GazioĞlu & Kolak: Journal of AOAC International Vol. 98, No.

4, 2015  939

FOOD CHEMICAL CONTAMINANTS

Method Validation for the Quantitative Analysis of Aflatoxins

(B1, B2, G1, and G2) and Ochratoxin A in Processed Cereal-
Based Foods by HPLC with Fluorescence Detection
Işil GazioǦlu
Bezmialem Vakif University, Faculty of Pharmacy, Department of Analytical Chemistry, Istanbul 34093, Turkey
Ufuk Kolak1
Istanbul University, Faculty of Pharmacy, Department of General and Analytical Chemistry, Istanbul 34116, Turkey

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Modified AOAC 991.31 and AOAC 2000.03 methods carcinogen (Group 1), and AFB2, AFG1, and AFG2 as possibly
for the simultaneous determination of total carcinogenic to humans (Group 2B). AFs also have toxigenic,
aflatoxins (AFs), aflatoxin B1, and ochratoxin mutagenic, and teratogenic effects (2).
A (OTA) in processed cereal-based foods by Ochratoxin A (OTA), which is produced mainly by
RP-HPLC coupled with fluorescence detection A.  ochraceus and Penicillium verrucosum, has the most toxic
were validated. A KOBRA® Cell derivatization effects among all ochratoxins. Because OTA biosynthesis
system was used to analyze total AFs. One of the requires complex fungi-substrate interactions, its production
modifications was the extraction procedure of and accumulation are difficult under normal conditions (7).
mycotoxins. Both AFs and OTA were extracted with Scientific investigations indicate that food may be mainly
methanol–water (75 + 25, v/v) and purified with an contaminated with OTA during storage, and it is stable
immunoaffinity column before HPLC analysis. The during most food processing stages  (8–10). OTA has been
modified methods were validated by measuring commonly found in cereals and starch rich foods with spices,
the specificity, selectivity, linearity, sensitivity, coffee, dried fruits, grapes, wines, beer, and meat (11). OTA
accuracy, repeatability, reproducibility, recovery, can be nephrotoxic, hepatotoxic, teratogenic, mutagenic, and
LOD, and LOQ parameters. The validated methods carcinogenic and can show fertility inhibition effects of an
were successfully applied for the simultaneous immunosuppressive nature in a variety of laboratory animals.
determination of mycotoxins in 81 processed cereal- It was considered to be responsible for a chronic kidney
based foods purchased in Turkey. These rapid, disease that had been observed in Balkans’ people (Balkan
sensitive, simple, and validated methods are suitable Endemic Nephropathy). OTA was classified by the IARC as a
for the simultaneous determination of AFs and OTA possible carcinogen for humans (Group 2B; 12). Recent studies
in the processed cereal-based foods. indicated that neurodegenerative diseases such as Parkinson’s
and Alzheimer’s could be related to OTA (13, 14).
Foods can be contaminated with mycotoxins preharvest

