Analysis of Nivalenol and Deoxynivalenol in Wheat Using Prominence-i

Importance of the Topic

Nivalenol and deoxynivalenol (DON) are trichothecene mycotoxins produced by Fusarium species that commonly contaminate cereal grains. Monitoring these toxins in wheat is critical for food safety and regulatory compliance, as high exposure can pose health risks to consumers. The Japanese provisional limit for DON in food products is 1.1 ppm, making sensitive and robust analytical methods essential for routine screening and quality control in the food industry.

Objectives and Study Overview

This application note evaluates the performance of the Shimadzu Prominence-i high-performance liquid chromatography (HPLC) system with UV detection for simultaneous quantification of nivalenol and deoxynivalenol in wheat samples. The study aims to verify method repeatability, linearity over a practical concentration range, and suitability of the system’s column washing capability, following the Japanese Food Safety Bureau test method (Notification No. 0717001).

Methodology and Instrumentation

The analytical procedure combines isocratic separation for target analytes with a post-run column wash using a high organic mobile phase. Key parameters include:

• Column: Shim-pack GIS C18, 250 mm × 4.6 mm, 5 μm
• Mobile phases: A (Water/ACN/MeOH 90/5/5 v/v/v), B (ACN/MeOH 50/50 v/v)
• Gradient program: 0–20 min at 0% B; 20.01–25 min at 50% B; 25.01–45 min returning to 0% B
• Flow rate: 1.0 mL/min; Column temperature: 40 °C
• Injection volume: 10 µL; Detection wavelength: 220 nm with cell held at 45 °C

Prominence-i’s integrated low-pressure gradient unit facilitates automated column washing after analyte elution, enhancing throughput and maintaining column performance.

Main Results and Discussion

Analysis of a 4.0 ppm standard mixture yielded clear, well-resolved peaks for both toxins. Repeatability at 0.1 ppm (approximately one-tenth of the regulatory limit) was excellent, with retention time RSDs below 0.1% and peak area RSDs under 0.8% for both compounds. Calibration curves exhibited outstanding linearity (R2 > 0.9999) over 0.1–4.0 ppm. Two different wheat varieties spiked at 1.0 ppm each showed clean chromatograms after cleanup with multifunction SPE columns (MultiSep #227 and Autoprep MF-T), demonstrating method robustness against matrix interferences.

Benefits and Practical Applications

This HPLC protocol offers food safety laboratories a reliable workflow for routine mycotoxin analysis with:

• High sensitivity to meet or exceed regulatory thresholds
• Excellent precision and linearity across relevant concentration ranges
• Automated column cleaning for consistent performance and reduced downtime
• Compatibility with standard UV detection at short wavelengths due to temperature-controlled optics

Future Trends and Possibilities

Advancements may include coupling to tandem mass spectrometry for even greater selectivity in complex matrices, further miniaturization of HPLC systems for field-deployable testing, and integration of online sample preparation modules. Emerging stationary phases may shorten analysis time without sacrificing resolution, supporting high-throughput screening in large-scale monitoring programs.

Conclusion

The Shimadzu Prominence-i HPLC system with UV detection delivers a robust, precise, and linear method for simultaneous quantification of nivalenol and deoxynivalenol in wheat. Incorporating a post-run column wash simplifies maintenance and ensures reproducible performance. This approach meets regulatory requirements and provides a practical solution for food safety laboratories.

Used Instrumentation

• Shimadzu Prominence-i HPLC with temperature‐controlled flow cell and optical system
• Shim-pack GIS C18 analytical column
• MultiSep #227 and Autoprep MF-T multifunction SPE cleanup columns