min, while GC/MS provided an alternative with <9 min analysis.

Benefits and Practical Applications

• Single SPE extraction for both analytes simplifies workflow and reduces consumables.
• Flexible analytical options (LC/MS/MS or GC/MS) accommodate laboratory instrumentation and expertise.
• Rapid methods enhance throughput in quality control, food safety, and regulatory testing.

Future Trends and Opportunities

Advances in core-shell and sub-2 µm HILIC stationary phases may further decrease analysis time and improve resolution. Integration with high-throughput automation and coupling with high-resolution MS can expand applicability to complex matrices and multi-analyte panels. Emerging portable MS technologies may enable on-site screening of melamine adulteration.

Conclusion

The combined Strata Melamine SPE and rapid HILIC or GC/MS methods deliver a robust, efficient workflow for simultaneous analysis of melamine and cyanuric acid in food products, achieving low detection limits and high precision while significantly reducing analysis time.

References

1. Litzau J, Mercer G, Mulligan K. GC-MS method for melamine and cyanuric acid analysis. FDA Center for Veterinary Medicine, 2007.
2. Tosoh Bioscience. TSK-GEL Amide-80 HILIC columns for melamine and cyanuric acid analysis in milk by LC-MS/MS. Application Note AN191108.
3. U.S. Department of Health and Human Services, FDA. Guidance for Industry Bioanalytical Method Validation. 2001.
4. World Health Organization. Toxicological and health aspects of melamine and cyanuric acid: report of a WHO expert meeting, 2009.