Natural Resources, 2011, 2, 250-257
doi:10.4236/nr.2011.24032 Published Online December 2011 (http://www.SciRP.org/journal/nr)
Effect of Matrix Clean-Up for Aflatoxin Analysis
in Corn and Dried Distillers Grains
A. McDaniel1,2, W. E. Holmes3, P. Williams4, K. L. Armbrust2, D. L. Sparks1,2*, A. E. Brown1,2*
1Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Starkville, USA;
2Office of the State Chemist, Mississippi State University, Starkville, USA; 3Swalm School of Chemical Engineering, Mississippi State
University, Starkville, USA; 4USDA ARS Corn Host Plant Resistance Research Unit, Washington DC, USA.
Received September 24th, 2011; revised October 15th, 2011; accepted October 28th, 2011.
ABSTRACT
Aflatoxins are a group of highly carcinogenic mycotoxins that contaminate a wide variety of agricultural crops and have
a detrimental econ omic impact on industries, such as corn and ethanol production. They are regulated by the FDA, and
therefore, rapid, reliable cleanup techniques with lo w detection limits are need ed for aflato xins in a wide array of matri-
ces. In this study the effect of usin g an immunoaffinity colu mn versus simp le filtering as a clea nup was tested for aflato x-
ins extracted from corn and Dried Distillers Grains (DDG). The aflatoxins were analyzed by liquid chromatography
tandem mass spectrometry (LC-MS/MS). The use of an immunoaffinity column resulted in greater signal-to-noise ratios
(S/N), S/N of 70 vs S/N of 5 for corn, as well as fewer non-target peaks in the ana lysis. Recoveries of aflatoxin usin g im-
munoaffinity ranged from 40% to 104.5% (spiked substrate) and 49% to 120% (spiked extract) while percent recoveries
of filtered samples ranged from 84% to 119 % (spiked substrate) and 88% to 119% (spiked extract). This comparison
study showed that filtering is acceptable for small sample sets or where rapid throughput is needed. However, for larger
sample sets a more stringent cleanup method is necessary to ens ure instrument performance.
Keywords: Aflatoxin, LC-MS/MS, Immunoaffinity, SPE
1. Introduction
Aflatoxins are a group of mycotoxins that are produced
by several fungal species including the genus Aspergillus,
most notably A. flavus and A. parasiticus [1-2]. The main
aflatoxins are B1, B2, G1, and G2 (AFB1, AFB2, AFG1,
and AFG2) [2]. A. flavus is a ubiquitous fungus that has
been found worldwide. It is a host pathogen known to
infect such crops as corn, peanuts, and cotton [2]. This
creates a regulatory issue with selling contaminated food
products as aflatoxin B1 has been found to be a potent
carcinogen [3]. The FDA has set action levels for afla-
toxins at 20 ppb (total aflatoxins) for foods designated
for human consumption [4].
Aflatoxins are found worldwide [5-7], and contamin-
ation has a significant economic impact on corn crops
within the United States. Southern states are especially
impacted with losses due to aflatoxin contamination of
corn each year [8] due to conditions that favor A. flavus
growth. These conditions include drought stress, high
temperatures during growing season, and insect damage
that allows an entrance for the fungi [9].
Another economic sector impacted by aflatox ins is the
ethanol industry, specifically the selling of dried dis-
tillers grains (DDG), which are a co-product of ethanol
produced by fermentation of corn [10]. Ethanol produc-
tion plants sell DDG as feed additives to increase profit
margin. However, if contaminated corn is used as the
feedstock, aflatoxins can be retained within the DDG
[11]. This causes the DDG to be unsalable if addition to
feedstuffs causes the combined product to exceed FDA
limits, leading to a profit loss for the ethanol distillery
[12].
