Determination of Aflatoxins (B1, B2, G1

and G2) in Corn and Peanut Butter by

HPLC-FLD Using Pre-column
Immunoaffinity Cleanup and Post-Column
Electrochemical Derivatization

Application Note

Author Abstract
Xinlei Yang, Rong An A method for simultaneous determination of four aflatoxins B1, B2, G1 and G2 in
corn and peanut butter using HPLC-FLD was developed in the work. After extracted
Application Engineer of Liquid by methanol/water (60/40, v/v), the sample was cleaned up and concentrated on
Chromatography an immunoaffinity column and then the sample solution was separated and quanti-
Life Science and Chemical Analysis fied by HPLC-FLD with KOBRA cell for derivatization. Each aflatoxin got linear range
from 0.1 ~ 10 µg/L with the square of correlation coefficient (R2) > 0.99998. The
Group limit of detection (LOD, S/N = 3) was in the range of 0.004 ~ 0.007 µg/L (equivalent
Agilent Technologies to 0.008 ~ 0.014 µg/kg content in samples). The precision was validated by six
shanghai, China sequential injections of 10 µg/L standard solution, the relative standard deviations
(RSDs) obtained of retention time and peak area were less than 0.1% and 0.3% for
each aflatoxin, respectively. It was proved that the proposed method could be uti-
lized to determine aflatoxins in corn and peanut butter effectively and accurately.

Introduction
Aflatoxins are produced as metabolites by the Aspergillus Flavus and Aspergillus
Parasiticus and exist in nature world widely. The common aflatoxins are B1, B2, G1
and G2. Among these mycotoxins the aflatoxin B1 is of most toxicity followed by G1,
the toxicities of B2 and G2 are relative weak. Due to the high toxicity and carcino-
genicity to human and animals, many countries and regulatory agencies impose
strict limits on aflatoxins. The European Commission has set maximum levels for
aflatoxin B1 between 2.0 and 8.0 µg/kg and for the sum total of all four aflatoxins
(B1, B2, G1 and G2) between 4.0 and 15.0 µg/kg in crops such as nuts, grains and
dried fruits [1]. The action level was set of 20 and 300 µg/kg for human food and ani-
mal feed by U.S. FDA, respectively [2]. Therefore, it is important to develop an accu-
rate and effective method for aflatoxins determination.
HPLC with fluorescence detection was utilized for aflatoxins Sample Preparation
determination in many matrices increasingly. HPLC analysis Weigh 25g of sample and 2g of sodium chloride into high-
was frequently required with immunoaffinity clean up for speed blender jar. Add 125ml of HPLC Grade methanol:dis-
sample preparation and derivatization for high sensitivity tilled water (60:40, v:v) into the jar, cover and blend for 1
detection due to the influence of matrix components and the minute at high speed. Dilute the extract with 125ml of distilled
trace level of target compounds. In this work, a fast HPLC-FLD water. Mix well by swirling followed by filtering approximately
method with immunoaffinity clean up and post-column elec- 40-50 ml of sample extract through Whatman No. 4 filter
trochemical bromo-derivatization was developed considering paper immediately. Transfer 10ml of the filter (equivalent to 1g
combined influences of post-column band broadening, analy- of sample) into the glass syring barrel for passage through the
sis speed, resolution and sensitivity and applied to determine prepared immunoaffinity column (ref. to AFLAPREP® column
contaminated corn and peanut butter successfully. preparation manual)[3] at a flow rate of 2-3 ml/min. Then add
10ml of distilled water to the glass syring for washing the col-
Experimental umn. Expel the residual water from the column and transfer
accurate 1ml of HPLC-grade methanol to elute aflatoxins from
Instrument and Equipment the column. It could be back-flushed with the methanol solu-
Agilent 1260 Infinity system, consisting of solvent rack, qua- tion for completely release of aflatoxins from the monoclonal
ternary pump (with built-in degasser), standard autosampler, antibody if necessary. Collect all of the methanol elution and
column compartment and fluorescence detector, was used for dilute with 1 ml of distilled water before injection into HPLC
separation and quantification. R-Biopharm electrochemical system.
derivatization kit, including KOBRA® cell, variable control cur-
Method Linear Range, Limit of Detection and Precision
rent source, 0.5 mm i.d. peek tubing (at least 34 cm long) and
Prepare a series of mixture solutions with the concentration
so on, was employed for derivatization.
of each aflatoxin at 0.1、0.25、0.5、1.0、5.0、10.0 µg/L
HPLC Conditions with MeOH/H2O=50/50 as dilution. Each solution was
Column: Zorbax Eclipse Plus C18, injected as HPLC conditions described above and the peak
4.6 x 150 mm x 5 µm area of each aflatoxin obtained was plotted against concen-
Column Temp.: 40 °C tration (See Figure 1). The linearity was maintained over the
Mobile Phase A: 1L water containing 238 mg KBr and concentration range of 0.1 ~ 10.0 µg/L for each aflatoxin
700 µL 4M HNO3 with the square of correlation coefficient (R2) > 0.99998. The
Mobile phase B: MeOH limit of detection (LOD), defined as signal-to-noise (S/N) =
Isocratic: A：B = 50 : 50, 12min 3, was 0.007、0.004、0.006 and 0.005 µg/L for B1、B2、G1
Flow rate: 1.0 mL/min and G2, respectively. Method precision was validated with
Detection: Ex: 362 nm, Em: 455 nm, gain = 15 relative standard deviation (R.S.D.), which was <0.3% of
Injection: 20 µL peak area and <0.1% of retention time for each aflatoxin by
Electrochemical Current: 100 µA setting six sequential injections of 10 µg/L standard solutions. The
Reaction coil: 0.5 mm i.d.*34 cm long peek tubing overlay of six injections of 10 µg/L standard solutions and
(from the exit of KOBRA cell to the the chromatogram of 0.1 µg/L standard solution were
entrance of FLD) shown in Figure 2. The LODs of this work and frequently-
used Chinese criterions were compared in Table 1. It was
evident that the method proposed in the work could provide
lower LOD than that of current Chinese SN and GB (/T)s.

