A Novel Method for Pre-Column Derivatization of Aflatoxin B1, B2, G1, and G2 Prior to HPLC Analysis using the XcelVap® System as a Thermostatically-Controlled Heated Water Chamber

Key Words

Aflatoxin, Mycotoxins, Derivatization, Enzyme-Linked Immunosorbent Assay (ELISA), ultra High Performance Liquid Chromatography (uHPLC)

Toni Hofhine, Horizon Technology Inc., Salem, NH USA

Elizabeth Krantz, Dr. Cheri A. Barta, and Dr. Pamela Doolittle,

University of Wisconsin, Madison, WI, USA

Robert Buco, Richard Koeritz, and Zachary Lilla, Shimadzu Scientific Instruments, Marlborough, MA, USA

Jennifer Claus, Kenneth Espenschied, and Michael Ye, Sigma-Aldrich, Bellefonte, PA, USA

Introduction

Aflatoxins, a mold largely produced by Aspergillus flavus and Aspergillus parasiticus1 are commonly tested mycotoxins found

naturally in a wide range of agriculture crops and food products. Due to their harmful effects on human health, animal health, and

global trade, aflatoxins are regulated in most countries and have established global limits in a wide variety of matrices2.

Regulations for the maximum limits vary for the reported aflatoxin B1 and total aflatoxins (sum of B1, B2, G1, and G2); however,

most countries importing food and agriculture products perform testing to approve the safety of products. Testing may often

reveal aflatoxin levels above the maximum limits allowed, creating a trade restriction for certain agriculture and food products

from certain countries3. The tests are performed according to their sampling methods and the results are measured against their

established limits.

There are several methods for detecting and quantifying aflatoxins; however, detecting all aflatoxins using the same method can

be challenging. The limited response for B1 and G1 to naturally absorb UV light or fluoresce at the levels many countries need to

quantify has created the need to add a derivatization step. To assist with detection at lower levels, derivatization of the aflatoxin

standards using an acid solution aids in the fluorescence of both aflatoxin B1 and G1. Fluorescence is the more preferred reverse

phase HPLC detection method for its ability to offer increased sensitivity at lower levels of aflatoxin1.

With the requirement to increase testing of agriculture and food products for the presence of aflatoxins, reliable and convenient

testing methods that utilize readily available standard laboratory tools are in demand to assist technicians with simplified testing

procedures that consistently generate accurate results. For the general laboratory, newer technologies for aflatoxin analysis (i.e.,

ELISA) may be financially unattainable. A novel method was developed using an enclosed, dark, and moist heated environment to

allow consistent linearity results to be obtained for all four Aflatoxin standards (B1, B2, G1, G2). This application focuses on the

successful use of general equipment to accurately detect and report a linear seven-point calibration curve of aflatoxin B1, B2, G1,

and G2 using the XcelVap as a thermostatically-controlled heated water chamber for derivatization.

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Sigma-Aldrich, Aflatoxin Mix 4 Solution

0.5 µg/mL B2 and G2 plus 2 µg/mL B1 and G1 in acetonitrile (HPLC grade)

Sigma-Aldrich, Titan™ C18 uHPLC Column

1.9 µm particle size, 10 cm x 2.1 mm

Sigma-Aldrich, Titan ™C18 HPLC Guard Cartridge

1.9 µm particle size, 5 mm x 2.1 mm I.D

Sigma-Aldrich, Trifluoroacetic Acid (TFA), 99%

Glacial Acetic Acid, JT Baker, >=99.7%

Laboratory Milli-Q™ Water

Acetonitrile, Sigma-Aldrich

Trifluoroacetic Acid (TFA), Sigma-Aldrich 99%

Shimadzu, Nexera XR uHPLC System with Fluorescence Detector

Horizon Technology, XcelVap® Automated/Concentration System Standard Preparation

Seven levels of aflatoxin standards were prepared using manual pipettes (Figure 1) at concentrations listed in Table 1. The TFA

derivatization solution was prepared using Milli-Q water:trifluoroacetic acid:glacial acetic acid in a 70:20:10 volume ratio.

Instrumentation

Figure 1: Aflatoxin B1, B2, G1, and G2 Standard Preparation Process

Extra precautions were taken to pre-heat the XcelVap to 65°C and fill the cavity with Milli-Q water to 75% of the vial height prior to derivatization. Following derivatization, standards were kept loosely covered with aluminum foil when possible to prevent light exposure.

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Aflatoxin B1

(µg/kg)

Aflatoxin B2

(µg/kg)

Aflatoxin G1

(µg/kg)

Aflatoxin G2

(µg/kg)

Level 1 0 0 0 0

Level 2 0.8 0.2 0.8 0.2

Level 3 1.6 0.4 1.6 0.4

Level 4 3.2 0.8 3.2 0.8

Level 5 4 1 4 1

Level 6 8 2 8 2

Level 7 12 3 12 3

Table 1: Aflatoxin B1, B2, G1, and G2 Standard Concentrations

HPLC Analysis

Duplicate 50 µL HPLC standard injections were performed at each level using the conditions outlined in Table 2. The average area

response of each level was used to calculate the linear regression for each aflatoxin standard.

