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Wageningen Academic P u b l i s h e r s
World Mycotoxin Journal, 2016; 9 (3): 465-474
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
Occurrence and exposure assessment of multiple mycotoxins in dried fruits based on
liquid chromatography-tandem mass spectrometry
Z. Han1,2#, M. Dong1#, W. Han1, Y. Shen1, D. Nie1, W. Shi1 and Z. Zhao1*
1Institute
for Agri-food Standards & Testing Technology, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road,
Shanghai 201403, China, P.R.; 2Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University,
Ottergemsesteenweg 460, 9000 Gent, Belgium; zhao9912@hotmail.com; # These authors contributed equally to this work
Received: 14 September 2015 / Accepted: 23 December 2015
© 2016 Wageningen Academic Publishers
RESEARCH ARTICLE
Abstract
A reliable analytical method based on liquid chromatography-tandem mass spectrometry was developed for
simultaneous determination of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1, aflatoxin G2, ochratoxin A
(OTA), deoxynivalenol, T-2 and HT-2 toxin, and zearalenone (ZEA) in various dried fruits. A simple one-step sample
extraction without using clean-up cartridges made the established method less labour consuming and less expensive,
while optimisation of the several important MS/MS parameters, i.e. the scan time and run segments, ensured its
sensitivity and selectivity. After careful validation of the method by determining the linearity (R2>0.99), recovery
(77.8-115.9%), precision (relative standard deviation ≤19.5%) and sensitivity (limits of quantification in the range of
0.1-10 μg/kg), a survey of 125 dried fruit samples including 25 pistachios, 28 dried longans, 32 raisins and 40 dried
dates randomly collected from different markets in Shanghai, China, was performed. Results revealed that 32.0% of
samples were contaminated with different mycotoxins, among which, OTA was the most frequent contaminant with
the incidence of 29.6% attaining the concentration levels in the range of 0.4-212.6 μg/kg. ZEA was positively found
in 2 pistachio samples with the concentrations of 84.9 μg/kg and 426.9 μg/kg. Trace amounts of AFB1 (0.8 μg/kg)
and AFB2 (0.2 μg/kg) were also observed in one pistachio sample and one dried longan sample, respectively. To the
best of our knowledge, this is the first report to reveal the real situations of mycotoxin contaminations in various
dried fruits in China.
Keywords: mycotoxins, pistachios, dried longans, raisins, dried dates
1. Introduction
Mycotoxins, a series of toxic secondary metabolites, are
produced by various fungal species primarily including
Aspergillus, Penicillium and Fusarium (Abarca et al., 2003;
Escobar et al., 2013; Romero et al., 2005). They are classified
into different groups on the basis of their different sources
and toxicities. Aflatoxins, mainly consisting of aflatoxin
B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and
aflatoxin G2 (AFG2), are produced by the spoilage of
Aspergillus fungi, which have been classified in group I
as human carcinogens by the International Agency for
Research on Cancer (IARC). Ochratoxin A (OTA) can
elicit nephrotoxic, hepatotoxic and immunotoxic effects
and is classified in group 2B (possible human carcinogen)
by IARC (Han et al., 2013). T-2 toxin (T-2) and HT-2 toxin
(HT-2), both belonging to type A trichothecenes, have
immunosuppressive and cytotoxic effects on humans
and animals. Deoxynivalenol (DON), the most frequently
detected type B trichothecene, can cause a variety of
toxic effects, i.e. feed refusal, weight loss and vomiting.
Zearalenone (ZEA) is a phenolic compound produced by
Fusarium moulds, of which acute and chronic ingestion can
lead to the enlargement of mammary glands and uterus,
infertility and atrophy of testicles and ovaries (Han et al.,
2014a). Mycotoxins are frequently reported to occur in
different cereal crops, i.e. maize, wheat, rice (Almeida et
al., 2012; Kabak, 2009). They could also be detected in a
variety of dried fruits when the samples are not fully dried
or improperly stored during preparation (Azaiez et al.,
ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2015.1983465
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
Z. Han et al.
2015). Due to their widespread occurrence and harmful
health effects, the maximum residue levels (MRLs) of these
mycotoxins have been regulated in different dried fruits to
protect public health. In European Union (EU), the MRLs
are set as 2-12 μg/kg for AFB1, 4-15 μg/kg for aflatoxins in
pistachios and other dried fruits, and 10 μg/kg for OTA in
raisins (EC, 2010).