M
or postharvest, during processing or preparation, or in
ycotoxins of different chemical structures and modes storage  (12,  15). Cereals and processed cereal-based products
of action are produced as secondary metabolites represent a serious health risk for consumers because of their
by various fungal species (1). There are more than sensitivity against mycotoxin contamination (16). The European
300 known mycotoxins classified as hepatotoxins, nephrotoxins, Union has established maximum legal limits as 4 µg/kg for total
neurotoxins, and immunotoxins by clinicians, and as teratogens, AFs, 2 µg/kg for AFB1, and 5 µg/kg for OTA in cereals (17).
mutagens, carcinogens, and allergens by cell biologists (1, 2). Numerous methods based on HPLC analysis with either
Common mycotoxins include aflatoxins, ochratoxin A, precolumn or postcolumn derivatization have been developed
ergot alkaloids, fumonisins, patulin, trichothecenes, and for determination of AFs and OTA in cereals (18–21).
zearalenone  (3). Aflatoxins (AFs) have the most acute toxic There are several methods for simultaneous determination
effects in humans and carcinogenic effects in susceptible of AFs and OTA. For example, EN 15851 and EN ISO 16050
animals among all mycotoxins (4). methods specify an RP-HPLC method with immunoaffinity
AFs, which are difuranocoumarin derivatives, are the column cleanup and postcolumn derivatization for the
toxic metabolites generated by the genus Aspergillus that determination of aflatoxins in cereals, nuts, and their derived
include A.  flavus, A. parasiticus, A. nomius, A. tamari, and products, and EN 15835 method specifies determination of
A. bombycis (5,  6). The major AFs, B1 (AFB1), B2 (AFB2), G1 OTA in cereal-based foods for infants and young children using
(AFG1), and G2 (AFG2), are mainly present in cereals, peanuts, HPLC with immunoaffinity column cleanup and fluorescence
corn, nuts, and cottonseeds, and the order of their toxicity is detection (FLD). Unlike these methods, the aim of the present
AFB1>AFG1>AFB2>AFG2. The International Agency for work was to validate the modified AOAC 991.31 and AOAC
Research on Cancer (IARC) classified AFB1 as a human 2000.03 methods for the simultaneous determinations of total
AFs (B1, B2, G1, and G2), AFB1, and OTA by RP-HPLC-FLD
Received October 9, 2014. Accepted by AP March 4, 2015. in 81 processed cereal-based food samples from Turkey. Total
1
Corresponding author’s e-mail: [email protected] AF analysis of the samples was carried out using a KOBRA®
DOI: 10.5740/jaoacint.14-211 Cell (KOk BRomine Apparatus Cell) derivatization system.
940  GazioĞlu & Kolak: Journal of AOAC International Vol. 98, No. 4, 2015

The validation procedure was applied according to Eurachem an RP C18 column Cronusil-S ODS2 (4.6 × 250 mm, 100 Å,
Guide  (22). In this study, the AOAC 991.31  (23) and AOAC and 5 µm particle size; Gloucester, UK).
2000.03  (24) methods that have been currently used for the To enhance the fluorescence activity of AFs, a KOBRA
simultaneous analyses of AFs and OTA in cereal samples, Cell electrochemical postcolumn derivatization system
respectively, were modified to obtain high sensitivity and reduce (R-Biopharm) was applied before the fluorescence detector.
time of analysis and consumption of solvents. In addition, the This derivatization method includes the addition of potassium
extraction procedure that was developed in this study was used bromide and nitric acid to the mobile phase. Once they reach
to determine both AFs and OTA in the samples. the KOBRA cell, electrolysis occurs and bromine is released.
Bromine reacts with AFB1 and gives derivatives that fluoresce
Experimental in the RP solvents (25).
All samples were ground using a blender (Waring 8011,
Materials and Reagents Stamford, CT).

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The analytical standards of total AFs (AFB1, AFB2, AFG1, Chromatographic Conditions
and AFG2), OTA, and Aflaprep and Ochraprep immunoaffinity
columns were purchased from R-Biopharm (Darmstadt, Acetonitrile–methanol–water for AFs, and methanol–water–
Germany). The catalog numbers of total AFs, OTA and Aflaprep acetic acid for OTA were used as mobile phases to equilibrate RP
and Ochraprep immunoaffinity columns are RBRP22, RBRP11, HPLC columns before analyses. The wavelengths of excitation
RBRP04, and RBRP14B, respectively. Total AF and OTA and emission were 365 and 435 nm for AFs, and 333 and 460 nm
standards were produced and certified in accordance with ISO for OTA, respectively. The injection volume was 100 µL, and
Guide 34:2009 and ISO/IEC 17025:2005). Potassium bromide flow rate was 1.00 mL/min. Separation was achieved at 30°C
(≥99 purity, 104907), HPLC grade acetonitrile (≥99.93% under isocratic elution with the following mobile phases:
purity, 600030.2500), HPLC grade methanol (≥99.9% purity, acetonitrile–methanol–water (8 + 38 + 54, v/v/v) with 0.2 g/L
106007.2500), nitric acid (65% purity, 100456), and glacial potassium bromide, acidified with nitric acid (300 µL/L, 65%)
acetic acid (100% purity, 100056) were purchased from Merck for AF analysis and methanol–water–acetic acid (68.5 + 29 +
(Darmstadt, Germany). All eluents were filtered through 2.5, v/v/v) for OTA analysis.
0.45 µm filters (Chromafil, Düren, Germany). Whatman No. 4
filters were purchased from GE Healthcare (Buckinghamshire,
Samples
UK). An ultrasonic cleaner bath (24 × 14 × 10 cm) was purchased
from Bandelin Sonorex (Berlin, Germany). Phosphate-buffered
Eighty-one processed cereal-based foods consisting of
saline (PBS) was prepared by dissolving PBS tablets (79382,
wheat (n = 12), bread (n = 9), starch (n = 9), semolina (n = 5),
Sigma-Aldrich, Steinheim, Germany) in distilled water.
cake flour (n = 7), pasta (n = 7), cake (n = 8), biscuit (n = 13),
Deionized distilled water was obtained from a Human water
chips (n = 5), and rusk (n = 6) were purchased from different
purification system (Seoul, South Korea).
markets in Istanbul (Turkey) in February 2013. All samples
were kept in suitable containers and stored at +4°C until initial
Safety Precautions sample preparation. In this study, one of the wheat samples not
contaminated with mycotoxins was used as a blank sample for
AFs and OTA are toxic substances; therefore, were always spiking.
manipulated in solution, avoiding the formation of dust and
aerosols. Nitrile gloves were used for all procedures. Sample Preparation for Analysis