HPLC Conditions

Flow Rate 0.4 mL/min

Column Titan™ C18 UHPLC Column, 10 cm x 2.1 mm I.D.,

1.9 μm particle size

Guard Column Titan™ C18 HPLC Guard Cartridge, 5 mm x 2.1 mm I.D.,

1.9 μm particle size

Column Temperature 45°C

Mobile Phase

0 – 3.75 minutes: 5% acetonitrile in Milli-Q water

3.75 – 15.5 minutes: 20% acetonitrile in Milli-Q water

18 – 25 minutes: 5% acetonitrile in Milli-Q water

Injection Volume 50 µL

Run Time 25 minutes

Wavelength Excitation: 360 nm/Emission: 440 nm

Table 2: HPLC Conditions for Aflatoxin Analysis

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Results and Discussions

A seven point linearity curve was used for all aflatoxin sample calculations. The linearity for all four aflatoxin standards was > 0.995

(Figures 2-5).

Figure 2: Aflatoxin B1 Linearity Figure 3: Aflatoxin B2 Linearity

Figure 4: Aflatoxin G1 Linearity Figure 5: Aflatoxin G2 Linearity

R2 = 0.9966 R2 = 0.9965

R2 = 0.9963 R2 = 0.9958

Derivatization was first performed at 65°C for 20 minutes, where results provided linearity values at <0.990. With a shorter deri-

vatization time, the chromatography for aflatoxin B1 visibly showed a small fronting peak that did not allow for consistent inte-

gration across the standard levels (Figures 6 and 7). Derivatization was then performed at 65°C for 25 minutes. This additional 5

minutes of derivatization provided chromatography that did not show the fronting peak, which may have been incomplete deri-

vatized aflatoxin B1 (Figures 8 and 9). Table 3 outlines the improvements the derivatization process had on individual aflatoxin

peak responses.

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Figure 6: Level 2 Aflatoxin Standard Chromatogram with Derivatization at 65°C for 20 Minutes

Figure 7: Level 5 Aflatoxin Standard Chromatogram with Derivatization at 65°C for 20 Minutes

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on

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ilk

Figure 8: Level 2 Aflatoxin Standard Chromatogram with Derivatization at 65°C for 25 Minutes

Figure 9: Level 5 Aflatoxin Standard Chromatogram with Derivatization at 65°C for 25 Minutes

Data collected compared the 20 minute and 25 minute derivatization peak responses for the low and medium standards, and the

area responses clearly indicated that full derivatization was not complete in 20 minutes. The additional 5 minutes of moist heat

improved peak area responses and improved baselines, eliminating the peak fronting that was visible in the 20 minute derivatized

aflatoxin B1. The additional 5 minutes of derivatization time also improved the aflatoxin B2, G1, and G2 peak area responses.

Derivatization using an aqueous TFA solution for reverse phase chromatography after optimizing the method to include 25 minutes

of moist heat produces sharp distinguishable peaks for quantitation, but does limit stability of aflatoxin standards1.

Conclusion

The XcelVap Evaporator/Concentrator System was successfully used to optimize pre-column derivatization of all four aflatoxin

standards (B1, B2, G1, G2). The enclosed, dark, and moist heated environment allowed for consistent linearity results to be

obtained prior to fluorescence HPLC detection. Elaborate technology and post-column derivatization should not be a requirement

for derivatization when common laboratory equipment can be used effectively and at a lower cost. Many journal resources have

researched and reported a variety of times and temperatures for performing derivatization; however, for the highest confidence

and most efficient quantitation, it is recommended to test a few derivatization times. In this application note, the longer

derivatization time produced optimal results. Chromatography was used as an indicator of full derivatization, and peak fronting

was been shown to be a reasonable factor in considering whether the derivatization process was complete.

References

1. W.Th. Kok, Derivatization reactions for the determination of aflatoxins by liquid chromatography with fluorescence detection,

Journal of Chromatography B: Biomedical Applications, 659 (1994) 127 – 137 (http://dare.uva.nl/document/37272).

2. European Mycotoxins Awareness Network Site. http://services.leatherheadfood.com/eman/FactSheet.aspx?ID=79

3. Devesh Roy, International Food Policy Research Institute, Aflatoxins: Finding Solutions For Improved Food Safety, Focus 20,

Brief 12, November 2013. http://www.ifpri.org/sites/default/files/publications/focus20_12.pdf

4. Food and Drug Administration. Federal Register: May 22, 1997 (Volume 62, Number 99), http://www.gpo.gov/fdsys/pkg/FR-

1997-05-22/pdf/97-13677.pdf.

Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2

20 Minute Derivatization

Level 2

Area Response 4079 1527 1362 527

25 Minute Derivatization

Level 2

Area Response 5677 2051 1872 663

20 Minute Derivatization

Level 5

Area Response 30137 9841 10203 3727

25 Minute Derivatization

Level 5

Area Response 39366 13250 13674 4975

Table 3: Derivatization Comparison of Aflatoxin B1, B2, G1, and G2 Peak Area Responses for Level 2 and Level 5 Standards

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