Multiclass Mycotoxin Analysis in Cheese Using Agilent Captiva EMR—Lipid Cleanup and LC/MS/MS

Significance of the Topic

Certain molds growing on cheese can produce mycotoxins that pose mutagenic, carcinogenic, teratogenic and immunogenic risks even at very low concentrations. The high lipid content in cheese complicates trace-level analysis by LC/MS/MS, as fats co-extract and cause matrix effects, poor reproducibility and instrument contamination. Effective lipid cleanup is therefore critical for accurate food safety screening and quality control.

Objectives and Study Overview

This work aimed to develop and validate a streamlined method for simultaneous quantitation of 13 mycotoxins (including aflatoxins B1, B2, G1, G2, M1; ochratoxins A, B; fumonisins B1, B2, B3; zearalenone; mycophenolic acid; sterigmatocystin) in blue and Parmesan cheese. A QuEChERS extraction was combined with Agilent Captiva EMR–Lipid cartridges to selectively remove lipids, enabling sensitive LC/MS/MS detection down to 0.5 ng/g.

Methodology and Instrumentation

Sample Preparation:

• 2 g cheese was hydrated, spiked with standards and internal standard (13C-AF-B1), extracted by acetonitrile/2 % formic acid.
• QuEChERS salts (MgSO₄, NaCl) were added, shaken and centrifuged.
• Supernatant diluted with water and loaded onto 3 mL Captiva EMR–Lipid tube for gravity pass-through cleanup.
• Eluate dried under nitrogen, reconstituted in ammonium formate/acetonitrile for injection.

Instrumentation:

• LC: Agilent 1290 Infinity II system with Poroshell 120 EC-C18 column (2.1×100 mm, 2.7 µm), 40 °C, 0.5 mL/min, 5 µL injection.
• Mobile phases: 5 mM ammonium formate + 0.1 % formic acid in water (A) and 1:1 acetonitrile:methanol + 0.1 % formic acid (B) with a gradient from 5 % to 98 % B.
• MS/MS: Agilent 6460 Triple Quadrupole with Jet Stream, dynamic MRM in positive/negative mode, optimized gas flows and voltages.

Main Results and Discussion

Calibration curves for all 13 toxins were linear (R² > 0.99) with ±10 % accuracy. Recovery in Parmesan and blue cheese at low, mid and high spikes ranged from 70.7 % to 111.8 %, with RSD < 20 % (most < 10 %). Parmesan cheese showed slightly better precision due to lower matrix complexity. Fumonisins required 2 % formic acid to enhance solubility.
Matrix cleanup efficiency was confirmed by:

• GC/MS full-scan: 61 % and 68 % total co-extractive reduction in blue and Parmesan cheese.
• LC/MS/MS precursor ion scan (m/z 184): 92 % removal of phospholipids in blue cheese.
• Gravimetric analysis: 51 % and 74 % reduction of residue mass.
• Lipid freeze-out test: Captiva EMR–Lipid treated extracts showed no visible fat precipitate, unlike untreated samples.

Competitive testing against another pass-through cleanup cartridge demonstrated higher recovery for more hydrophobic analytes (zearalenone, OTA, STC) when using Captiva EMR–Lipid, attributed to its size-exclusion and hydrophobic sorbent design.

Benefits and Practical Applications

The combined QuEChERS and Captiva EMR–Lipid workflow delivers:

• High lipid removal while preserving diverse analytes.
• Lower detection limits (down to 0.5 ng/g).
• Robust recoveries and precision in complex dairy matrices.
• Time-saving pass-through cleanup without conditioning or elution steps.

This method is readily implementable in food testing laboratories for routine multiclass mycotoxin surveillance in high-fat samples.

Future Trends and Potential Applications

Advancements may include adapting EMR sorbents for larger sample sizes to further lower quantitation limits, extending the approach to other fatty foods (e.g., nuts, meats), and integrating online cleanup modules for increased throughput. Combining selective lipid removal with high-resolution MS could broaden target and non-target screening capabilities.

Conclusion

Agilent Captiva EMR–Lipid provides a novel, efficient cleanup for multiclass mycotoxin analysis in cheeses, achieving selective lipid removal, reliable quantitation and simplified workflow. The validated method supports sensitive, reproducible screening of 13 toxins, addressing key challenges in high-fat food analysis.

Reference

1. Richard JL. Some major mycotoxins and their mycotoxicoses—An overview. Int J Food Microbiol. 2007;119:3–10.
2. Hymery N, et al. Filamentous fungi and mycotoxins in cheese: A review. Compr Rev Food Sci Food Saf. 2014;13:437–456.
3. Bueno D, et al. Determination of mycotoxins in food: A review of bioanalytical to analytical methods. Appl Spectrosc Rev. 2015;50:728–774.
4. Vaclavik L, et al. Determination of multiple mycotoxins in dietary supplements containing green coffee bean extracts using UHPLC-MS/MS. J Agric Food Chem. 2013;61:4822–4830.
5. Zhang K, et al. Determining mycotoxins in baby foods and animal feeds using isotope dilution and LC-MS/MS. J Agric Food Chem. 2014;62:8935–8943.
6. Zhao L, Lucas D. Comparative evaluation of lipid cleanup products for multiclass mycotoxin analysis. Agilent Technologies application note. 2017.