Different analytical methods based on thin layer
chromatography, ELISA, gas or liquid chromatography
have been developed for the detection of mycotoxins (CanoSancho et al., 2013; Corcuera et al., 2011; Yogendrarajah
et al., 2013). Among them, liquid chromatography tandem
mass spectrometry (LC-MS/MS) is the most promising
approach for obtaining an accurate picture of mycotoxin
in contamination pattern due to its high sensitivity and
superior selectivity (Arroyo-Manzanares et al., 2013a,b;
Diana Di Mavungu et al., 2009). However, LC-MS/MS
frequently suffers from the interferences of the impurities
in the sample matrix. In order to improve the sensitivity
and selectivity, many studies have been devoted to reducing
or eliminating interferences by development of adequate
sample extraction and efficient purification methods. Since
the mycotoxins are small and polar compounds, they are
usually extracted with the polar solvents or mixtures of
several polar solvents, i.e. methanol, acetonitrile and water.
The proportion of the solvent in the extraction mixture is
dependent on the physical and chemical characteristics of
the targets and the selected analytical systems (ArroyoManzanares et al., 2013a,b; Diana Di Mavungu et al., 2009;
Ren et al., 2007; Sulyok et al., 2006). The most frequently
used solvents for the extraction of multiple mycotoxins are
acetonitrile:water:acetic acid (79:20:1, v/v/v) (Sulyok et al.,
2006) and acetonitrile:water (84:16, v/v) (Ren et al., 2007),
which have been proven to result in less coextraction of the
interfering compounds. Following the extraction, the raw
extracts are subsequently subjected to a further purification
step to remove interferences. Different purification methods
have been developed based on liquid-liquid separation (Lai
et al., 2014; Sheijooni-Fumani et al., 2011), solid-phase
extraction (SPE) (Almeida et al., 2012; Berthiller et al.,
2009), immunoaffinity column (IAC) (Brenn-Struckhofova
et al., 2007; Desmarchelier et al., 2014), and single-step
clean-up cartridges (Mycosep©, Romer Labs, Tulln, Austria)
solid-phase extraction (Ren et al., 2007). Compared to
those approaches, direct injection after simple extraction
is more efficient, rapid, economical and convenient for
the determination of multiple mycotoxins, especially for
the screening purpose of high throughput samples if the
sensitivity and selectivity are satisfactory (Sulyok et al.,
2006). In the previous studies, the LC conditions for the
separation of the targeted compounds have been extensively
optimised. Unfortunately, the optimisation of the MS/MS
was easily overlook and confined only on the precursorproduct transitions. So far it is still rarely reported to
466
increase the sensitivity and selectivity by adjustment of
the useful parameters on the MS/MS itself.
Dried fruits, major consumptions of which are pistachios,
dried longans, raisins and dried dates in China, can be
simultaneously infected by different toxigenic moulds,
which potentially results in the co-occurrence of multiple
mycotoxins (Azaiez et al., 2015; Drusch and Ragab, 2003;
Romero et al., 2005; Trucksess and Scott, 2008). The
previous studies were only directed toward a single class or a
very small fraction of the mycotoxins, primarily aflatoxins in
dried figs and OTA in dried vine fruits (Bircan, 2009; Feizy et
al., 2012; Iamanaka et al., 2005; Janati et al., 2012; Zinedine
et al., 2007), while the other important mycotoxins, i.e.
ZEA, DON, T-2 and HT-2, are neglected. Consequently,
the same sample needs to be analysed multiple times to
cover all relevant analytes. Therefore, within the field of
mycotoxin, analysis in dried fruits, a clear trend toward the
use of multi-analyte methods can be seen. Recently, a LCMS/MS method based on a modified QuEChERS procedure
has been developed for multiple mycotoxins in raisins, figs,
apricots, plums and dates purchased from local markets in
Spain (Azaiez et al., 2015). However, the absence of the two
important mycotoxins, i.e. DON and ZEA, and the different
sample matrices to be investigated, make this established
method unsuitable for screening mycotoxins in dried fruits
in China. In addition, data available on the exposure and
occurrence of mycotoxins in dried fruits, except dried figs,
dried vine grapes and raisins are still limited.
In the present study, a reliable LC-MS/MS method was
developed for simultaneous determination of 9 frequently
found mycotoxins, i.e. AFB1, AFB2, AFG1, AFG2, OTA,
DON, T-2, HT-2 and ZEA in different dried fruits based
on a simple one-step sample extraction. Several important
parameters regarding the MS/MS instrument itself, i.e. the
scan time and run segments, were carefully investigated
to filter out the interferences in the sample matrices and
make one chromatographic peak contain 10-20 scan
points, so as to ensure the sensitivity and selectivity of the
established method. Based on the methodological advances,
the exposure of all frequently contaminated mycotoxins
in pistachios, dried longans, raisins and dried dates was
investigated for the first time to reveal the real situation
of contamination in China.