Traditionally, aflatoxins have been detected using classi-
cal analytical methods such as thin layer chromatography
[13]. Recently, the detection of aflatoxins has been mov-
ing towards analytical methods that can provide a higher
throughput of samples such as enzyme-linked immuno-
sorbent assay (ELISA) [14] and high-performan ce liquid
chromatography (HPLC) coupled to fluorescence dete-
ction [15] or mass spectrometry (MS) [16]. With the ad-
vent of new column technology for HPLC systems, ultra
high-pressure liquid chromatography (UHPLC) results
can be achieved on a regular HPLC system, resulting in

Effect of Matrix Clean-Up for Aflatoxin Analysis in Corn and Dried Distillers Grains251
faster analysis time. The use of these analytical methods,
however, often requires sample cleanup. Immunoaffinity
solid phase extraction (SPE) columns have become po-
pular [17], as these columns are capable of greatly mini-
mizing background detector noise while also reducing
the chance of damaging a HPLC column. When coupled
to LC-MS/MS, a sensitive and reliable detection method
of aflatoxins is possible. The objective of this investi-
gation was to compare the matrix removal capabilities of
immunoaffinity SPE columns versus standard filter paper
and determine when the use of each cleanup technique is
appropriate.
2. Materials
2.1. Standards, Solvents, and Materials
Aflatoxin standards (AFB1, AFB2, AFG1, AFG2 and afla-
toxin M1 (AFM1); >98% purity) were purchased from
Sigma-Aldrich (Saint Louis, MO). Aflatoxin-free corn
was obtained from the Mississippi State Chemical Labo-
ratory. AflaCLEAN Immunoaffinity SPE columns and
PBS buffer were obtained from Pickering Laboratories
(Mountain View, CA). Optima grade methanol, acetoni-
trile, and water were purchased from Fisher Scientific
(Fair Lawn, NJ). DDG were purchased from Sigma Al-
drich (St. Louis, MO). Formic acid was purchased from
Sigma Aldrich (St. Louis, MO). Whatman filter paper no.
1 was purchased from Fisher Scientific (Fair Lawn, NJ).
BD 3 mL Luer-Lok Tip Syringes were purchased from
Fisher Scientific (Fair Lawn, NJ). PTFE filters (0.45 m)
were purchased from Fischer Scientific (Fair Lawn, NJ).
2.2. LC-MS
An Agilent 1100 Liquid Chromatograph system (Santa
Clara, CA) with a Phenomenex Kinetex Column (C18
150  4.6 mm i.d. wit h a particle size of 2.6 m and a pore
size of 100 Å) was used. Additionally, a HPLC Krud-
Katcher Ultra Column In-Line Filter (0.5 m Porosity 
0.004 in. ID) purchased from Phenomenex (Torrance,
CA) was installed for added system protection. The mass
spectrometer used was a Bruker Esquire (Billerica, MA)
with an electrospray ionization (ESI) interface and ion
trap.
3. Methods
3.1. Stock Solution
A stock solution of aflatoxins AFB1, AFB2, AFG1, and
AFG2 was prepared at a concentration of 5 ppm and
stored at 4˚C. A spiking solution was made from this
stock solution by diluting an aliquot of the stock solution
to 1 ppm. Spiked substrates were achieved by adding 1
mL of the spiking solution onto 5 g of corn/DDG and
adjusting the extraction solvent to a final volume of 25
mL. Sp iked extract s amples were pr epared by addi ng 0.2
mL spiking solution to 4.8 mL extract. A stock internal
standard solution of AFM1 was made by diluting an ali-
quot of the purchased standard (10 ppm) to 1 ppm and
stored at 4˚C.
3.2. Cleanup Techniques
Spiked substrate samples (corn or DDG) were extracted
using a modified method provided by Pickering Labora-
tories, Inc. Ground corn provided by the Mississippi
State Chemical Laboratory or DDG were weighed out (5
g) into a 50-mL Falcon centrifuge tube and mixed with
25 mL of the extraction solvent, 80:20 methanol:water
(v:v). This mixture was shaken for 15 min before being
centrifuged at 3000 RPMs for 10 minutes. Samples were
cleaned by either AflaCLEAN SPE or Whatman filter
paper. For the AflaCLEAN samples, the extracted super-
natant (1.4 mL) was mixed with 8.6 mL of PBS Buffer
and passed through the SPE column on a vacuum mani-
fold at a flow rate of 1 - 2 drops per second. After col-
umn loading, the immunoaffini ty SPE column was washed
with 10 mL of water before being eluted with 2  1 mL
of methanol.
