Selectivity of Aflatoxin Analogs on Multiple Supelco Ascentis Stationary Phases

By: Sean W. Linder, Reporter US Volume 26.1

Contributed Article

The following was generated by an outside source using Sigma-Aldrich products. Technical content provided by:

Sean W. Linder1

1 Arkansas Veterinary Diagnostic Laboratory, #1 Natural Resources Drive, Little Rock, AR 72205.


The differences in selectivity and retention characteristics of four HPLC stationary phases in Supelco’s Ascentis product line has been demonstrated using multiple aflatoxin analogs. Baseline chromatographic resolution of four aflatoxin analogs with a total analysis time of less than ten minutes, using an isocratic mobile phase consisting of methanol, acetonitrile, and water was obtained.

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Aflatoxins are naturally occurring mycotoxins that are produced by a variety of species of Aspergillus, most commonly A. flavus and A. parasiticus. During Aspergillus growth, four structurally unique aflatoxin analogs, B1, B2, G1, and G2 are produced as secondary metabolites. These analogs can be found in many agriculturally important commodities such as: grains, nuts, milk products, and dried fruit. Aflatoxin quantitation in commodities is of major interest because of their carcinogenic impact to both human and animal health. Due to the substantial health impact, both the United States Food & Drug Administration (FDA) and European Union (EU) strictly regulate acceptable levels for human and animal consumption.

Quantitation of aflatoxin analogs is often reported as a total aflatoxin quantitation, which corresponds to the sum of the four most common analogs B1, B2, G1, and G2. Screening for total aflatoxins is commonly performed by ELISA due to high sample throughput, however, confirmation and quantitation is often performed by high-performance liquid chromatography (HPLC). Therefore, the ability to separate and detect the most common analogs of aflatoxins in an efficient and cost-effective manner is of great interest to agricultural-based analytical laboratories.

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In this report, four unique HPLC stationary phases were evaluated for selectivity and retention characteristics of the aflatoxin analogs B1, B2, G1, and G2. The goal of the evaluation was to determine if baseline separation was obtainable for all four analogs with an isocratic HPLC mobile phase and fluorescence detection. Furthermore, it was desirable to obtain the separation with a total run time of less than 15 minutes without using a gradient system. For these experiments, post-column photochemical derivatization (PHRED) was used to enhance the fluorescence detector response of Aflatoxin G1 and B1 (1).

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columns: Ascentis C8, C18, RP-Amide, and Phenyl,
15 cm x 4.6 mm I.D., 3 µm particle size
mobile phase: 20:20:60 acetonitrile:methanol:water
flow rate: 1.0 mL/min
temp.: 30 °C or 50 °C
detector: fluorescence, 360 nm excitation, 455 nm emission
photo derivatization: PHRED (25 m x 0.25 mm ID coil)
injection: 20 mL
sample: Alfatoxin Standard Mix (diluted 1:1 with water)

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Results & Discussion

Selectivity between chromatographic stationary phases is often not well understood. As a chromatographer, it is often impossible to predict how a particular stationary phase will interact with a given analyte or group of analytes, such as the aflatoxins. Figure 1 demonstrates the selectivity and retention for the four analogs of aflatoxin using various Ascentis stationary phases with an isocratic mobile phase composed of methanol, acetonitrile, and water.

Figure 1. Evaluation of Multiple Stationary Phases for the Separation of Common Analogs of Aflatoxin

Selectivity and retention between the four aflatoxin analogs was markedly similar between the C8, C18, and RP-Amide stationary phases. The only stationary phase to provide different selectivity and retention characteristics between the four analogs was the phenyl phase. It is speculated that this difference can be attributed to the increased π-π interactions between the phenyl phase and the additional carbon-carbon double bond that is present in the structures for both aflatoxin B1 and G1. Phenyl based chromatographic stationary phases are well known to offer unique selectivity and retention characteristics to compounds that contain polycyclic aromatic structures due to interactions between the analytes of interest and the benzene ring of the stationary phase. These subtle interactions allow the reversal in selectivity for analogs G1 and B2, while also increasing the retention of all analogs with respect to the other stationary phases.

Baseline separation was only achieved when using the C18 stationary phase, however, all of the stationary phases included in this study exhibited some resolution. Further exploration of mobile phase conditions on these other phases can be expected to result in baseline separation.

The secondary goal of this study was to determine separation conditions that could produce an analysis time of less than 15 minutes. The C18 stationary phase was utilized in this experiment because it exhibited the highest resolution for the four analogs. Figure 2 demonstrates the effect of column temperature on the retention characteristics of the C18 stationary phase. The analysis time for all aflatoxin analogs was decreased when performing analytical analysis at 50 °C, as compared to 30 °C, with baseline resolution for all four analogs. By increasing the temperature of the chromatography column, the quantitation of the four analogs can be performed in less than 10 minutes total analysis time. Furthermore, due to the utilization of an isocratic mobile phase, equilibration time between analytical samples and/or standards should be minimal, thus increasing the overall efficiency of the analysis.

Figure 2. Effects of Column Temperature on Retention Time and Resolution for Common Analogs of Aflatoxins using Ascentis C18

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Many chromatography vendors offer specialized aflatoxin HPLC stationary and mobiles phases to achieve baseline resolution between various aflatoxin analogs, however the present study demonstrates that baseline resolution can be obtained between all four analogs within 15 minutes at ambient temperature using commonly available chromatographic stationary and mobile phases. Furthermore, the total analysis time can be reduced to less than 10 minutes by heating the column to 50 °C.

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  1. Henry Joshua, Determination of Aflatoxins by Reversed-Phase High Performance Liquid Chromatography with Post-Column In-Line Photochemical Derivatization and Fluorescence Detection, Journal of Chromatography, 654, 247-254 (1993).

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