2. Material and methods
Materials
The standards of AFB1, AFB2, AFG1, AFG2, OTA, DON,
T-2, HT-2 and ZEA were all purchased from Sigma-Aldrich
(St. Louis, MO, USA). Acetonitrile and methanol, both
HPLC grade, were from Merck (Darmstadt, Germany).
The Milli-Q quality water (Millipore, Billerica, MA, USA)
was used throughout the whole analysis. Other solvents
World Mycotoxin Journal 9 (3)
and chemicals from local suppliers were all analytical or
HPLC grade.
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
Instrument
HPLC analysis was performed using a Quaternary Surveyor
HPLC system (Thermo Finnigan, San Jose, CA, USA).
The separated compounds were detected with a tandem
triple quadrupole mass spectrometer (TSQ Quantum
Ultra, Thermo Scientific, San Jose, CA, USA). The mass
spectrometer was operated with a heated electrospray
source both in positive ionisation (ESI+) and negative
ionisation (ESI-) modes. An Agilent Poroshell 120 EC-C18
column (100×3 mm, 2.7 µm) (Agilent, Santa Clara, CA,
USA) was utilised for the separation of the targeted analytes.
The mobile phase consisted of water containing 5 mmol/l
ammonium acetate (A) and methanol (B). Linear gradient
elution program was applied as follows: 20% B (0-2 min), 2060% B (2-6 min), 60-90% B (6-10 min), 90% B (10-12 min),
90-20% B (12-13 min), 20% B (13-16 min). The flow rate was
0.3 ml/min and the injection volume was 5 μl. The following
settings were employed for MS/MS conditions: vaporiser
temperature, 300 °C; capillary temperature, 350 °C; sheath
gas pressure, 30 psi; aux valve flow, 30 arb. The total run
time was divided into three segments: 0-6 min for segment 1
with the scan time of 0.2 s; 6-8.5 min for segment 2 with the
scan time of 0.05 s; 8.5-16 min for segment 3 with the scan
time of 0.002 s, respectively. Quantification was performed
in selected reaction monitoring mode. Data were acquired
and processed by Xcalibur software (Thermo Scientific,
Brookfield, WI, USA).
Preparation of standard solutions
Accurately weighted solid portions of each mycotoxin
standard were dissolved in acetonitrile to prepare 100 μg/ml
of stock solution. Then, an aliquot of 100 μl of each stock
solution was mixed together and diluted with water to 10
ml, to prepare a mixed stock solution with the concentration
of 1 μg/ml for each mycotoxin. Then, the mixed stock
solution was diluted step by step with the combined solution
(acetonitrile:water containing 5 mmol/l ammonium acetate,
20:80, v/v) to prepare a sequence of working solutions.
The stock solutions should be stored at -20 °C in the dark.
Working solutions were prepared immediately before use.
Samples
A total of 125 samples consisting of pistachios (25), dried
longans (28), raisins (32) and dried dates (40) were randomly
collected from different markets in Shanghai, China during
2014-2015. The collected dried fruits were prepared
according to the previous studies (Arroyo-Manzanares et
al., 2013a; Muhammad et al., 2015): the samples were cut
into small pieces, ground with a grinding mill (Retsch ZM
200; Retsch GmbH, Haan, Germany) and then maintained
World Mycotoxin Journal 9 (3)
Occurrence of multiple mycotoxins in various dried fruits
in zipper top paper bags to prevent humidity changes and
stored at -20 °C until analysis. Information about geographic
origins and brands of samples were required and registered.
Sample preparation
Samples (2.0 g) were weighted into 50 ml centrifuge tubes
and brought into suspension with 10 ml acetonitrile:water
solution (86:14, v/v). The mixture was ultrasonicated for 1 h
and an aliquot (5 ml) of supernatant was dried by nitrogen
gas at 40 °C. The residues were re-dissolved with 1 ml
acetonitrile:water containing 5 mmol/l ammonium acetate
(20:80, v/v), passed through a 0.22 μm nylon filter and
ready for analysis.
Method validation
The method was thoroughly validated on a series of
characteristics including linearity, sensitivity, accuracy
and precision. Matrix effects for the 9 targeted analytes
in pistachios, dried longans, raisins and dried dates were
also evaluated.