Spiked extract samples were obtained by spiking
AFB1, AFB2, AFG1, and AFG2 into the extract of afla-
toxin-free corn or DDG. The extracts were obtained ac-
cording to the method described above, and collected
after centrifugation. The spiked extract (1.4 mL) was
mixed with 8.6 mL of PBS Buffer and passed through
the AflaCLEAN column as described above.
Whatman spiked extract and spiked substrate samples,
were gravity filtered using Whatman filter paper # 1. All
samples, whether cleaned via SPE or Whatman, were
filtered with a 0.45 m PTFE filter prior to LC-MS/MS
analysis. PTFE filtering showed no effective aflatoxin
loss (data not shown). AFM1 was used as an internal
standard at a concentration of 50 ppb. AFM1 was chos en
as the internal standard as it is the metabolite of AFB1
found in milk thus, there should be no AFM1 found in
these matrices [18].
3.3. LC-MS/MS Protocol
A solvent gradient program was used to maximize the
signal-to-noise ratio (sensitivity). Th e solvents used were
as follows: Solvent A-water, Solvent B-acetonitrile. Both
solvents contained 0.1% formic acid by volume. The gra-
dient used was: 0 min - 0.5 min—90% A, 0.51 min
—50% A, 2.0 min - 9.0 min—20% A, 9.01 min -10 min
—90% A. Mass Spectrometer settings were adjusted so
that sensitivity was maximized. The conditions were as
follows: Capillary: –4000 V, End Plate Offset: –500 V,
Nebulizer: 30 p si, Dry G as: 12 L/min, Dry Temp: 300 ˚C,
scan range: 200 - 360 m/z, averages: 3. MS/MS was used

Effect of Matrix Clean-Up for Aflatoxin Analysis in Corn and Dried Distillers Grains 252
for absolute identification of aflatoxins and to further in-
crease sensitivity. This can be seen in Table 1.
3.4. Standard Curves
In-matrix standard curves (corn Whatman, DDG What-
man, corn SPE, and DDG SPE) were produced with con-
centrations at 5, 25, 75, 250, and 500 ppb (3 replicates at
each level). Each standard curve had AFM1 added to
each point for a final concentration of 50 ppb. Before
AFM1 was added, each point in each standard curve was
filtered through a 0.45-m PTFE filter.
3.5. Data Analysis
Limit of Detection (LOD) was determined by following
the guidelines outlined in Code of Federal Regulations,
Part 136, Appendix B [19]. LOD calculations were de-
termined from a replicate set of n = 7 at a concentration
of 5 ppb for each standard curve. Percent recoveries were
calculated by dividing the amount of aflatoxin in each
sample by aflatoxin amount calculated from spiked ma-
trix. Signal-to-noise ratios were calculated by dividing
the analyte signal by the background noise signal. Statis-
tics were calculated for ANOVA Table (  = 0.05) and
statistical difference was determined using least squares
means analysis in Statistical Analysis Software (SAS)
9.2.
4. Results
Figure 1 shows the chromatographic separation and ana-
lysis of AFB1, AFB2, AFG1, and AFG2, and AFM1 (in-
ternal standard) at a concentration of 75 ppb (50 ppb
AFM1) by LC-MS/MS. While baseline separation was
not achieved for the aflatoxins, this was not a concern
since this method was designed to be a rapid detection
method and each had unique precursor and daughter ions.
The elution order for the aflatoxins is (Table 1): AFB1
(3.5 min), AFB2 (3.4 min), AFG1 (3.4 min), and AFG2
(3.2 min), and AFM1 (3.2 min). Limit of Detection (LOD)
studies were performed for each of the four aflatoxins.
These are instrument LODs and not method LODs. This
can be seen in Table 2. LODs for Afla-CLEAN SPE
columns (0.53 - 6.47 ppb for corn and 4.37 - 14.36 ppb
for DDG) were generally lower than Whatman LODs
(5.00 - 21.84 ppb for corn and 4.81 - 20.90 ppb for
DDG). The pH was checked for each matrix extract
(corn and DDG) and both were found to be in a range
from 6.5 to 7.5. This is important because pH can play a
role in ion enhancement or suppression in mass spec-
trometry. Since the pH was essentially neutral, no ion
enhancement or suppression was thought to have oc-
curred.
