Sensitive Detection of Aflatoxins by the Agilent 1290 Infinity III Fluorescence Detector

Significance of the Topic

This application note addresses the critical need for ultrasensitive detection of aflatoxins in food and beverage matrices. Aflatoxins, produced by mold on crops, pose serious health risks due to their toxicity and carcinogenicity, requiring monitoring at extremely low concentrations. Implementing high-performance fluorescence detection under UHPLC conditions enhances food safety and regulatory compliance without time-consuming derivatization steps.

Objectives and Study Overview

The main aim was to demonstrate the capability of the Agilent 1290 Infinity III Fluorescence Detector (FLD) for direct measurement of aflatoxins B1, B2, G1, G2, and their metabolites M1 and M2 at sub-ng/L levels. The study evaluated sensitivity, linearity, and repeatability under optimized UHPLC conditions, comparing performance across a five-decade calibration range (1 to 100,000 ng/L).

Methodology and Instrumentation Used

Analyses were performed using UHPLC-FLD with the following key parameters and components:

• Mobile phase: isocratic 40% organic (methanol:acetonitrile 1:1) in water; flow rate 0.3 mL/min; run time 5 min.
• Column: Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 × 100 mm, 1.8 µm.
• Injection volume: 5 µL; column temperature: 40 °C; needle wash time: 3 s in ACN.
• Detector settings: excitation 365 nm, emission 445 nm; 13 µL low-dispersion flow cell; 10 Hz response time.

Instrumentation list:

• Agilent 1290 Infinity III High-Speed Pump (G7120A)
• Agilent 1290 Infinity III Multisampler (G7167B)
• Agilent 1290 Infinity III Multicolumn Thermostat (G7116B)
• Agilent 1290 Infinity III Fluorescence Detector (G7123B) with 13 µL cell (p/n G7123-60500)
• Software: Agilent OpenLab CDS v2.8

Main Results and Discussion

Calibration curves exhibited exceptional linearity (R2 ≥ 0.99998) across 1–100,000 ng/L for all six aflatoxins. Limits of quantitation ranged from 0.4 to 1.8 ng/L and limits of detection from 0.12 to 0.78 ng/L, determined at S/N ratios of 10 and 3, respectively. Repeatability tests (n = 10) produced retention time RSDs below 0.15% and area RSDs below 0.38%, with symmetry factors between 0.83 and 1.04. The results confirm that direct FLD under UHPLC conditions meets strict sensitivity requirements without derivatization.

Benefits and Practical Applications

By eliminating post-column derivatization, the method reduces analysis time, reagent consumption, and potential sources of error. The low-dispersion flow cell preserves peak shape at high flow rates, making the approach suitable for high-throughput screening in food safety laboratories, quality control, and regulatory compliance testing.

Future Trends and Opportunities

Advances in detector technology and UHPLC instrument design are expected to push detection limits even lower, enabling real-time monitoring and on-site testing. Integration with automated sample preparation and coupling with mass spectrometric confirmation could further strengthen analytical workflows for mycotoxin surveillance.

Conclusion

The Agilent 1290 Infinity III FLD provides an efficient, accurate, and derivatization-free solution for detecting aflatoxins at single-digit ng/L levels. The approach offers excellent linearity, sensitivity, and repeatability within a rapid 5-minute UHPLC method, supporting enhanced food safety analysis.

Reference

• Naegele, E. "Sensitive Detection of Aflatoxins by the Agilent 1290 Infinity III Fluorescence Detector," Agilent Technologies Application Note DE-011925, February 2026.