The matrix matched calibration curves were constructed
in the matrices of pistachio, dried longan, raisin and
dried date, respectively. The sensitivity was estimated by
determining the matrix-dependent limit of detection (LOD)
and limit of quantification (LOQ), which were defined as the
concentrations of the targets that provided signal to noise
(S/N)=3 and S/N=10 in different matrices, respectively. The
matrix effects were evaluated according to the following
formula: the matrix effects (%) = [(the response of the target
compound in matrix –the response of the target compound
in solvent)/the response of the target compound in solvent]
× 100% (He et al., 2015). The accuracy of the established
method was investigated using standard addition assays
with the low, intermediate and high fortification levels of 1,
50 and 100 μg/kg for AFB1, AFB2, AFG1, AFG2 and 10, 50
and 100 μg/kg for the other mycotoxins in sextuplicate. The
precision tests were also performed in the uncontaminated
samples (n=6) employing the method of standard addition
with the intermediate spiked concentration of 50 μg/kg for
all mycotoxins. The relative standard deviations (RSDs)
in the same day were used for evaluation of the intra-day
precision, while the values in three consecutive days were
for inter-day precision.
3. Results
MS/MS parameters
The selection of MS/MS conditions was firstly performed
by direct injection of the standard solutions with the
concentration of 500 ng/ml for all mycotoxins. The
precursor ions were identified by recording the m/z from
100 to 800 under full scan both ESI+ and ESI- modes. The
467
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
Z. Han et al.
results showed that AFB1, AFB2, AFG1, AFG2, OTA, T-2
and HT-2 were suitable in the ESI+ mode, and DON and
ZEA were ionized in ESI− mode. The precursor ions were
[M+H]+ for AFB1, AFB2, AFG1, AFG2, OTA and [M-H]for ZEA, while for the three trichothecenes, i.e. DON, T-2
and HT-2, the responses of [M+CH3COO]− generated for
DON and [M+NH4]+ for T-2, HT-2 were the highest among
different precursor ions. Based on the confirmation of the
precursor ions, the collision energies for the quantifier
and qualifier fragments were automatically optimised
in the Xcalibur Tune program and two product ions for
each precursor ion were selected according to the highest
response and optimal selectivity. The final selections of
MS/MS parameters are shown in Table 1.
mycotoxins was observed in raisins, followed by pistachios
(28%) and dried dates (22.5%), while the lowest incidence
(21.4%) was found in dried longans (Figure 2).
Among the 9 investigated mycotoxins, OTA, ZEA, AFB1
and AFB2 were positively found. A total of 37 samples
were contaminated by OTA with the concentration
levels in the range of 0.4-212.6 μg/kg. Only one pistachio
sample was contaminated with a trace amount of AFB1
(0.8 μg/kg), and one dried longan sample with AFB2
(0.2 μg/kg). ZEA was also presented in 2 pistachio samples
with the concentrations of 84.9 μg/kg and 426.9 μg/kg,
respectively (Table 4).
4. Discussion
Method validation
For screening high throughput samples, it is much more
appealing to develop a less labour consuming and less
expensive sample pretreatment method without using SPE
or mixed toxin IAC cartridges. To enhance the sensitivity
and selectivity of the established method, the parameters
of the MS spectrometer itself were carefully optimised in
the present study. Since the positive and negative ionised
compounds were simultaneously determined, the sensitivity
could be affected by the additional polarity switching time
(0.025 s) in the present study. Therefore, the total run time
was divided into three segments: 0-6 min monitoring for
DON, 6-8.5 min for AFB1, AFB2, AFG1 and AFG2, 8.5-16
min for the other four mycotoxins, to compensate for this
disadvantage. Compared to the unsegmented detection
(Supplementary Figure S1B), the responses of the 9
mycotoxins determined with the segments were greatly
improved (Supplementary Figure S1A). After confirmation
of the detection segments, the scan time was further
investigated for each segment to obtain a suitable scan
point number (10-20) for each chromatographic peak,
which made large contributions to the sensitivity of the
The linearity was evaluated using matrix-matched
calibration curves. As shown in Table 2, nice linear
relationships with the coefficients (R2) higher than 0.99
were obtained for all targets in four different matrices.
With regard to the sensitivity of the established method, the
values of LOD and LOQ were in the range of 0.03-3 μg/kg
and 0.1-10 μg/kg, respectively. The extent of matrix effects
ranged from -81.9 to 6.2% for pistachios, -63.4 to 6.9% for
dried longans, -67.1 to 5.7% for raisins and -89 to -19.8% for
dried dates, respectively (Figure 1). The recoveries of the 9
mycotoxins were in the range of 88.5-115.6% for pistachios,
77.8-113.2% for dried longans, 89.5-115.9% for raisins and
79.4-105.7% for dried dates, respectively (Table 3). The
RSDs for intra- and inter-day precision were in the range
of 1.6-18.6% and 3.6-19.5%, respectively.
Occurrence of mycotoxins
Among the 125 samples, 40 (incidence of 32.0%) were
contaminated by different mycotoxins, ranging in levels
up to 426.9 μg/kg (Table 4). Highest occurrence (56.2%) of
Table 1. MS/MS parameters for the 9 targeted mycotoxins.