Method efficiencies (spiked substrate) were calculated
Table 1. MS/MS results for aflatoxins B1, B2, G1, G2, and
M1 (internal standard).
Aflatoxin Time
(min) Precursor m/z Product m/z
B1 3.5 313 285.0, 298.0
B2 3.4 315 259.0, 287.0, 29 7.0
G1 3.4 329 243.0, 283.0, 30 1.0, 311.0
G2 3.2 331 285.1, 303.1, 313.1
M1 3.2 329 259.1, 273.1
Table 2. Percent Recoveries for Aflatoxins B1, B2, G1, and
G2.
B1 B2 G1 G2
Corn
Whatman
spiked
extracta 102 ± 15.4994 ± 0.71 91 ± 7.07 101 ± 2.83
spiked
substrateb 110 ± 0.0793 ± 18.38 99 ± 1 8.68 101 ± 2.4
LODc 8.08 21.84 10.56 5.00
DDG
Whatman
spiked
extracta 119 ± 8.49105 ± 18.38 88 ± 43.72 108 ± 23.33
spiked
substrateb 90 ± 1.41118 ± 14.14 84 ± 1.4 1 119 ± 10.61
LODc 10.62 20.90 4.81 16.19
Corn
Afla-CLEAN
spiked
extracta 120 ± 21.7874 ± 9.07 108 ± 24.58 54 ± 6.11
spiked
substrateb 104.5 ± 7.7867 ± 9.9 88.5 ± 2.12 63 ± 5.66
LODc 4.81 3.11 0.53 6.47
DDG
Afla-CLEAN
spiked
extracta 75 ± 23.9749 ± 11.06 72 ± 27.07 54 ± 31.66
spiked
substrateb 74 ± 10.0740 ± 5.86 57 ± 3.00 59 ± 20.22
LODc 7.98 14.36 9.93 4.37
aColumn efficiency, bMethod efficiency, cInstrument Limit of Detection in
ppb [19].
from an n = 3 for each aflatoxin (AFB1, AFB2, AFG1,
and AFG2) and can be seen in Table 2. These are a
measure of how effective the method is from beginning
to end of extracting aflatoxins from a particular matrix.
They were from 84% to 119% (Whatman) and 40% to
104.5% (AflaCLEAN). Column efficiencies (spiked ex-
tract) were calculated from an n = 3 for each aflatoxin
and can be seen in Table 2. These are a measure of how
effective the column is releasing the aflatoxin s during the
elution step. They were from 88% to 119% (Whatman)

Effect of Matrix Clean-Up for Aflatoxin Analysis in Corn and Dried Distillers Grains 253
Figure 1. LC-MS/MS analysis of aflatoxin standard. Each aflatoxin (top to bottom: M1, G2, G1, B2, and B1) is at a concentra-
tion of 75 ppb (M1 is at 50 ppb). The elution is as follows: M1-3.2 min, G2-3.2 min, G1-3.4 min, B2-3.4 min, B1-3.5 min Scales
are different due to each aflatoxin has a different response factor.
and 49% to 120% (AflaCLEAN). From the results, it can
be seen that corn SPE (spiked extract and spiked sub-
strate) have higher percent recoveries (54% to 120%)
when compared to DDG SPE spiked extract and spiked
substrate (40% to 75%). From the statistical analysis, it
was shown that there was a significant difference (
 =
0.05) in the percent recoveries (corn and DDG SPE) for
AFB1 and AFB2 but not AFG1 and AFG2 for spiked ex-
tract and spiked substrate. Statistical analysis for What-
man samples (spiked extract and substrate) showed no
significant difference for AFG1 and AFG2 and no clear
significant difference for AFB1 and AFB2. AFB2 and
AFG2 Whatman samples were all significantly different
(  = 0.05) from the SPE samples.
This comparative study was performed between the
capabilities of Whatman filter paper and the AflaCLEAN
immunoaffinity SPE column for cleanup of aflatoxins
extracted from corn and DDG. Data for this can be seen
in Figures 2 and 3. The corn matrix showed higher
background noise (approximately 14-fold increase for
AFB2) for the Whatman cleanup when compared to the
immunoaffinity SPE cleanup. Additionally, for the DDG
matrix, higher background noise (approximately 35-fold
increase for AFG2) was seen for DDG cleaned with
Whatman filter paper versus the immunoaffinity SPE
column. Overall, samples cleaned with the immunoaffi-
nity SPE column had a higher signal-to-noise ratio (S/N)
over Whatman filter paper (S/N of 70 versus S/N of 5 for
corn and S/N of 70 versus S/N of 2 for DDG, respec-
tively).
