A High Sensitivity LC/MS/MS Method with QuEChERS Sample Pre-treatment for Analysis of Afatoxins in Peanut Butter Samples

Importance of the Topic

Aflatoxins are highly toxic fungal metabolites commonly found in nuts and processed foods under warm and humid conditions. Their potent carcinogenicity and strict regulatory limits worldwide make sensitive and reliable detection in food matrices essential for public health and compliance.

Objectives and Overview of the Study

This work aimed to develop and validate a high-sensitivity LC/MS/MS method combined with a simplified QuEChERS sample pre-treatment for quantifying aflatoxins B1, B2, G1 and G2 in peanut butter. The study sought to replace costly immunoaffinity procedures with a more economical and efficient workflow.

Methodology and Sample Preparation

• Weigh 2 g peanut butter and defat with 15 mL hexane.
• Extract aflatoxins using acetonitrile/water (86:14) fortified with formic acid and QuEChERS salt pack (MgSO4, NaCl, trisodium citrate, disodium hydrogen citrate).
• Clean up with dispersive SPE tubes containing MgSO4, PSA and C18.
• Concentrate, reconstitute in acetonitrile, filter through 0.2 µm PTFE, then transfer to vial for analysis.

Instrument Used

The analysis was conducted on a Shimadzu LCMS-8050 triple quadrupole system equipped with electrospray ionization. Chromatographic separation was achieved on a Kinetex C18 column (2.1 × 100 mm, 1.7 µm) using a gradient of ammonium acetate/formic acid in water and methanol.

Main Results and Discussion

• Calibration was linear (r² > 0.999) over 10 pg/mL to 10 ng/mL for B1 and G1, and up to 3 ng/mL for B2 and G2.
• Limits of detection ranged from 1.5 to 7.4 pg/mL and LOQs from 4.6 to 22.4 pg/mL.
• Intra-day repeatability (%RSD, n=6) was below 7.5% for all except aflatoxin G2 (12.1%).
• Matrix effects in peanut butter ranged from 71% to 81%, indicating moderate signal suppression.
• Recoveries across spiking levels (30–200 pg/mL) varied from 65% to 95%, meeting typical validation criteria.
• Analysis of three commercial peanut butter samples found aflatoxin B1 above EU limits (2.09 ng/g) in one sample, while others were compliant.

Benefits and Practical Applications

The method offers rapid, cost-effective sample preparation without immunoaffinity columns, high sensitivity and robust quantification suitable for routine food safety screening and regulatory monitoring of aflatoxins in fatty matrices.

Future Trends and Potential Uses

Integration of high-throughput automation and on-line sample cleanup may further increase efficiency. Expansion to other mycotoxins and complex matrices, as well as coupling with high-resolution mass spectrometry, could broaden the scope of food safety analyses.

Conclusion

A sensitive and streamlined LC/MS/MS approach using modified QuEChERS extraction has been demonstrated for reliable determination of aflatoxins in peanut butter. Validation results confirm the method’s suitability for compliance testing and quality control in the food industry.

Reference

• Pereira V., Fernandes J., Cunha S. Trends in Food Science & Technology 2014, 36, 96–136
• Liu Y., Han S., Lu M., Wang P., Han J., Wang J. Journal of Chromatography B 2014, 970, 68–76