Mycotoxins
Precursor ion
(m/z)
Quantification ion
(m/z)
Collision energy
(eV)
Diagnostic ion
(m/z)
Collision energy
(eV)
ESI1 mode
Aflatoxin B1
Aflatoxin B2
Aflatoxin G1
Aflatoxin G2
Ochratoxin A
Deoxynivalenol
T-2 toxin
HT-2 toxin
Zearalenone
313
315
329
331
404
355
484
442
317
241
259
243
313
239
295
305
215
175
37
28
25
25
25
14
13
13
26
285
287
200
189
358
265
215
263
273
22
26
38
40
14
19
18
15
22
ESI+
ESI+
ESI+
ESI+
ESI+
ESIESI+
ESI+
ESI-
1
ESI = electrospray isonisation.
468
World Mycotoxin Journal 9 (3)
Occurrence of multiple mycotoxins in various dried fruits
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
method. The number of scan point was calculated according
to the following formula:
0.2 s (Figure S1A) and 0.5 s (Figure S1D), were compared.
The peak width is about 6 s and two transitions were
selected for DON, therefore, 0.2 s (about 15 points) was
the best choice leading to the highest responses compared
to the others. For the second segment, four different
compounds were detected. After comparison of different
scan times including 0.01 s (Figure S1C), 0.05 s (Figure S1A)
and 0.1 s (Figure S1D), 0.05 s (about 15 points) seemed to
be more suitable than the others resulting in an improved
sensitivity for the mycotoxins in this segment. The third
Number of scan point (n) = peak width (s) / [number of
transitions × (transition switching time + scan time (s) +
polarity switching time (s) × 2)]
The transition switching time could not be set and was
neglected in the present study. For the first segment, three
different scan times, i.e. 0.02 s (Supplementary Figure S1C),
Table 2. The matrix-matched calibration curves and sensitivities of the 9 mycotoxins in pistachio, dried longan, raisin and dried
date samples.1
Mycotoxins
Pistachio
Dried longan
Raisin
Dried date
AFB1
AFB2
AFG1
AFG2
OTA
DON
T-2
HT-2
ZEA
AFB1
AFB2
AFG1
AFG2
OTA
DON
T-2
HT-2
ZEA
AFB1
AFB2
AFG1
AFG2
OTA
DON
T-2
HT-2
ZEA
AFB1
AFB2
AFG1
AFG2
OTA
DON
T-2
HT-2
ZEA
Slopes
183.77
153.47
38.35
171.48
521.87
412.28
1,704.01
886.84
2,982.80
224.90
193.99
47.16
177.92
558.81
392.65
1,981.88
846.08
6,751.31
217.96
149.51
55.67
200.34
541.14
487.22
2,018.91
880.84
3,858.13
171.62
172.60
54.05
198.61
531.51
500.36
1,667.99
801.46
1,914.74
Intercepts
R2
Linear range (ng/ml)
LODs (μg/kg)
LOQs (μg/kg)
570.94
2,474
488.03
1,413.04
4,765.71
7,047.20
17,411.9
7,155.62
105.556
-12.50
80.69
1,542.38
5,060.82
4,823.11
3,589.22
9,546.58
20,800.4
-633.94
2,938.01
1,917.98
1,290.19
1,162.44
7,895.24
12,599.7
-17,325.5
8,374.16
22,697
2,992.71
1,689.39
322.94
1,266.35
3,738.97
1,675.41
21,877.5
19,018.6
29,076.5
0.9915
0.9911
0.9996
0.9905
0.9940
0.9961
0.9930
0.9961
0.9957
0.9968
0.9965
0.9906
0.9907
0.9997
0.9948
0.9974
0.9978
0.9957
0.9967
0.9980
0.9930
0.9951
0.9984
0.9945
0.9951
0.9982
0.9932
0.9900
0.9966
0.9975
0.9955
0.9975
0.9918
0.9963
0.9906
0.9986
1-200
1-200
1-200
1-200
1-500
10-500
10-500
10-500
10-500
1-200
1-200
1-200
1-200
1-500
10-500
10-500
10-500
10-500
1-200
1-200
1-200
1-200
1-500
10-500
10-500
10-500
10-500
1-200
1-200
1-200
1-200
1-500
10-500
10-500
10-500
10-500
0.03
0.2
0.2
0.3
0.3
3
1
1
1
0.1
0.1
0.2
0.3
0.3
3
1
1
1
0.1
0.3
0.3
0.3
0.3
3
1
1
1
0.1
0.1
0.3
0.3
0.3
3
1
1
1
0.1
0.5
0.6
0.8
1.0
10
3
3
3
0.5
0.4
0.6
0.8
1.0
10
3
3
3
0.5
0.8
0.8
0.8
1.0
10
3
3
3
0.4
0.4
1.0
1.0
1.0
10
3
3
3
1 AFB
1
= aflatoxin B1; AFB2 = aflatoxin B2; AFG1 = aflatoxin G1; AFG2 = aflatoxin G2; OTA = ochratoxin A; DON = deoxynivalenol; T-2 = T-2 toxin; HT-2
= HT-2 toxin; ZEA = zearalenone; LOD = limit of detection; LOQ = limit of quantification.
