Screening and Quantitation of 191 Mycotoxins and Other Fungal Metabolites in Almonds, Hazelnuts, Peanuts, and Pistachios Using UHPLC/MS/MS

Significance of the Topic

Mycotoxins are toxic fungal metabolites that can contaminate nuts during cultivation and storage, posing health risks and trade concerns. A comprehensive screening of multiple mycotoxins and metabolites in nuts is critical to ensure food safety, comply with regulations, and expand occurrence data beyond the few regulated compounds.

Objectives and Study Overview

This study aimed to develop and validate a multitarget UHPLC-MS/MS method for simultaneous screening and quantitation of 191 mycotoxins and fungal metabolites in almonds, hazelnuts, peanuts, and pistachios. The method’s performance was characterized for 65 priority analytes, including all EU-regulated mycotoxins and commonly encountered fungal metabolites, and then applied to naturally contaminated market samples.

Methodology and Used Instrumentation

Sample preparation and analysis were designed for simplicity and throughput:

• Extraction: 5 g ground nuts with 20 mL acidified acetonitrile–water (79:20:1, v/v/v), shaken 90 min, 8× dilution of the raw extract.
• Chromatography: Agilent 1290 UHPLC, ZORBAX RRHD Eclipse Plus C18 column (2.1×150 mm, 1.8 µm), 21 min gradient with ammonium acetate in methanol–water–acetic acid mobile phases.
• Detection: Agilent 6460 Triple Quadrupole MS with Jet Stream ESI, dynamic MRM acquisition in positive and negative modes, two transitions per analyte.
• Calibration: solvent standards over at least three orders of magnitude, 1/x-weighted linear curves.

Main Results and Discussion

Validation for 65 analytes demonstrated:

• Linear responses (R2>0.997) over broad concentration ranges.
• Limits of quantitation down to 0.04 µg/kg for sensitive analytes.
• Apparent recoveries of 70–120 % for ~60 % of compounds; variation driven by extraction efficiency and matrix effects.
• Extraction recoveries above 50 % for ~50 % of analytes; lower values for highly polar toxins (e.g., fumonisins).
• Matrix effects within ±20 % for 57 % of analytes; significant suppression for early eluters and enhancement for certain metabolites.

Application to 53 market samples revealed over 40 fungal metabolites:

• Hazelnuts exhibited the highest incidence (36 analytes), with one sample containing 26 mycotoxins.
• Most frequent contaminants: beauvericin, enniatin B, macrosporin, 3-nitropropionic acid, alternariol methyl ether.
• Regulated aflatoxins exceeded EU limits in multiple hazelnut and peanut samples; sterigmatocystin, T-2, and HT-2 toxins detected in hazelnuts for the first time.

Benefits and Practical Applications

This method offers a rapid, cost-effective workflow for broad mycotoxin surveillance in nuts, supporting quality control, regulatory compliance, and risk assessment. Its high throughput and comprehensive coverage make it suitable for routine monitoring and research into co-occurrence patterns.

Future Trends and Possibilities for Application

Enhancements may include stable isotope dilution or matrix-matched calibration to correct matrix effects, integration of more sensitive detectors, extension to other commodities, and coupling with advanced data-processing tools. Such developments will improve quantitation accuracy and expand knowledge of mycotoxin co-exposure risks.

Conclusion

A robust UHPLC-MS/MS method was established for simultaneous screening of 191 mycotoxins in nuts, with validated quantitation of 65 priority analytes. It combines simple sample preparation, fast UHPLC separation, and sensitive dynamic MRM detection. Application to commercial samples revealed widespread contamination by nonregulated metabolites, underscoring the need for comprehensive monitoring.

Reference

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