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, Derivazaon, Enzyme-Linked Immunosorbent Assay (ELISA), ultra High Performance Liquid Chromatography (uHPLC) Toni Hoine, Horizon Technology Inc., Salem, NH USA Elizabeth Krantz, Dr. Cheri A. Barta, and Dr. Pamela Doolile, University of Wisconsin, Madison, WI, USA Robert Buco, Richard Koeritz, and Zachary Lilla, Shimadzu Scienfic 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 parasicus 1 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 matrices 2 . Regulaons for the maximum limits vary for the reported aflatoxin B1 and total aflatoxins (sum of B1, B2, G1, and G2); however, most countries imporng food and agriculture products perform tesng to approve the safety of products. Tesng may oſten reveal aflatoxin levels above the maximum limits allowed, creang a trade restricon for certain agriculture and food products from certain countries 3 . The tests are performed according to their sampling methods and the results are measured against their established limits. There are several methods for detecng and quanfying aflatoxins; however, detecng 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 quanfy has created the need to add a derivazaon step. To assist with detecon at lower levels, derivazaon of the aflatoxin standards using an acid soluon aids in the fluorescence of both aflatoxin B1 and G1. Fluorescence is the more preferred reverse phase HPLC detecon method for its ability to offer increased sensivity at lower levels of aflatoxin 1 . With the requirement to increase tesng of agriculture and food products for the presence of aflatoxins, reliable and convenient tesng methods that ulize readily available standard laboratory tools are in demand to assist technicians with simplified tesng procedures that consistently generate accurate results. For the general laboratory, newer technologies for aflatoxin analysis (i.e., ELISA) may be financially unaainable. 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 applicaon focuses on the successful use of general equipment to accurately detect and report a linear seven-point calibraon curve of aflatoxin B1, B2, G1, and G2 using the XcelVap as a thermostacally-controlled heated water chamber for derivazaon.
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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
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