World Mycotoxin Journal 9 (3)
469
Z. Han et al.
40
Pistachio
Dried longan
Raisin
Dried dates
0
Matrix effects (%)
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20
- 20
- 40
- 60
- 80
AFB1
AFB2
AFG1
AFG2
- 100
OTA
DON
T-2
HT-2
ZEA
Mycotoxins
Figure 1. The matrix effects of 9 mycotoxins in different dried fruits. Acceptable extents are in the range of two dashed lines (-20%
to 20%) with the spiked levels of 100 μg/kg. AF = aflatoxin; OTA = ochratoxin A; DON = deoxynivalenol; T-2 = T-2 toxin; HT-2 =
HT-2 toxin; ZEA = zearalenone.
Zearalenone
8% Ochratoxin A and
aflatoxin B1
4%
Undetected
44%
Ochratoxin A
16%
Ochratoxin A
56%
Undetected
72%
Raisin
32%
Pistachio
25%
Dried longan Dried dates
28%
40%
Ochratoxin A
Aflatoxin B2
18%
4%
Undetected
78%
Ochratoxin A
23%
Undetected
77%
Figure 2. The variability in type and relative proportions of 9 targeted mycotoxins in the dried fruit samples (pistachios, dried
longans, raisins and dried dates).
segment was a special one, in which, both positive ions
and negative ions were included. Consequently, the scan
times were significantly decreased and 0.001 s (Figure S1C),
470
0.002 s (Figure S1A) and 0.02 s (Figure S1D) were tested.
As expected, 0.002 s showed highest sensitivity and nice
peak shapes. Finally, the scan times were selected as 0.2 s
World Mycotoxin Journal 9 (3)
Occurrence of multiple mycotoxins in various dried fruits
Table 3. Recovery, intra- and inter-day precision of the 9 mycotoxins in dried fruits (%, n=6).
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
Mycotoxins
Pistachio
Fortified Recovery
levels
± SD
(μg/kg)
Aflatoxin B1
1
50
100
1
Aflatoxin B2
50
100
1
Aflatoxin G1
50
100
1
Aflatoxin G2
50
100
Ochratoxin A
10
50
100
Deoxynivalenol 10
50
100
T-2 toxin
10
50
100
HT-2 toxin
10
50
100
Zearalenone
10
50
100
1
2
95.6±3.2
103.6±11.1
110.2±3.3
99.3±5.6
106.4±17.7
113.2±10.5
105.8±4.5
106.4±5.7
115.6±7.3
105.6±7.2
108.7±16.6
110.2±10.1
90.5±5.6
97.7±12.4
91.2±3.5
95.6±4.6
91.4±3.4
94.3±3.2
94.1±7.9
102.7±19.1
110.3±10.1
93.2±7.4
107.6±11.1
89.6±7.7
88.5±4.6
98.3±3.4
91.5±4.6
Dried longan
Raisin
IntraRSD1
InterRSD2
Recovery
± SD
IntraRSD
Inter- Recovery
RSD ± SD
10.8
5.9
6.8
7.6
16.7
17.6
4.3
5.6
5.3
6.8
12.5
14.9
15.3
16.8
7.0
8.9
12.7
13.7
15.7
12.1
3.7
4.5
4.3
9.3
18.6
19.5
1.6
4.5
10.3
12.3
12.8
15.6
3.5
4.3
95.3±4.4
99.7±6.7
105.9±4.3
97.5±6.4
107.8±4.6
105.2±3.9
110.3±10.2
107.6±13.4
92.5±3.6
105.6±7.5
108.5±7.6
102.3±4.7
99.6±3.4
101.3±15.8
105.6±7.5
95.6±3.2
98.9±4.2
99.1±3.6
102.6±7.1
107.8±1.6
113.2±4.6
95.2±3.5
111.6±14.2
99.6±7.4
89.6±7.9
93.9±11.2
77.8±3.6
11.9
16.7
Dried date
IntraRSD
92.4±3.9
7.2
111.4±8.0
99.6±4.8
112.5±3.6
115.9±8.7
7.5
107.5±4.5
99.6±4.9
9.9
97.4±9.6
89.5±4.9
94.2±8.8 10.9
96.9±10.6
89.6±11.2
105.6±7.2
1.2
111.5±1.2
113.2±4.5
95.6±3.5
4.2
101.8±4.3
96.5±4.6
91.5±3.2
2.6
98.5±2.5
105.2±4.3
90.5±4.6
3.2
95.0±3.0
105.4±4.6
95.6±4.6 11.3
112.8±12.7
99.7±7.6
Inter- Recovery
RSD ± SD
8.6
9.5
11.3
15.4
4.5
5.6
3.6
4.5
15.4
90.3±6.5
87.8±12.5
95.6±3.2
95.6±5.6
89.4±13.6
104.3±8.6
95.8±8.9
91.5±10.1
89.6±7.3
88.6±6.9
85.4±12.2
79.4±10.6
105.6±3.6
102.7±5.7
105.7±7.2
90.9±4.6
94.1±6.7
99.6±7.6
95.6±3.2
100.5±2.2
94.6±4.4
88.9±7.2
91.0±16.3
95.6±7.1
88.2±4.6
91.2±10.7
95.6±7.5
IntraRSD
InterRSD
14.3
12.6
15.2
12.3
11.0
15.6
14.4
17.6
5.6
6.8
7.1
10.2
2.2
4.5
18.0
19.3
11.8
15.6
Intra-RSD: The relative standard deviations (RSDs) in the same day.