Stock and Working Standard Preparation Analyses of replicate spiked samples provided applicability
and verification of the method. Blank wheat samples were
Stock solutions that contained 1000 ng/mL total AFs (AFB1, spiked with a suitable amount of mycotoxins to achieve 6.0 and
AFB2, AFG1, and AFG2) and 1000 ng/mL OTA were prepared 8.0 µg/kg total AFs, and 3.0 and 8.0 µg/kg OTA. After 30 min at
from the purchased AF and OTA certified standards. The room temperature, the spiked samples were extracted, cleaned
working standard solutions were prepared by diluting these up, and analyzed using the modified AOAC 991.31 and AOAC
stock solutions with methanol or methanol–water (60 + 40, v/v) 2000.03 methods. All samples were ground with a blender. Each
to achieve different concentrations of mycotoxin mixtures. The ground and spiked sample (25 g) was extracted with 125  mL
calibration curve ranged from 0.08 to 10.00 ng/mL for total AFs methanol (75%). After blending vigorously for 30 min, the
and from 0.125 to 10.00 ng/mL for OTA. All prepared solutions extract was filtered through Whatman No. 1 filter paper. For AF
were stored at -4°C and kept at room temperature in the dark analysis, 15 mL filtrate was diluted with 30 mL distilled water and
for 30 min before their use. 15 mL of the supernatant was passed through an immunoaffinity
column without preconditioning. For OTA analysis, 5 mL of the
Instrumentation filtrate was diluted with 40  mL distilled water and 45 mL of
the supernatant was passed through an immunoaffinity column
A Shimadzu LC-20AT liquid chromatographic system without preconditioning. After the sample passed, the column
(Shimadzu, Kyoto, Japan) equipped with a fluorescence was washed with 10 mL distilled water. Then, the column was
detector (RF-20A), autosampler system (SIL-20A), pump air-dried and AFs were eluted with 1 mL methanol and OTA
(LC-20AT), and column oven (CTO-10AS) and controlled by was eluted with 1.5 mL of methanol–acetic acid (98 + 2, v/v).
Lab Solutions software was used. Separation was achieved on Finally, 1 mL and 1.5 mL distilled water were added for AFs and
 GazioĞlu & Kolak: Journal of AOAC International Vol. 98, No. 4, 2015  941

Table  1.  Linearity and sensitivity data of AFs and OTA using the optimal HPLC conditions
In spiked wheat, µg/kg
2
Analytes Range, µg/kg Slope ± SD Intercept ± SD R LOD LOQ U, %a

AFB1 0.02–2.50 1.9747e-006 0.00664189 ± 0.004 0.9995828 0.019 0.078 6.9

AFB2 0.02–2.50 1.33944e-006 0.00393259 ± 0.005 0.9995975 0.016 0.064 6.2
AFG1 0.02–2.50 3.30819e-006 0.0117839 ± 0.005 0.9997416 0.021 0.085 5.2
AFG2 0.02–2.50 3.0326e-006 0.0107589 ± 0.006 0.9997093 0.021 0.086 8.0
OTA 0.25–10.00 1.23559e-005 0.0763268 ± 0.003 0.9997438 0.230 0.922 8.2
a
  U = Percentage relative uncertainty at the 95% confidence level (k = 2; k= coverage factor).

