Development of Automated Screening and Quantitation Approach on Novel On-Line SFE-SFC-MS/MS Platform – (II) For Five Afatoxins in Powdered Food Samples

Importance of the Topic

The presence of aflatoxins in food products poses significant health risks and requires sensitive, reliable analytical methods. Automated platforms that integrate sample extraction and separation with mass spectrometric detection can enhance throughput, reduce solvent consumption, and improve reproducibility in routine food safety testing.

Objectives and Study Overview

This study aimed to develop and evaluate an on-line supercritical fluid extraction–supercritical fluid chromatography–tandem mass spectrometry (SFE-SFC-MS/MS) approach for simultaneous screening and quantitation of five aflatoxins (B1, B2, G1, G2, M1) in powdered food matrices such as corn and wheat flour.

Methodology and Instrumentation

Sample Preparation and Extraction:

• Powdered samples (60 mg) spiked with mixed aflatoxin standards dried on filter paper for on-line loading.
• Dynamic extraction with supercritical CO₂ (sfCO₂) and methanol modifier for 2 minutes per injection.

Chromatographic Separation:

• Shim-pack UC-X Sil column (250 mm × 2.1 mm, 3 µm) on a Nexera UC system.
• Gradient elution using CO₂ (mobile phase A) and methanol-based modifiers (B: 5 mM ammonium formate; C: 0.1% formic acid in methanol).
• Flow rates: 2.0 mL/min SFC, 0.2 mL/min makeup.

Mass Spectrometry:

• Shimadzu LCMS-8050 with electrospray ionization in positive MRM mode.
• Two transitions per analyte for quantitation and confirmation.
• Instrument settings: block 400 °C, interface 350 °C, DL 250 °C, Ar collision gas, N₂ nebulizing/drying gases.

Main Results and Discussion

Calibration and Sensitivity:

• Linear calibration (r² > 0.995) over 0.15–12.5 pg on-column (5 µL injection).
• Limits of quantitation between 0.125 pg and 0.325 pg for the five aflatoxins.
• Intra-day precision RSD ≤ 10% at higher levels; one analyte showed RSD ~17.8% at low level.

On-line SFE Performance:

• System recovery estimated by comparing SFE-SFC-MS/MS areas to direct SFC injections: 74–89% across analytes.
• Dynamic extraction delivered < 20% efficiency in first run, cumulative recovery improved over successive runs.

Analysis of Spiked Flour Samples:

• Corn flour spiked at 0.167 µg/kg and 0.050 µg/kg levels yielded recoveries of 23–61% in the second extraction run.
• Wheat flour displayed increased matrix background and detectable aflatoxin levels around 1.0–1.2 µg/kg.

Benefits and Practical Applications

The integrated SFE-SFC-MS/MS workflow offers:

• Fully automated sample handling, eliminating offline cleanup.
• Reduced solvent usage and waste compared to traditional LC methods.
• High sensitivity suitable for screening and quantitation at µg/kg levels.
• Potential for high-throughput monitoring in QA/QC and research laboratories.

Future Trends and Opportunities

Further improvements may include:

• Incorporation of a static extraction phase to boost recovery.
• Optimization of modifier composition and extraction parameters for complex matrices.
• Extension to additional mycotoxins and other food commodities.
• Integration with data-processing software for fully automated reporting.

Conclusion

The Shimadzu Nexera UC SFE-SFC-MS/MS platform enables direct, automated analysis of aflatoxins in powdered food samples with high sensitivity and minimal prep. While extraction efficiency requires enhancement, the approach demonstrates promise for routine food safety screening and quantitation.

Reference

• Shimadzu Application News No LAAN-A-LC-E273 (2015). Online SFE-SFC-MS for pesticide residues.
• Xing J., Lee J.X., Zeng P., Zhan Z. ASMS 2016 Poster MP 283. On-line SFE-SFC-MS/MS for restricted PFCs.