High Speed, High Resolution Analysis of Aflotoxins

Aflatoxins are toxins produced by several aspergillus fungus species. As well as being highly toxic, they are also known to be carcinogenic, so the inspection of foods for their presence is necessary (1). In Europe the maximum allowed concentrations in food and food compositions are regulated; limits have been described for the totals and for the species aflatoxin B₁, B₂, G₁ and G₂. This application describes two cases of analysis of these 4 aflatoxins (B₁, B₂, G₁ and G₂), using the prominence RF-20A_XS high-sensitivity fluorescence detector while applying trifluoroacetic acid derivatization and direct detection.

Derivatization of Aflatoxins with Trifluoroacetic Acid

Typically, aflatoxins B₁ and G₁ are converted to the hydroxylderivative aflatoxins B_2a and G_2a using trifluoroacetic acid (TFA) to increase their fluorescence intensity in HPLC analysis. Figure 1 shows the structures of the 4 aflatoxins and the B_2a and G_2a TFA derivatives. When analysing aflatoxins in food, TFA derivatization is done for both the standard solution and the sample solution.

Figure 1: Structures of aflatoxins B1, B2, G1, G2 and trifluoroacetic acid-derivatized forms (B2a, G2a).

Analysis of Standard Solution after Derivatization with Trifluoroacetic Acid

Figure 2 shows chromatograms of standard solutions of the 4 aflatoxins (B₁, B₂, G₁ and G₂) obtained following TFA derivatization (Figure 5) with 20 µL injections, and Table 1 shows the analytical conditions. A peak area repeatability (n = 6) of 1.2% relative standard deviation (RSD) was obtained for the B_2a peak in the chromatogram on the right in Figure 2, and the detection limit (S/N ratio = 3.3, 20 µL injection) was calculated to be 0.4 ng/L (8 fg). Figure 3 shows the calibration curves (B₁ and G₁: 0.004–20 µg/L, B₂ and G₂: 0.001–5 µg/L). Excellent linearity was obtained for all 4 components, with an R2 value greater than 0.9999. Ultra-trace levels of aflatoxins can therefore be detected accurately with high sensitivity using the RF-20A_XS.

Figure 2: Chromatograms of aflatoxin standard solutions after derivatization with trifluoroacetic acid (20 µL injected); (left) B1 and G1: 2.0 µg/L, B2 and G2: 0.5 µg/L, (right) B1 and G1: 20 ng/L, B2 and G2: 5 ng/L.

Table 1: Analytical conditions.

Column: Shim-pack FC-ODS (150 mm L. × 4.6 mm i.d., 3 µm)

Mobile phase: Water/Methanol/Acetonitrile = 6/3/1 (v/v/v)

Flow rate: 0.8 mL/min

Column temp.: 40 °C

Detection : RF-20AXS, Ex at 365 nm, Em at 450 nm

RF cell : Conventional cell

Cell temp.: 25 °C

Injection volume: 20 µL

Figure 3: Calibration curves of aflatoxin standard solutions after derivatization with trifluoroacetic acid (B1 and G1: 0.004–2.0 µg/L, B2 and G2: 0.001–5 µg/L, 20 µL injected).

Apart from using derivatization methods, direct detection of aflatoxins is also possible using the highly sensitive RF-20A_XS fluorescence detector.

Analysis of Standard Solution by Direct Detection

Figure 4: Chromatograms of aflatoxin standard solutions by direct detection (20 µL injected) (left) B1 and G1: 2.0 µg/L, B2 and G2: 0.5 µg/L, (right) B1 and G1: 20 ng/L, B2 and G2: 5 ng/L.

Figure 4 shows the chromatograms of standard solutions of the 4 aflatoxins (B₁, B₂, G₁, G₂) obtained with 20 µL injections without TFA derivatization. Analytical conditions were the same as those shown in Table 1. Regarding the B₁ peak in the chromatogram on the right in Figure 4, a peak area repeatability (n = 6) of 2.7% RSD was obtained, and the detection limit (S/N ratio = 3.3, 20 µL injection) was calculated to be 3 ng/L(60 fg). This demonstrated that testing for aflatoxin B₁ and G₁ can be done with sufficient sensitivity using direct detection with the RF-20A_XS, without TFA derivatization.

Analysis of Food

For a food application example, aflatoxin standard solution was added to commercially available cornflour so that the aflatoxin concentrations in the sample became 0.8 µg/kg for B₁ and G₁, and 0.2 µg/kg for B₂ and G₂, respectively. The pseudo-contaminated sample was then analysed using both TFA derivatization and direct detection without derivatization. Figure 5 shows the chromatograms obtained from analysis of commercially available cornflour, unspiked (blank) and spiked with the aflatoxin standard solution, in this case using TFA derivatization.

Figure 5: Chromatograms of cornflour using derivatization with TFA (20 µL injected) (upper) spiked with aflatoxin standard, (lower) unspiked.

Reference

(1) Shimadzu - LC Application News L 428, Shimadzu Corp. Kyoto, Japan.

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