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
by methanol/water (60/40, v/v), the sample was cleaned up and concentrated on
an immunoaffinity column and then the sample solution was separated and quantified 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
limit of detection (LOD, S/N = 3) was in the range of 0.004 ~ 0.007 µg/L (equivalent
to 0.008 ~ 0.014 µg/kg content in samples). The precision was validated by six
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 utilized to determine aflatoxins in corn and peanut butter effectively and accurately.
Application Engineer of Liquid
Chromatography
Life Science and Chemical Analysis
Group
Agilent Technologies
shanghai, China
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 carcinogenicity 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 animal feed by U.S. FDA, respectively [2]. Therefore, it is important to develop an accurate and effective method for aflatoxins determination.
HPLC with fluorescence detection was utilized for aflatoxins
determination in many matrices increasingly. HPLC analysis
was frequently required with immunoaffinity clean up for
sample preparation and derivatization for high sensitivity
detection due to the influence of matrix components and the
trace level of target compounds. In this work, a fast HPLC-FLD
method with immunoaffinity clean up and post-column electrochemical bromo-derivatization was developed considering
combined influences of post-column band broadening, analysis speed, resolution and sensitivity and applied to determine
contaminated corn and peanut butter successfully.
Sample Preparation
Weigh 25g of sample and 2g of sodium chloride into highspeed blender jar. Add 125ml of HPLC Grade methanol:distilled water (60:40, v:v) into the jar, cover and blend for 1
minute at high speed. Dilute the extract with 125ml of distilled
water. Mix well by swirling followed by filtering approximately
40-50 ml of sample extract through Whatman No. 4 filter
paper immediately. Transfer 10ml of the filter (equivalent to 1g
of sample) into the glass syring barrel for passage through the
prepared immunoaffinity column (ref. to AFLAPREP® column
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 column. Expel the residual water from the column and transfer
accurate 1ml of HPLC-grade methanol to elute aflatoxins from
the column. It could be back-flushed with the methanol solution for completely release of aflatoxins from the monoclonal
antibody if necessary. Collect all of the methanol elution and
dilute with 1 ml of distilled water before injection into HPLC
system.
Experimental
Instrument and Equipment
Agilent 1260 Infinity system, consisting of solvent rack, quaternary pump (with built-in degasser), standard autosampler,
column compartment and fluorescence detector, was used for
separation and quantification. R-Biopharm electrochemical
derivatization kit, including KOBRA® cell, variable control current source, 0.5 mm i.d. peek tubing (at least 34 cm long) and
so on, was employed for derivatization.
Method Linear Range, Limit of Detection and Precision
Prepare a series of mixture solutions with the concentration
of each aflatoxin at 0.1、0.25、0.5、1.0、5.0、10.0 µg/L
with MeOH/H2O=50/50 as dilution. Each solution was
injected as HPLC conditions described above and the peak
area of each aflatoxin obtained was plotted against concentration (See Figure 1). The linearity was maintained over the
concentration range of 0.1 ~ 10.0 µg/L for each aflatoxin
with the square of correlation coefficient (R2) > 0.99998. The
limit of detection (LOD), defined as signal-to-noise (S/N) =
3, was 0.007、0.004、0.006 and 0.005 µg/L for B1、B2、G1
and G2, respectively. Method precision was validated with
relative standard deviation (R.S.D.), which was <0.3% of
peak area and <0.1% of retention time for each aflatoxin by
six sequential injections of 10 µg/L standard solutions. The
overlay of six injections of 10 µg/L standard solutions and
the chromatogram of 0.1 µg/L standard solution were
shown in Figure 2. The LODs of this work and frequentlyused 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.
HPLC Conditions
Column:
Zorbax Eclipse Plus C18,
4.6 x 150 mm x 5 µm
Column Temp.:
40 °C
Mobile Phase A:
1L water containing 238 mg KBr and
700 µL 4M HNO3
Mobile phase B:
MeOH
Isocratic:
A：B = 50 : 50, 12min
Flow rate:
1.0 mL/min
Detection:
Ex: 362 nm, Em: 455 nm, gain = 15
Injection:
20 µL
Electrochemical Current: 100 µA setting
Reaction coil:
0.5 mm i.d.*34 cm long peek tubing
(from the exit of KOBRA cell to the
entrance of FLD)
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);
ELISA (the secondary method)
5.0;
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.
G1, FLD1 A
Area = 38.9685647*Amt -0.0275005
G2, FLD1 A
Area = 42.8029125*Amt -0.0094133
Area
Area
400
Rel. Res%(1): 10.828
6
400
G2
Rel. Res%(1): 11.809
350
300
300
6
G1
250
5
5
200
200
150
100
100
3
12
4
50
R2 : 0.99999
0
0
5
3
12
0
Area
Rel. Res%(1): 10.879
6
B2
Amount [ng/µl]
Rel. Res%(1): 13.291
400
6
B1
350
300
500
400
250
5
5
200
300
150
200
100
5
B1, FLD1 A
Area = 41.3441001*Amt -0.3309725
700
600
R2 : 0.99999
0
Amount [ng/µl]
B2, FLD1 A
Area = 70.4480546*Amt -0.6390655
Area
4
100
3
12
4
50
R2 : 0.99998
0
0
5
Amount [ng/µl]
3
12
4
R2 : 0.99998
0
0
5
Figure 1. The linear fitted curve and the relative coefficient of each aflatoxin
3
Amount [ng/µl]
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)
(AFLATOXINS\AFLATOXINS 20110715 2011-07-15
FLD1 A
Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715
21-11-30\1107150000017.D)
11 30\1107150000017 D)
A, Ex=362
2011 07 15 21
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)
B1
1.2
25
4.965 - G1
1.3
G1
30
4.1522 - G2
1.4
G2
35
0.1 ppb STD
6.122 - B2
1.5
10 ppb STD
40
7.481 - B1
B2
LU
45
FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000001.D)
LU
1.1
20
1
15
0.9
10
5
0.8
0
0.7
0
2
4
6
8
10
min
0
2
6
4
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\1107160000036.D)
FLD1 A, Ex=362, Em=455 (AFLATOXINS\AFLATOXINS-20110715 2011-07-15 21-11-30\1107150000020.D)
LU
7
3.5
7.482 -B1
Corn
6
Peanut butter
3
1
0
2
4
6.124 - B2
4167 - G2
4.969 - G1
4.233 - G2
2
1.5
1
6.107 - B2
2
4
3
4.956 - G1
2.5
5
0
7.463 - B1
LU
0.5
6
8
10
0
min
Figure 3. Chromatograms of corn (left) and peanut butter (right)
4
0
2
4
6
8
10
min
Conclusion
References
The limit of detections of aflatoxin G1 and B1 were improved
by post-column electrochemical bromo-derivatization and fluorescence detector. The analysis was shortened into 8 minutes 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 sample 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.
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.
5
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August 22, 2011
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