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Determination of Aflatoxins in Pistachio Samples

Significance of the Analysis

Peanuts and tree nuts such as pistachios are prone to contamination by Aspergillus molds during cultivation and storage. These molds produce aflatoxins—potent carcinogenic and mutagenic secondary metabolites—that resist typical food‐processing steps. Aflatoxin exposure from nuts can pose serious health risks, making reliable detection and quantification methods essential for consumer safety.

Sample Preparation Protocol

• Grind 50 g of pistachio kernels to a fine powder.
• Suspend in 20 mL methanol:water (17:3, v/v) and shake for 30 min.
• Centrifuge and collect the supernatant; repeat extraction.
• Degrease combined extracts twice with n-hexane.
• Extract aqueous layer twice with chloroform.
• Evaporate the organic phase to ~3 mL under nitrogen.
• Perform silica SPE: condition with n-hexane and chloroform; load extract; wash off lipids; elute aflatoxins with chloroform:acetone (9:1, v/v).

Chromatographic Conditions

• System: AZURA HPLC Plus with LPG pump, cooled autosampler, column thermostat, and RF-20Axs fluorescence detector.
• Column: Eurospher II C₁₈ 100-3, 150 × 4.6 mm.
• Mobile phase: isocratic acetonitrile:water (25:75) at 2.4 mL/min.
• Post‐column: Photochemical derivatization at 254 nm (UVE reactor) followed by FLD at Ex 365/Em 460 nm.
• Run time: 15 min including elution (0–3 min purge, 3–12 min read, 12–15 min re-equilibration).

Analytical Performance

• Baseline separation of aflatoxins B₁, B₂, G₁, G₂ (resolution >1.5).
• Limits of detection: 0.05 ng/mL (B₁/G₁), 0.015 ng/mL (B₂/G₂), several‐fold below regulatory maximums.
• Recoveries: 78–87 % at spiking levels (LOQ, 2× LOQ, 20 ng/mL).
• Repeatability (n=8): RSD<0.1 % (retention), <0.5 % (peak area/height).
• Robust to ±2 °C, ±0.2 mL/min, and ±2 % mobile phase variation.

Conclusion

The described protocol combines simple SPE cleanup with photochemical post‐column derivatization and HPLC‐FLD to achieve highly sensitive, rapid (15 min) and robust aflatoxin analysis in pistachio matrices. Detection limits significantly surpass EU and FDA requirements, and the method’s automation reduces handling time and improves reproducibility, ensuring effective routine monitoring of nuts for food safety.