Inter-RSD: The relative standard deviations (RSDs) in three consecutive days were for inter-day precision.
for the first segment, 0.05 s for the second one and 0.002
s for the last.
For the method validation, matrix effect was considered to
be an important factor needing to be carefully investigated
for the development of the LC-MS/MS method. The values
higher than 20% or lower than -20% were defined as strong
matrix affects, leading to an inaccurate quantification.
The negative matrix effects with some of the mycotoxins
were rather extreme, especially for AFB1 and ZEA with the
largest extents of -74.7 and -89%, respectively. These large
and negative matrix effects were the price that was paid
for the minimal sample preparation. As a consequence, it
was necessary to use matrix matched calibration curves
to compensate for the matrix effects so as to guarantee
World Mycotoxin Journal 9 (3)
a reliable quantification. After application of the matrix
matched calibration curves, good linearity, satisfactory
recovery and precision were obtained. Compared to
the previously established LC-MS/MS method based
on QuEChERS techniques (Azaiez et al., 2014), the less
labour intensive method showed comparable sensitivity
and accuracy values for determination of the targeted 9
mycotoxins in different dried fruits, and even better results
for AFB1 in raisins. Based on all validation parameters,
the current analytical method can be regarded as
selective, robust, and accurate, and could be applied for
simultaneous determination of various mycotoxins in
different dried fruits.
471
Z. Han et al.
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
Table 4. Contamination levels of 9 mycotoxins in different dried
fruits in Shanghai, China (μg/kg).1,2
Pistachio
Raisin
Dried longan
Dried dates
1 AFB
OTA
ZEA
1.3
31.1
1.3
120.1
–
212.6
–
3.4
2.3
0.4
10.9 3
32.8 3
3.7
4.2
4.1
17.1 3
58.9 3
5
4.8
3.7
2.1
2.3
8.1
8.3
65.7 3
–
8.9
1
1.6
1.5
1
2.9
0.5
2.6
61.4
2.1
16.3
12.2
1.8
19.2
–
–
–
–
426.9 3
–
84.9 3
AFB1
AFB2
0.8
–
–
–
–
–
–
0.2
–
–
–
–
–
1 = aflatoxin B1; AFB2 = aflatoxin B2; OTA = ochratoxin A; ZEA =
zearalenone.
2 Undetected (–). i.e. the concentrations of mycotoxins were lower than
the limit of detection. For pistachio AFB2 was undetected; for dried longan
ZEA and AFB1 were undetected; and for raisin and dried dates ZEA, AFB1
and AFB2 were undetected in all samples.
3 The concentrations of the mycotoxins exceeded the maximum
concentration levels regulated by European Union.
472
Due to the dietary habits of the consumers in Shanghai,
China, different dried fruit samples, including pistachios,
dried longans, raisins and dried dates, were randomly
collected from different markets and analysed by the
established LC-MS/MS method. The exposure of
mycotoxins in raisin and pistachio samples were in good
agreement with the results reported by Azaiez et al.
