No. SSI-HPLC-014
High Performance Liquid Chromatography
No. HPLC-014
A Novel Method for Pre-Column Derivatization of
Aflatoxin B1, B2, G1, and G2 Prior to HPLC Analysis
using the XcelVap® System as a ThermostaticallyControlled 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
Key Words: Aflatoxin, Mycotoxins, Derivatization, Enzyme-Linked Immunosorbent Assay (ELISA), ultra High Performance Liquid
Chromatography (uHPLC)
■ 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.
■ Instrumentation
Sigma-Aldrich, Aflatoxin Mix 4 Solution
o 0.5 µg/mL B2 and G2 plus 2 µg/mL B1
and G1 in acetonitrile (HPLC grade)

Sigma-Aldrich, Titan™ C18 uHPLC Column
o 1.9 µm particle size, 10 cm x 2.1 mm

Sigma-Aldrich, Titan ™C18 HPLC Guard
Cartridge
o 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
No. SSI-HPLC-014
■ 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.
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.
Figure 1: Aflatoxin B1, B2, G1, and G2 Standard Preparation Process
Level 1
Level 2
Level 3
Level 4
Level 5
Level 6
Level 7
Aflatoxin B1
(µg/kg)
0
0.8
1.6
3.2
4
8
12
Aflatoxin B2
(µg/kg)
0
0.2
0.4
0.8
1
2
3
Aflatoxin G1
(µg/kg)
0
0.8
1.6
3.2
4
8
12
Aflatoxin G2
(µg/kg)
0
0.2
0.4
0.8
1
2
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
Run Time
Wavelength
50 µL
25 minutes
Excitation: 360 nm/Emission: 440 nm
Table 2: HPLC Conditions for Aflatoxin Analysis
No. SSI-HPLC-014
■ 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).
R2 = 0.9966
Figure 2: Aflatoxin B1 Linearity
R2 = 0.9963
Figure 4: Aflatoxin G1 Linearity
Derivatization was first performed at 65°C for 20
minutes, where results provided linearity values at
<0.990. With a shorter derivatization time, the
chromatography for aflatoxin B1 visibly showed a
small fronting peak that did not allow for consistent
integration across the standard levels (Figures 6 and
7). Derivatization was then performed at 65°C for 25
minutes.
R2 = 0.9965
Figure 3: Aflatoxin B2 Linearity
R2 = 0.9958
Figure 5: Aflatoxin G2 Linearity
This additional 5 minutes of derivatization provided
chromatography that did not show the fronting
peak, which may have been incomplete derivatized
aflatoxin B1 (Figures 8 and 9). Table 3 outlines the
improvements the derivatization process had on
individual aflatoxin peak responses.
No. SSI-HPLC-014
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
No. SSI-HPLC-014
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
No. SSI-HPLC-014
Table 3: Derivatization Comparison of Aflatoxin B1, B2, G1, and G2 Peak Area Responses for Level 2 and Level 5 Standards
20 Minute
Derivatization
25 Minute
Derivatization
20 Minute
Derivatization
25 Minute
Derivatization
Level 2
Area Response
Level 2
Area Response
Level 5
Area Response
Level 5
Area Response
Aflatoxin
B1
Aflatoxin
B2
Aflatoxin
G1
Aflatoxin
G2
4079
1527
1362
527
5677
2051
1872
663
30137
9841
10203
3727
39366
13250
13674
4975
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
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).
European Mycotoxins Awareness Network Site.
http://services.leatherheadfood.com/eman/FactSheet.
aspx?ID=79
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/fo
cus20_12.pdf
Food and Drug Administration. Federal Register: May
22, 1997 (Volume 62, Number 99),
http://www.gpo.gov/fdsys/pkg/FR-1997-0522/pdf/97-13677.pdf.
First Edition: April 2016
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