5. Discussion
While baseline sep aration of the aflatoxins was not ach i-
eved, coelution of compounds is acceptable when using
tandem mass spectrometry as this technique allows for
definitive identification [20,21] and has been used in
such fields as proteomics [22], pesticide analysis [23],
and forensics [24]. Compounds are detected after being
ionized and forming a specific mass-to-charge ratio (m/z).
The mass spectrometer is capable of detecting multiple
ions simultaneously, which are unique to each analyte.
LOD were calculated for how low the instrument can

Effect of Matrix Clean-Up for Aflatoxin Analysis in Corn and Dried Distillers Grains 254
Figure 2. Differential analy sis for immunoaffinity column cleanup for aflatoxins B1-peak 5, B2-peak 4, G1-peak 3, G2-peak 2,
and M1-peak 1 in corn (top) and Dried Distillers Grains (bottom). Both corn and DDG had a Signal-to-Noise ratio of 70.
Figure 3. Contrast for whatman paper cleanup for aflatoxins B1-peak 5, B2-peak 4, G1-peak 3, G2-peak 2, and M1-peak 1 in
corn (top) and Dried Distillers Grains (bottom). Corn had a Signal-to-Noise ration of 5 versus a Signal-to-Noise ratio of 2 for
DDG.

Effect of Matrix Clean-Up for Aflatoxin Analysis in Corn and Dried Distillers Grains255
detect, but not the method limits of detection, which
quantifies how low the method can detect. LOD for the
aflatoxins were comparable to other detection methods
designed for aflatoxins (LOD averaged among all four
aflatoxins): LC- MS: 0.467 ppb [25] and ELISA: 2 .5 ppb
[26].
Spiked corn extract efficiencies (column efficiency)
for AflaCLEAN SPE were calculated for all aflatoxins
and shown to be greater than 74% (except AFG2) indi-
cating that the immunoaffinty column is not retaining the
aflatoxins beyond the final elution step. It should be
noted that column efficiencies have to be calculated from
spiked matrix and not pure standards (made in methano l,
the eluting organic solvent) as p ure standards will not be
retained on column. Column effiencies achieved in this
study are comparable to other immunoaffinity SPE col-
umns such as AFLASCAN, AFLA-RHONE, AflaTest,
and AFLAPREP. For example, AflaTest column effici-
encies for corn were calculated through the use of spiked
sample extract and were: AFB1, AFG1  90%, AFB2 
85%, and AFG2  80% [27]. However, for the DDG SPE
spiked extract efficiencies, they were lower than 75%
suggesting that the aflatoxins are not being released from
the column. AFG2 spiked extract efficiency was signifi-
cantly lower than the other aflatoxins. This is because the
binding affinity of AFG2 to the antibodies within the im-
munoaffinity column seems to be lower than the other
aflatoxins. This trend is seen in other immunoaffinity
columns, not just the one produced by Pickering Labora-
tories: 17.7% (pH dependent) and 53.7% [28,29]. Spiked
substrate efficiencies (method efficiency) for DDG were
lower than those seen in corn, 57% vs 88.5% for afla-
toxin AFG1, respectively. Again, this could be due to
aflatoxins not being released by the column due to the
DDG matrix.
Before the inclusion of AFM1 as the internal standard,
percent recoveries for Whatman filter paper samples
were lower than 60%. An internal standard was used to
correct for ion suppression effects of the matrix. After
the inclusion of AFM1 as the internal standard, percent
recoveries for corn samples (spiked extract and spiked
substrate) rose to around 100%. However, for the DDG
samples, percent recoveries ranged from 100% to 450%
(data not shown for these recoveries). The theorized rea-
son for this is that ion suppression is occurring for the
aflatoxins in the DDG matrix and the intern al standard is
correcting for this. When this is coupled to using a stan-
dard curve without ion suppression (standards made up
in pure methanol), very large percent recoveries were
seen. To correct for this, the standard curves were swit-
ched from being made in pure methanol to matrix-match
standards (standards made up with the matrix extract
being used as the diluent). This solved the high percent
recoveries seen for the Whatman filter paper samples.
