Extraction of Aflatoxins and Ochratoxin from Dried Chili Using ISOLUTE® Myco Prior to LC-MS/MS Analysis

Importance of the Topic

Mycotoxin contamination in food products poses significant health risks and is tightly regulated worldwide. Chili peppers are particularly susceptible to contamination by aflatoxins and ochratoxin A, requiring reliable analytical methods to ensure food safety and comply with legislative limits.

Objectives and Study Overview

This application note details a streamlined sample preparation and LC–MS/MS protocol for simultaneous quantification of aflatoxin B1, B2, G1, G2 and ochratoxin A in dried chili (pimiento). The primary goals are to demonstrate high recovery, low detection limits, and reproducible results using a single SPE product across multiple mycotoxin classes.

Methodology and Instrumentation

A comprehensive workflow combines sample grinding, extraction with 80% aqueous acetonitrile, dilution, and ISOLUTE Myco SPE cleanup. Key steps include:

• Sample pre-treatment: grind 5 g chili, extract for 30 min, centrifuge and dilute.
• SPE protocol: condition with ACN and water, load matrix, perform two interference washes, dry, and elute in two steps (acidic ACN and basic methanol).
• Evaporation and reconstitution: concentrate eluates under nitrogen/air, reconstitute in acidified ACN/methanol/water and filter.

Instrumentation Used

• UHPLC: Shimadzu Nexera with Kinetex XB-C18 column (50 × 2.1 mm, 2.6 µm).
• Mobile phase: ammonium acetate/0.5% acetic acid in water and 95% methanol; gradient to 100% organic over 10 min.
• MS/MS: AB Sciex Triple Quad 5500 with Turbo V™ ESI source; positive mode MRM transitions optimized for each toxin.

Main Results and Discussion

The method achieved linear calibration across 0.5–80 µg/kg for aflatoxins and 5–80 µg/kg for ochratoxin A, with correlation coefficients >0.997. Limits of quantitation ranged from 2 µg/kg (aflatoxins) to 10 µg/kg (ochratoxin A). Recoveries were between 89% and 108% with %RSDr below 4%, meeting or exceeding EU criteria. Chromatographic separation provided retention times of 3.3–4.1 min for aflatoxins and 6.1 min for ochratoxin A, supporting a 10-minute run time.

Benefits and Practical Applications of the Method

• High throughput: single SPE column covers multiple toxin classes across various food matrices.
• Robust performance: consistent recoveries and low variability ensure regulatory compliance.
• Rapid analysis: 10 min LC–MS/MS cycle time accelerates sample turnaround.
• Versatility: applicable to dried chili and other cereals, nuts, spices and feed materials.

Future Trends and Applications

Ongoing enhancements in SPE sorbent chemistries and high-resolution mass spectrometry promise further improvements in sensitivity and multiplexing capability. Automation of bead-beating and SPE workflows will increase sample throughput. Emerging mycotoxin analogues and masked toxins will require expanded MRM libraries and improved cleanup strategies.

Conclusion

The presented SPE–LC–MS/MS protocol using ISOLUTE Myco offers a reliable, sensitive, and efficient approach for multi-mycotoxin analysis in dried chili. It meets stringent EU regulatory requirements and supports routine monitoring in food safety laboratories.

References

1. Biotage Application Note AN785: Extraction of Aflatoxins and Ochratoxin From Dried Chili Using ISOLUTE Myco Prior to LC–MS/MS Analysis. 2014.