Determination of Aflatoxins B₁, B₂, G₁, G₂ in Food and Grain Products

• Analyte: Aflatoxins B₁, B₂, G₁, G₂
• System: Agress 1100 or iChrom 5100 HPLC System with fluorescence detector and post-column photochemical derivatizer
• Column: SinoChrom ODS-BP (5 μm, 4.6 × 200 mm)
• Highlight: Enhanced sensitivity for B₁ and G₁ using photochemical post-column derivatization without additional reagents.

Aflatoxins are toxic metabolites produced by aspergillus flavus and Aspergillus parasiticus growing on food and feed. They are highly toxic and carcinogenic, with aflatoxin B₁ being the most potent. Contamination can occur in grains, nuts, and their derived products, especially under warm and humid conditions. Detection typically requires derivatization to enhance fluorescence. Traditional pre-column trifluoroacetic acid (TFA) or post-column iodine derivatization methods are complex and involve hazardous reagents. Photochemical derivatization offers a simpler, safer, and more environmentally friendly alternative. This solution, based on GB/T 5009.23-2006, provides a complete workflow for the analysis of aflatoxins B₁, B₂, G₁, G₂ in grain products using HPLC with post-column photochemical derivatization.

Aflatoxin standards: aflatoxins B₁, B₂, G₁, G₂ (purity appropriate for quantitative analysis); Methanol, HPLC grade;  acetonitrile, HPLC grade; water, deionized (18.2 MΩ·cm);

Other materials: immunoaffinity columns or solid-phase extraction cartridges (for sample cleanup, as per standard methods), glass fiber filter paper, centrifuge tubes, volumetric flasks, pipettes, etc.

Stock Solutions: Prepare individual stock solutions of aflatoxins B₁, B₂, G₁, G₂ in methanol at known concentrations (e.g., 1–10 μg/mL). Store protected from light at low temperature (≤4°C).

Mixed Working Solutions: Dilute appropriate volumes of stock solutions with mobile phase to prepare mixed standard working solutions at desired concentrations. For linearity studies, typical concentration ranges: B₁ and G₁ from 0.2 to 20.0 μg/L; B₂ and G₂ from 0.06 to 6.0 μg/L.

The general steps of sample preparation include:

• Extraction: Weigh a representative portion of the ground sample (e.g., corn, grain) and extract with methanol-water solution.
• Cleanup: Pass the extract through an immunoaffinity column or solid-phase extraction cartridge specific for aflatoxins. Wash to remove impurities.
• Elution: Elute aflatoxins with methanol or acetonitrile.
• Concentration (if needed): Evaporate the eluate under a gentle stream of nitrogen and reconstitute in mobile phase to a known volume.
• Filtration: Filter through a 0.45 μm membrane before HPLC injection.

System Configuration Options (Table 1):

Table 1. System Configuration Options

Chromatographic Conditions

• Column: SinoChrom ODS-BP (5 μm, 4.6 × 200 mm)
• Mobile Phase: Methanol / Acetonitrile / Water = 20 : 20 : 60 (V/V/V)
• Flow Rate: 0.8 mL/min
• Detection: Fluorescence detector, excitation wavelength 360 nm, emission wavelength 450 nm
• Post-Column Derivatization: Photochemical derivatizer (PD3110) installed between column and detector
• Injection Volume: 20 μL
• Column Temp.: Ambient

Standard Separation

A typical chromatogram of a mixed aflatoxin standard solution (20.0 μg/L) after photochemical derivatization is shown in Figure 1. The derivatization significantly enhances the signals of B₁ and G₁ while having minimal effect on B₂ and G₂, as summarized in Table 2.

Fig.1. Chromatogram of mixed standard solution (20.0 μg/mL) of aflatoxin

Table 2. Comparison of response before and after derivatization

Linearity and Detection Limit

Calibration curves were constructed using mixed standard solutions at various concentrations. Linearity and detection limits are summarized in Tables 3.

Table 3. Linear Equations and Correlation Coefficients

Sample Analysis

A commercially purchased corn sample was analyzed following the described procedure. Results are shown in Table 4 and Figure 2.

Table 4. Aflatoxin Content in Corn Sample

Fig.2. Chromatogram of mixed aflatoxin standard solution (20.0 μg/mL) and corn sample solution