(2015), from which, mycotoxins were detected in 28 out
of 53 sultana samples (incidence of 53%) and 34 out of
169 pistachio samples (incidence of 20%). However, with
respect to dried date, much more seriously contaminated
situations were found with the incidence more than 80%
in Turkey and Spain. From the world-wide available data
on mycotoxins in freshly produced and on derived (dried)
products, the incidences were 95.4% in raisin samples and
82.5% in pistachio samples, respectively (Van de Perre et al.,
2015). The relatively low contamination levels of mycotoxins
reported here may be due to the implementation of the
strict regulations in Shanghai, which prevents highly
contaminated samples from entering the markets, along
with the differences in the geographical and environmental
conditions in other countries when compared to China
(Han et al., 2014b). Worth noting is the fact that mycotoxins
were detected in dried longan samples for the first time,
indicating that this popular food also poses potential health
risks to consumers due to the contamination of mycotoxins,
and thus should be included in the monitoring systems.
Various mycotoxins with different concentration levels
have been detected in the collected samples. OTA was
the most frequently detected contaminant indicating that
the OTA producing fungi, notably Aspergillus ochraceus,
Aspergillus alliaceus, Aaspergillus auricomus and
Aaspergillus albertenses (Han et al., 2010b), could easily
infect the matrices of dried fruits. The highest incidence
of OTA was found in raisin samples, which was in good
agreement with the previous studies that showed that
raisin was easily contaminated by OTA with an incidence
range of 30-90% (Azaiez et al., 2015; Van de Perre et al.,
2015). Dried date, as another important food, has also been
investigated for the mycotoxin contamination around the
world. The data obtained in China in the present study were
similar to the reports from Tunisia and Spain, where OTA
occurred in 29 out of 75 samples with a mean concentration
of 1.26 μg/kg. With regard to pistachio samples, in contrast
to the values (undetected in 429 samples) reported in the
previous study (Van de Perre et al., 2015), the positive
samples found in the present study might be because of the
various climatic conditions during harvest and post-harvest
storage, as well as the different features of dried fruits (the
sugar concentration and water activity). The contamination
of AFB1 and AFB2 in one pistachio sample and one dried
longan sample indicated that either Aspergillus flavus or
Aspergillus parasiticus might infect these samples (Han
et al., 2010a). Comparing to the previous survey of AFB1
(incidence of 82.5%) in pistachio samples, the obvious
World Mycotoxin Journal 9 (3)
http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1983 - Wednesday, October 25, 2017 12:31:31 AM - Queen's University of Belfast IP Address:143.117.16.36
differences also verified the critical roles played by the
environmental conditions of the harvest and post-harvest
in the infection of the toxigenic fungi. In respect of ZEA,
the presence in pistachio samples revealed the prevalence
of Fusarium spp., a potential producer of ZEA (Tsakmakidis
et al., 2008).
According to the regulations set by EU (EC, 2010),
2-12 μg/kg were set as the MRLs for AFB1, 4-15 μg/kg for
aflatoxins in pistachios and other dried fruits, and 10 μg/kg
for OTA in raisins. Since there are no MRLs for ZEA in
dried fruits, 75 μg/kg, which is set as the MRL for ZEA in
cereals intended for direct human consumption, was utilised
as the reference. Among the 125 investigated samples,
the concentrations of mycotoxins in a total of 7 samples
exceeded the related regulations, including OTA in 5 raisins
and ZEA in 2 pistachios, emphasising the potential health
risks posed by the contamination of various mycotoxins in
different dried fruits to the consumers in Shanghai, China.
4. Conclusions
A reliable analytical method has been developed for
simultaneous determination of 9 mycotoxins in different
dried fruits, with the performance characteristics fully
meeting the established criteria. The sensitivity and
selectivity were improved by optimisation of the MS
spectrometer. A one step extraction was used for sample
preparation, which was simple, rapid, and thus quite suitable
for screening various mycotoxins in high throughput
samples. A great incidence of OTA was detected in
dried fruits, which was considered to be a major source
of human exposure. Trace amounts of AFB1 and AFB2
were also detected in pistachio and dried longan samples
proving the infection of Aspergillus fungi in these matrices.
Noteworthy, large amounts of ZEA were also found in
two pistachio samples for the first time. Considering
the highly toxic characteristics of all these mycotoxins
alongside their critical incidence and contamination levels,
this study underlined once more the potential health risks
to consumers by these mycotoxins, as a consequence, it is
recommended to include various mycotoxins in monitoring
and control programs of production, storage and safety
administration of dried fruits.
Supplementary material
Supplementary material can be found online at http://
dx.doi.org/10.3920/WMJ2015.1983.
Figure S1. Comparison of the ionization efficiency of nine
mycotoxins among different segments and scan times.
World Mycotoxin Journal 9 (3)
Occurrence of multiple mycotoxins in various dried fruits
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (31301493, 31301494), Shanghai
Science and Technology Innovation Action Plan Project
(15142201500), Shanghai Agriculture Commission Project
(2014, NO. 3-2; 2013, NO. 3-1) and Shanghai Academy of
Agricultural Sciences ‘Zhu Pao’ Project.
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