Due to this, AflaCLEAN SPE match standard curves
were made up as well.
The use of AflaCLEAN SPE for matrix removal has
been reported over a broad range of matrices: peanut
butter (HPLC/Fluorescence detection) [30], wheat bran
(HPLC/Fluorescence detection) [31], and sake and wheat
beer (LC-MS/MS) [32]. However, this is the first study
showing the effectiveness for DDG cleanup. Addition-
ally, this comparison study shows that the immunoaffin-
ity SPE column cleanup is superior in eliminating back-
ground noise versus Whatman filter paper. While the
difference between the two techniques was not as pro-
found for corn samples, there was a large amount of
background noise for the DDG not removed by the
Whatman filter paper. The immunoaffinity SPE column
was superior for DDG cleanup versus the Whatman filter
paper cleanup. This is most likely due to the removal of
matrix effects that the Whatman filter paper is unable to
capture. However, as can be seen from Table 2, percent
recoveries for the two methods are comparable. There-
fore, it was concluded that even though Whatman filter
paper had higher background versus the immunoaffinity
column, it was acceptable for cleanup of small sets of
samples. Another area that Whatman filter paper cleanup
would be useful is quick screening of samples for afla-
toxins as this is a faster cleanup method than immunoaf-
finity SPE. For larger sets of samples or for sensitive
instruments, it was concluded that using SPE was a bet-
ter choice. This is due to the continued analysis of sam-
ples using Whatman filter paper as cleanup led to the
instrument becoming extremely dirty, both on the LC
column (pressure increase was seen which returned to
normal after continued flushing of column) and the front
of the mass spectrometer. If larger sample sets using
Whatman filter paper for cleanup are to be an alyzed, it is
encouraged that frequent cleaning and flushing of the
system is to be performed. Fro m the results, bo th cleanup
methods are useful for sample cleanup for aflatoxin
analysis with the choice of which one to use depending
on several factors (sample set size, sensitivity of instru-
ment, time).
6. Safety
Aflatoxins are carcinogenic compounds that should be
handled carefully at all times. Any hand ling of aflatoxins
should be done using gloves and a lab coat. Any spills
should be neutralized with a 10% bleach solution.
7. Acknowledgements
The authors would like to thank Beth Thomas and Joey
Raines of the Mississippi State Chemical Laboratory for
their help in experimentation. They would also like to

Effect of Matrix Clean-Up for Aflatoxin Analysis in Corn and Dried Distillers Grains 256
thank the funding agencies that supported this research:
USDA, Mississippi Corn Promotion Board, and the Mis-
sissippi State Research Initiative.
REFERENCES
[1] M. A. Klich, “Aspergillus flavus: The Major Producer of
Aflatoxin,” Molecular Plant Pathology, Vol. 8, No. 6,
2007, pp. 713-722.
doi:10.1111/j.1364-3703.2007.00436.x
[2] K. K. Maggon, L. Viswanathan and T. A. Venki-tasubra-
manian, “The Chemistry of Aflatoxins,” The Journal of
Scientific and Industrial Research, Vol. 29, 1970, pp. 8-
17.
[3] K. A. Scheidegger and G. A. Payne, “Unlocking the Se-
crets behind Secondary Metabolism: A Review of Asper-
gillus flavus from Pathogenicity to Functional Genom-
ics,” Journal of Toxicology, Vol. 22, No. 2-3, 2003, pp.
423-459.
[4] F. Wu, “Mycotoxin Risk Assessment for the Purpose of
Setting International Regulatory Standards,” Environ-
mental Science and Technology, Vol. 38, No. 15, 2004,
pp. 4049-4055. doi:10.1021/es035353n
[5] L. Lewis, M. Onsongo, H. Njapau, H. Schurz-Rogers, G.
Luber, S. Kieszak, J. Nyamongo, L. Backer, A. M. Da-
hiye, A. Misore, K. DeCock and C. Rubin, “Kenya Afla-
toxicosis Investigation Group. Aflatoxin Contamination
of Commercial Maize Products during an Outbreak of
Acute Aflatoxicosis in Eastern and Central Kenya,” En-
vironmental Health Perspectives, Vol. 113, 2005, pp.