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OTA, respectively, into a glass vial, and 100 µL of these eluates 5.0 µg/kg for total AFs, 8.0 and 3.0 µg/kg for OTA) to indicate
was directly injected for HPLC. the selectivity of the modified methods. After the spiked samples
were analyzed by the modified methods, their recoveries were
Modifications of AOAC 991.31 and AOAC 2000.03 calculated.
Methods (c)  Linearity.—Calibration curves were used to determine
linearity. For this purpose, working standard solutions of
In this study, the AOAC 991.31 and AOAC 2000.03 total AFs and OTA were prepared in three replicates at
methods were modified by changing sample preparation steps different concentrations of 0.08–10.0 ng/mL for total AFs, and
in the simultaneous determination of AFs and OTA. The same 0.125–10.0 ng/mL for OTA. As a result of HPLC measurements,
extraction process was used for both AF and OTA analysis. the peak area ratio of the mycotoxin versus the nominal
The modified AOAC 991.31 and AOAC 2000.03 methods concentration of the analyte was used to obtain the calibration
consisted of a solvent mixture (methanol–water) for extraction, standard of each concentration (Table 1). The linearity was
and an extract cleanup with an immunoaffinity column. This evaluated by the correlation coefficient, y-intercept, and slope of
modification provided some advantages in the analysis, i.e., the calibration curve. Additionally, the use of external standards
reduction of the extraction solvent volume, analysis time, allowed us to evaluate the originality of the method. Also, it
and sample amount. In addition, the compositions of mobile provided removal of the interferences that come from matrix.
phases, flow rates, and column temperatures were changed An attempted validation of the method in wheat samples, in
in these methods. All of the changes reduced the analysis the concentration ranges 0.08–10.0 µg/kg for total AFs and
time. The new mobile phases [acetonitrile–water–methanol 0.25–10.0 µg/kg for OTA, gave the data presented in Table 2,
(8 + 54 + 38, v/v/v) and methanol–water–acetic acid (68.5 + 29 and the chromatographic parameters are given in Table 3.
+ 2.5, v/v/v)] provided the best peak resolution in AF and OTA (d)  Sensitivity.—LOD and LOQ values were used to
analysis, respectively. Potassium bromide was added into the determine sensitivity of the methods. LOD was calculated by the
mobile phase for derivatization of AFs with the KOBRA Cell. concentration of the analyte that produced a peak whose height
When the amount of potassium bromide (0.2 g/L) increased, was 3x the height of the noise from a blank sample (S/N = 3).
AF peaks got sharper. Since the recovery values were found LOQ was the lowest concentration at which the analyte could
to be <70% in the spiked samples below 30 min, the optimum not only be reliably detected but at which some predefined
incubation time was determined as 30 min in this study. goals for bias and imprecision were met. LOQ was calculated
by S/N = 10. Spiked sample that was used to calculate LOD and
Method Validation LOQ values contained a small amount of mycotoxin that could
be detected but not to be quantified.
The modified AOAC 991.31 and AOAC 2000.03 methods (e)  Precision.—The precision of the modified methods
were validated according to Eurachem guidelines. Validation was demonstrated as repeatability (RSDr) and reproducibility
of HPLC-FLD methods was based on the following criteria: (RSDR). RSDr and RSDR (Horwitz values) were determined
specificity, selectivity, linearity, sensitivity, precision (intraday by analyzing duplicates of each spiked sample at two
and interday, and analyst variability), accuracy, LOD, LOQ, and different levels (6.0 and 8.0 µg/kg for total AFs, and 3.0 and
recovery. 8.0  µg/kg for OTA). Within-day repeatability was determined
(a)  Specificity.—The retention times of standards were by duplicate determination on the same day by the same analyst.
compared with those of the samples to indicate the specificity Between-day repeatability was evaluated by using the same
of the modified methods. method on 5 different days (Table 4). The spiked samples at the
(b)  Selectivity.—One of the wheat samples was spiked respective concentration levels were used in the methods.
with total AFs, and OTA at different concentrations (8.0 and (f)  Accuracy and repeatability.—Accuracy and repeatability

