Determination of aflatoxins in pistachio samples - from extraction to high efficient detection

Significance of the Topic

Consumption of pistachios contaminated with aflatoxins carries significant health risks due to the potent carcinogenicity and stability of these fungal metabolites. Warm and humid conditions during cultivation and storage promote Aspergillus growth and aflatoxin production, making reliable analysis essential for food safety and compliance with regulatory limits.

Objectives and Study Overview

This study presents a streamlined workflow for extracting aflatoxins from pistachio kernels and quantifying all four major aflatoxins (B1, B2, G1, G2) in a single HPLC run. Emphasis is placed on simple sample preparation, efficient matrix removal, and sensitive photochemical post-column derivatization to meet stringent detection requirements.

Methodology and Instrumentation

Sample preparation involves grinding 50 g of shelled pistachios, extracting with methanol/water, degreasing with n-hexane, and liquid–liquid extraction into chloroform. Solid-phase extraction (SiOH cartridge) further reduces matrix interferences. The purified extract undergoes HPLC on a C18 column with post-column UV irradiation at 254 nm for in-situ derivatization, enhancing fluorescence detection at 460 nm.

Instrumentation

• Pump: AZURA P6.1L, LPG
• Autosampler: AZURA AS 6.1L
• Column Thermostat: AZURA CT 2.1 at 60 °C
• Column: Eurospher II 100-3 C18, 150 × 4.6 mm ID
• Fluorescence Detector: RF-20A with UVE Box photochemical reactor
• Software: ClarityChrom 8.1

Main Results and Discussion

The three-step extraction effectively reduced matrix fluorescence from over 1600 counts to below 45 counts in the critical retention window. Chromatograms of spiked extracts demonstrated clear separation of aflatoxins. Limits of detection were 0.05 ng/mL for B1/G1 and 0.015 ng/mL for B2/G2, exceeding European Commission requirements by factors of 3.4 and 11.3, respectively.

Benefits and Practical Applications

• High sensitivity allows detection well below regulatory limits.
• Robust sample cleanup minimizes matrix effects across diverse food matrices.
• Non-toxic photochemical derivatization avoids halogen waste.
• Single chromatographic run for four aflatoxins streamlines routine testing.

Future Trends and Opportunities

Advances may include integration with mass spectrometry for multi-mycotoxin profiling, automated sample preparation to increase throughput, and miniaturized photochemical reactors for field-deployable analysis. Ongoing developments in stationary phases and detection technologies will further enhance sensitivity and reduce analysis time.

Conclusion

The optimized extraction and HPLC method with photochemical derivatization provides a fast, reliable, and environmentally friendly approach for routine determination of aflatoxins in pistachios and related food matrices, ensuring consumer safety and regulatory compliance.

References

1. Hedayati MT, Pasqualotto AC, Bowyer P, Denning DW. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153(6):1677–1692 (2007).
2. Bui-Klimke TR, Guclu H, Kensler TW, Yuan JM, Wu F. Aflatoxin regulations and global pistachio trade: insights from social network analysis. PLoS One 9(3):e92149 (2014).
3. World Health Organization. Aflatoxins. Food Safety Digest, Department of Food Safety and Zoonoses, Ref. No. WHO/NHM/FOS/RAM/18.1 (2018).
4. U.S. FDA. Action levels for aflatoxins in animal food. CPG Sec. 683.100 (2019).
5. European Commission Regulation (EC) No 401/2006 laying down methods of sampling and analysis for mycotoxins in foodstuffs (2016).
6. European Commission Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs (2006).
7. Folmert K, Margraf M, Monks K. Quick and easy determination of aflatoxins in food matrices with photochemical post-column derivatization. KNAUER AppNote VFD0178:1–4 (2019).