1763-1767. doi:10.1289/ehp.7998
[6] Z. Liu, Z. Gao and J. Yu, “Aflatoxins in Stored Maize
and Rice Grains in Liaoning Province, China,” Journal of
Stored Products Research, Vol. 42, No. 4, 2006, pp. 468-
479. doi:10.1016/j.jspr.2005.09.003
[7] F. Peers, X. Bosch, J. Kaidor, A. Linsell and M. Plujimen,
“Aflatoxin Exposure, Hepatitis B Virus Infection and
Liver Cancer in Swaziland,” International Journal of
Cancer, Vol. 39, No. 5, 1987, pp. 545-553.
doi:10.1002/ijc.2910390502
[8] G. Wood, “Mycotoxins in Foods and Feeds in the United
States,” Journal of Animal Science, Vol. 70, No. 12, 1992,
pp. 3941-3949.
[9] M. A. Klich, “Environmental and Developmental Factors
Influencing Aflatoxin Production for Aspergillus flavus
and Aspergillus parasiticus,” Mycoscience, Vol. 48, No.
2, 2007, pp. 71-80. doi:10.1007/s10267-006-0336-2
[10] J. L. Firkins, L. L. Berger and G. C. Fahey, “Evaluation
of Wet and Dry Distillers Grains and Wet and Dry Corn
Gluten Feeds for Ruminants,” Journal of Animal Science,
Vol. 60, No. 3, 1985, pp. 847-860.
[11] E. B. Lillehoj, A. Lagoda and W. F. Maisch, “The Fate of
Aflatoxins in Naturally Contaminated Corn during the
Ethanol Fermentation,” Canadian Journal of Microbiol-
ogy, Vol. 25, No. 8, 1979, pp. 911-914.
doi:10.1139/m79-135
[12] K. A. Nelson, P. P. Motavalli and R. L. Smoot, “Utility of
Dried Distillers Grains as a Fertilizer Source for Corn,”
Journal of Agricultural Science, Vol. 1, No. 1, 2009, pp.
3-12.
[13] P. M. Scott, J. W. Lawrence and W. Van Walbeek, “De-
tection of Mycotoxins by Thin Layer Chromatography:
Application to Screening of Fungal Extracts,” Applied
Microbiology, Vol. 20, 1970, pp. 839-842.
[14] N. A. Lee, S. W. Wang, R. D. Allan and I. R. Kennedy,
“A Rapid Aflatoxin B1 ELISA: Development and Vali-
dation with Reduced Matrix Effects for Peanuts, Corn,
Pistachio, and Soybeans,” Journal of Agriculture and
Food Chemistry, Vol. 52, No. 10, 2004, pp. 2746-2755.
doi:10.1021/jf0354038
[15] J. Jaimex, C. A. Fente, B. I. Vazquez, A. Cepeda, G.
Mahuzler and P. Prognon, “Application of the Assay of
Aflatoxins by Liquid Chromatography with Fluorescence
Detection in Food Analysis,” Journal of Chromatography
A, Vol. 882, No. 1-2, 2000, pp. 1-10.
doi:10.1016/S0021-9673(00)00212-0
[16] M. Vahl and M. Jorgenson, “Determination of Aflatoxins
in Food using LC/MS/MS,” European Food Research
and Technology, Vol. 206, No. 4, 1998, pp. 243-245.
[17] J. Stroka, E. Anklam, U. Jörissen and J. Gilbert, “Immu-
noaffinity Column Cleanup with Liquid Chromatography
Using Post-Column Bromination for Determination of
Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste,
and Paprika Powder: Collaborative Study,” Journal of
AOAC International, Vol. 83, No. 2, 2000, pp. 320-340.
[18] F. Galvano, V. Galofaro and G. Galvano, “Occurrence
and Stability of Aflatoxin M1 in Milk and Milk Products:
A Worldwide Review,” Journal of Food Protection, Vol.
59, No. 10, 1996, pp. 1079-1090.