Table  2.  Precision and accuracy for AF and OTA determinations with matrix-matched method for the calibration curves
Analytes Nominal concn, μg/kg Mean calculated concn, μg/kg Accuracy, % Precision, %

Total AFs 0.08–10 0.082–10.87 102.5–108.7 101.98–110.54

OTA 0.25–10 0.26–9.98 104–99.8 97.6–100.02
942  GazioĞlu & Kolak: Journal of AOAC International Vol. 98, No. 4, 2015

Table  3.  Chromatography parameters of AFB1, AFB2, AFG1, AFG2, and OTA in spiked samples at 3 µg/kg
AFB1 AFB2 AFG1 AFG2 OTA

Retention time (tR), min 11.84 10.26 8.31 7.30 6.77

Tailing factor 1.383 1.380 1.384 1.402 1.244
Resolution (Rs) 2.077 2.854 1.624 3.024 6.916
Retention factor (k’) 3.095 2.549 1.875 1.527 0.794
Number of theoretical plates (N) 3578 3161 2750 2333 4558
Peak width at half height (Wh) 0.274 0.257 0.239 0.227 0.230
HETPa 25.256 30.543 35.444 42.658 32.909
a
  HETP = Height equivalent to a theoretical plate.

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were determined by analyzing spiked samples at low and high Grubbs’ critical value table. The statistical significance was set
levels of each calibration curve (8.0 and 5.0 µg/kg for total AFs, at the level of 95% (P = 0.05).
8.0 and 3.0 µg/kg for OTA) in duplicate on 5 different days and
by two different analysts. A 25 g portion of each sample was Results and Discussion
spiked with adequate volumes of stock and working standard
solutions. They were extracted for 30 min with methanol–water Selectivity and Specificity
(75 + 25, v/v). Recovery was determined from the peak areas of
mycotoxins. The accuracy has been calculated as the SE of the AFB1, AFB2, AFG1, AFG2, and OTA showed good
mean of the data obtained during the precision study, and the chromatography with an acceptable baseline and resolution of
repeatability was RSD (Table 5). each mycotoxin (Figure 1). In the chromatograms, the AF and
The calculated and expected concentrations (C) of the spiked OTA peaks of blank and spiked samples were well separated
sample were compared to determine the recovery values using from each other. There were no foreign peaks that interfered
following equation: with analytes at the retention times of the AFs and OTA, which
were 7.0, 7.9, 9.8, and 11.4 min for AFG1, AFG2, AFB1,
Recovery, % = [Cspiked sample/Cexpected] × 100 and AFB2, respectively, and 6.7 min for OTA. The modified
methods exhibited good selectivity and specificity.

Statistical Analysis Linearity and Sensitivity

Concentration levels and analysis results were expressed Three replicates of eight calibration samples were analyzed
as the average of AFs and OTA values (µg/kg) ± SD. The for each mycotoxin and range. Correlation coefficient of
precision parameters RSDr, RSDR (Horwitz values) were determination (R2) was > 0.9995 for all calibration curves. The
calculated according to the International Union of Pure and slope of the linear calibration curve was statistically different
Applied Chemistry/AOAC Harmonized Protocol using an from 0 (P = 95%), and the intercept was not statistically
Excel® template (26). Horwitz values were used to compare different from 0 (P = 95%). LODs of the spiked samples were
the between-analysts variability (RSDR) at different levels. 0.019, 0.016, 0.021, 0.021, and 0.230 µg/kg for AFB1, AFB2,
The difference of the mean of the sample and the most extreme AFG1, AFG2, and OTA, respectively, and LOQs of the spiked
data considering the SD were based on the Grubbs’ test using samples were 0.078, 0.064, 0.085, 0.086, and 0.922 µg/kg for