2
Table 1. Comparison the LODs in this work with current Chinese SN and GB(/T)s

Method in Aflatoxins Method Abstraction LOD (µg/kg)

This work B1、B2、G1、G2 Post-column electrochemical bromo-derivatization HPLC-FLD 0.008 ~ 0.014
SN 0277-93 B1、B2、G1、G2 HPLC-FLD 0.12 ~ 0.50
GB/T 5009.22-2003 B1 Thin layer chromatography (the primary method); 5.0;
ELISA (the secondary method) 0.01
GB/T 18979-2003 B1、B2、G1、G2 Post-column derivatization (I2)-HPLC-FLD 1 (total sum)
GB/T 5009.23-2006 B1、B2、G1、G2 Pre-column derivatization (TFA)-HPLC-FLD (the third method) 0.05 ~ 0.5
GB/T 23212-2008 B1、B2、G1、G2、M1、M2 Post-column derivatization (PBPB)-HPLC-FLD 0.001 ~ 0.003*
GB 5009.24-2010 B1、M1 Thin layer chromatography 0.1 ~ 0.5
*：Injection volume was 100 µL.

G2, FLD1 A G1, FLD1 A

Area = 42.8029125*Amt -0.0094133 Area = 38.9685647*Amt -0.0275005
Area Rel. Res%(1): 10.828 Area Rel. Res%(1): 11.809
400
400 6 6
350
G2 300
G1
300
250
5 5
200
200
150

100 100
4 50 4
3 3
12 12
0 R2 : 0.99999 0 R2 : 0.99999
0 5 Amount [ng/µl] 0 5 Amount [ng/µl]

B2, FLD1 A B1, FLD1 A

Area = 70.4480546*Amt -0.6390655 Area = 41.3441001*Amt -0.3309725
Area Rel. Res%(1): 10.879 Area Rel. Res%(1): 13.291
700 400
6 6
600 B2 350
B1
500 300

400 250
5 5
200
300
150
200
100
100 4 4
3 50 3
12 12
0 R2 : 0.99998 0 R2 : 0.99998
0 5 Amount [ng/µl] 0 5 Amount [ng/µl]

Figure 1. The linear fitted curve and the relative coefficient of each aflatoxin

3
 FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000014.D)
FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000015.D)
FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000016.D)
FLD1 A
A, Ex=362 (AFLATOXINS\AFLATOXINS 20110715 2011-07-15
Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 21-11-30\1107150000017.D)
2011 07 15 21 11 30\1107150000017 D) FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000001.D)
FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000018.D)
FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000019.D) LU
LU

B2
45 1.5 0.1 ppb STD

6.122 - B2
40 10 ppb STD 1.4

4.1522 - G2
35 1.3
G2

4.965 - G1

7.481 - B1
G1

30
1.2

B1
25
1.1
20
1
15

10 0.9

5 0.8

0 0.7
0 2 4 6 8 10 min 0 2 4 6 8 10 min

Figure 2. The overlay of six sequential injections of 10 µg/L standard solution (Left) and the chromatogram of 0.1 µg/L standard solution (Right)

Sample Analysis
Aflatoxins in Corn and peanut butter were determined by the
method after using described sample preparation procedure
for cleanup. Chromatograms were shown in Figure 3.

FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000020.D) FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107160000036.D)
LU LU
7.463 - B1
7 3.5
Corn
7.482 -B1

Peanut butter
6 3

5 2.5
6.107 - B2

4 2
4.956 - G1
6.124 - B2

3 1.5
4167 - G2
4.969 - G1
4.233 - G2

2 1

1 0.5

0 0
0 2 4 6 8 10 min 0 2 4 6 8 10 min

Figure 3. Chromatograms of corn (left) and peanut butter (right)

4
Conclusion
The limit of detections of aflatoxin G1 and B1 were improved
by post-column electrochemical bromo-derivatization and flu-
orescence detector. The analysis was shortened into 8 min-
utes without compromise of resolution of aflatoxins (in order
to completely elute all the components in sample the analysis
time was set for 12 minutes). Meanwhile, it was shown from
the chromatograms for real samples that the cleanup of sam-
ple was effective by pre-column immunoaffinity column. It
was proved that the method in the work could be applied for
determination of aflatoxins at trace level in real samples, and
method is fast and accurate.
5
References
1. Commission Regulation (EC) No 1881/2006 of 19
December 2006 Setting Maximum Levels for Certain
Contaminants in Foodstuffs. Official Journal of the
European Union 2006, 49, L364, 5-24.
2. Action Levels for Poisonous or Deleterious Substances in
Human Food and Animal Feed. Industry Activities Staff
Booklet. U.S. Food and Drug Administration, Washington,
DC, 2000. http://www.cfsan.fda.gov/~lrd/fdaact.html
3. Application of Immunoaffinity Columns for Sample
Clean-up Prior to the HPLC Analysis for Aflatoxins.
Instruction of AFLAPREP® Immunoaffinity Columns
provided by R-BIOPHARM RHÔNE LTD.
http://www.r-biopharm.com.
www.agilent.com/chem
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© Agilent Technologies, Inc., 2011

Printed in the USA
August 22, 2011
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