[19] Code of Federal Regulations Title 40, Part 136, Appendix
B, Guidelines Establishing Test Procedures for the Ana-
lysis of Pollutants, U.S. Government Printing Office,
Washington, DC, 2009.
www.epa.gov/earth1r6/6pd/qa/qadevtools/mod4reference
s/analytical_references/40cfr136_03.pdf
[20] T. M. Annesley, “Ion Suppression in Mass Spectrome-
try,” Clinical Chemistry, Vol. 49, No. 7, 2003, pp. 1041-
1044. doi:10.1373/49.7.1041
[21] K. Biemann, “Contributions of Mass Spectrometry to
Peptide and Protein Structure,” Biological Mass Spec-
trometry, Vol. 16, No. 1-12, 1988, pp. 99-111.
doi:10.1002/bms.1200160119
[22] R. Aebersold and M. Mann, “Mass Spectrometry Based
Proteomics,” Nature, Vol. 422, 2003, pp. 198-207.
doi:10.1038/nature01511
[23] F. Hernandez, J. V. Sancho and O. J. Pozo, “Critical Re-
view of the Application of Liquid Chromatography to the
Determination of Pesticide Residues in Biological Sam-
ples,” Analytical Bioanalytical Chemistry, Vol. 382, No.
4, 2005, pp. 934-946. doi:10.1007/s00216-005-3185-5
[24] H. H. Maurer, “Liquid Chromatography-Mass Spectro-
metry in Forensic and Clinical Toxicology,” Journal of
Chromatography B: Biomedical Sciences and Applica-
tions, Vol. 713, No. 1, 1998, pp. 3-25.

Effect of Matrix Clean-Up for Aflatoxin Analysis in Corn and Dried Distillers Grains 257
doi:10.1016/S0378-4347(97)00514-8
[25] H. S. Chun, H. J. Kim, H. E. Ok, J. Hwang and D. Chung,
“Determination of Aflatoxin Levels in Nuts and Their
Products Consumed in South Korea,” Food Chemistry,
Vol. 102, No. 1, 2007, pp. 385-391.
doi:10.1016/j.foodchem.2006.05.031
[26] Z. Zheng, C. W. Humphrey, R. S. King and J. L. Richard,
“Validation of an ELISA Test Kit for the Detection of
Total Aflatoxins in Grain and Grain Products by Com-
parison with HPLC,” Mycopathologia, Vol. 159, No. 2,
2005, pp. 255-263. doi:10.1007/s11046-004-8666-0
[27] H. Z. Senyuva and J. Gilbert, “Immunoaffinity Column
Clean-Up Techniques in Food Analysis: A Review,”
Journal of Chromatography B, Vol. 878, No. 2, 2010, pp.
115- 132. doi:10.1016/j.jchromb.2009.05.042
[28] S. Ip and C. Che, “Determination of Aflatoxins in Chi-
nese Medicinal Herbs by High-Performance Liquid Chro-
matography Using Immunoaffinity Column Cleanup: Im-
provement of Recovery,” Journal of Chromatography A,
Vol. 1135, No. 2, 2006, pp. 241-244.
doi:10.1016/j.chroma.2006.10.025
[29] V. A. Vega, “Rapid Extraction of Aflatoxins from Crea-
my and Crunchy Peanut Butter,” Journal of AOAC Inter-
national, Vol. 88, 2005, pp. 1383-1386.
[30] M. Ofitserova, “Analysis of Aflatoxins in Ground Pea-
nuts and Peanut Butter by HPLC with Photochemical De-
rivatization,” LC/GC North America Supplement, Vol. 46,
2010.
[31] M. Muscarella, M. Iammarino, D. Nardiello, S.L. Magro
and C. Palermo, “Validation of a Confirmatory Analytical
Method for the Determination of Aflatoxins B1, B2, G1,
and G2 in Foods and Feed Materials by HPLC with
On-Line Photochemical Derivatization and Fluorescence
Detection,” Food Addicitves & Contaminants: Part A,
Vol. 26, No. 10, 2009, pp. 1402-1410.
doi:10.1080/02652030903107906
[32] M. Rudranhatia, “A Rapid LC/MS/MS Method for the
Analysis of Aflatoxins in Complex Matrixes with Immu-
noaffinity Clean-Up,” Application Note 00927, Varian,
Inc. Palo Alto.