Table  4.  Precision for AF and OTA determinations in the optimal HPLC conditions for spiked wheat
Spiked wheat

Within-day (n = 2) Between-day (n = 10)

Mycotoxins Spiked concn, µg/kg Recovery, % RSD, % Recovery, % RSD, %

AFB1 1.25 84.16 6.20 105.32 1.03

2.0 73.52 1.12 109.18 6.05
AFB2 1.25 98.63 10.01 89.25 6.92
2.0 97.27 9.43 98.42 4.04
AFG1 1.25 73.36 7.56 111.14 6.18
2.0 76.42 3.47 116.30 1.52
AFG2 1.25 74.13 9.80 74.82 6.45
2.0 85.17 6.43 82.76 9.40
OTA 3.0 100.30 1.85 104.13 12.00
8.0 96.73 2.40 78.10 11.14
 GazioĞlu & Kolak: Journal of AOAC International Vol. 98, No. 4, 2015  943

Table  5.  Accuracy for AF and OTA determinations in the optimal HPLC conditions for mycotoxin standard solution
Mycotoxin standard solution

Within-day (n = 2) Between-day (n = 10)

Mycotoxin Spiked concn, µg/kg Accuracy, % RSD, % Accuracy, % RSD, %

AFB1 1.25 100.01 0.02 100.50 1.56

2.0 99.67 0.50 108.42 1.67
AFB2 1.25 101.24 0.45 100.24 2.34
2.0 100.87 0.88 98.34 1.05
AFG1 1.25 90.44 3.48 102.0 0.89
2.0 93.26 4.20 106.52 2.45

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AFG2 1.25 87.48 2.56 94.45 2.56
2.0 86.32 1.80 92.66 4.80
OTA 3.0 99.64 0.76 104.66 0.10
8.0 97.80 1.00 98.10 1.44

AFB1, AFB2, AFG1, AFG2, and OTA, respectively. The R2, 95% confidence level (k = 2). Calculated uncertainties are given
LOD, and LOQ values are summarized in Table 1. in Table 1.

Accuracy and Precision Conclusions

The accuracy and precision of the methods were determined In the present study, the rapid, reproducible, sensitive, and
by analyzing duplicate samples of standards and spiked samples simple modified methods were validated for the simultaneous
at various concentrations. The methods were applied on the determinations of AFs and OTA in 81 processed cereal-based
same day and over 5 different days (Table 4). The precisions food samples. The modified AOAC 991.31 and AOAC 2000.03
(RSD) were all less than 10% for AFs, and 15% for OTA. methods consisted of a solvent mixture [methanol–water
Recoveries of all mycotoxins were determined by analysis of (75 + 25, v/v)] for extraction, and an extract cleanup with an
spiked wheat samples at two different concentrations. For total immunoaffinity column in the sample preparation step. The
AFs the mean recovery at 8.0 and 5.0 µg/kg was 98%, and for LODs of the spiked samples were found to be 0.019, 0.016,
OTA the mean recovery at 8.0 and 3.0 µg/kg was 91% (Table 5). 0.021, 0.021, and 0.230 µg/kg for AFB1, AFB2, AFG1, AFG2,
and OTA, respectively, and their LOQ values were 0.078,
The RSD for repeatability was found to be 1.03–9.34% for total
0.064, 0.085, 0.086, and 0.922 µg/kg for AFB1, AFB2, AFG1,
AFs and 11.15–15.05% for OTA. The methods had acceptable
AFG2, and OTA, respectively. The within-day and between-day
within-laboratory and between-analyst precision for processed
accuracy study indicated 86.32–101.24 and 92.66–108.42%
cereal-based products at two different levels.

Application of the Validated Methods to the Processed (a)

Cereal-Based Products

In this work, the validated methods were successfully

applied for the simultaneous determination of AF and OTA in
the processed cereal-based products. AF and OTA levels were
detected in 81 processed cereal-based products, and the results
are exhibited in Table 6. Our results indicated that 31 (38.2%)
of 81 analyzed samples were contaminated with AFs, and nine
samples (11.1%) with OTA. Only pasta samples were found to
(b)
be not contaminated with AFs or OTA.

Estimation of Uncertainty

The Eurachem Guide report was used to evaluate and quantify

the uncertainty sources of the applied methods (22). Purity of
reference standards (pur), sample weights (w), sample volumes
(vol), calibration curves (cal), repeatability (rep), and recoveries
(rec) were used to calculate the uncertainties. Calibration curves
and the purity of standards were defined as the main sources Figure  1.  Chromatograms of spiked wheat samples: (a) AFG2,
of uncertainty. The relative uncertainties were calculated at the AFG1, AFB2, and AFB1 and (b) OTA.
944  GazioĞlu & Kolak: Journal of AOAC International Vol. 98, No. 4, 2015

Table  6.  Total AFs, AFB1, and OTA concentration ranges in processed cereal-based products
No. of contaminated samples by mycotoxin

Sample groups No. of analyzed samples Total aflatoxin AFB1 OTA

Wheat 12 4 (0.033–3.606)a 4 (0.016–3.250) NDb

Bread 9 2 (0.018–0.014) 1 (0.018) 3 (0.313–0.373)
Starch 9 2 (0.116–0.191) 2 (0.100–0.168) ND
Semolina 5 2 (0.056–0.132) 2 (0.056–0.108) 2 (0.361–0.409)
Cake flour 7 4 (0.017–0.04) 2 (0.017–0.027) 1 (0.282)
Pasta 7 ND ND ND
Cake 8 2 (0.018) 2 (0.018) ND

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Biscuits 13 10 (0.017–0.072) 10 (0.017–0.072) ND
Chips 5 2 (0.052–0.137) 2 (0.052–0.137) ND
Rusk 6 3 (0.017–0.041) 3 (0.017–0.028) 3 (0.380–1.157)
a
  Values in parentheses represent the range of mycotoxin level (µg/kg).
b
 ND = Not detected.

Table  7.  Comparison of AOAC methods with the modified methods

Aflatoxin analysis Ochratoxin A analysis

AOAC method Modified method AOAC method Modified method

Extraction solvent Methanol–water (70 + 30, v/v) Methanol–water (75 + 25, v/v) Acetonitrile–water (60 + 40, v/v) Methanol–water
(75 + 25, v/v)
Mobile phase Water–methanol–acetonitrile Water–methanol–acetonitrile Acetonitrile–water–acetic acid Methanol–water–acetic acid
(6 + 3 + 2, v/v/v) (54 + 38 + 8, v/v/v) (51 + 47 + 2, v/v/v) (68.5 + 29 + 2.5, v/v/v)
+ 0.132 g KBr + 300 µL + 0.2 g KBr + 360 µL
HNO3 (for 1 L) HNO3 (for 1 L)
Flow rate, mL/min 1 1 1 1
Column temperature, °C 40 45 35 45
Analysis time, min 10 12 9 7.5

recoveries for AFS and OTA, respectively, in the spiked wheat Acknowledgments
samples, and the RSD values were 0.02–4.80%, respectively.
The modified methods were successfully applied to the This study is a part of Işıl Gazioğlu’s PhD thesis. The authors
processed cereal-based food samples purchased from different are grateful to the Research Fund of Istanbul University (Project
markets in Istanbul, Turkey. Among 81 processed cereal-based No. 22522).
food samples, AFs were detected in 31 samples (38.2%) and
OTA in nine samples (11.1%). The highest AF level was found Conflict of Interest
in a wheat sample (3.606 µg/kg), the highest AFB1 level in a
wheat sample (3.250 µg/kg), and the highest OTA level in a Işıl Gazioğlu declares that she has no conflict of interest. Ufuk
Kolak declares that she has no conflict of interest. This article
rusk sample (1.157 µg/kg), while no contamination was found
does not contain any studies with human or animal subjects.
in pasta samples.
The current AOAC and modified AOAC methods for the
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