Food Anal.

Methods
DOI 10.1007/s12161-013-9785-3

Multi-mycotoxins Analysis in Dried Fruit by LC/MS/MS

and a Modified QuEChERS Procedure
Ines Azaiez & Federica Giusti & Gianni Sagratini &
Jordi Mañes & Mónica Fernández-Franzón

Received: 3 September 2013 / Accepted: 9 December 2013

# Springer Science+Business Media New York 2014

Abstract A sensitive and reliable multi-mycotoxin method Introduction

was developed for the simultaneous determination of 16 tox-
icological important mycotoxins, such as aflatoxins B1, B2, Mycotoxins, secondary metabolites of filamentous fungi, rep-
G1, and G2; enniatins A, A1, B, and B1; beauvericin; ochra- resent a very large group of different substances produced by
toxin A; fumonisin B1, B2, and B3; diacetoxyscirprenol; HT-2; different mycotoxigenic species. These compounds with great
and T-2 toxin in dried fruits using liquid chromatography structural variation result in diverse physicochemical proper-
combined with electrospray ionization-triple quadrupole ties (Köppen et al. 2010). Hundreds of mycotoxins have been
tandem-mass spectrometry. Mycotoxins have been extracted recognized, although only a few of them present a significant
from the samples using a modified quick, easy, cheap, effec- toxic effect and are of major concern. These substances are
tive, rugged, and safe procedure. The method was based on a associated with carcinogenic, teratogenic, nephrotoxic, and
single extraction with acidified acetonitrile, followed by hepatotoxic properties; besides toxic effects, mycotoxins can
partitioning with salts, avoiding any further clean-up step. cause tremendous economic losses derived from the contam-
Limits of detections ranged from 0.08 to 15 μg kg−1 and limits ination of the world’s crop production.
of quantification ranged from 0.2 to 45 μg kg−1, which were Moulds of the genera Aspergillus and Penicillium occur in
below the legal limit set by the European Union for the different fruits. Fruits become increasingly susceptible to fun-
legislated mycotoxines. The recoveries in spiked samples gal invasion during ripening as the pH of the tissue increases,
ranged from 60 to 135 % except for beauvericin using skin layers soften, soluble carbohydrates build up, and defense
matrix-matched calibration curves for quantification, with barriers weaken (Fernández-Cruz et al. 2010). Furthermore,
good inter- and intraday repeatability (respective relative stan- varying processing and storage conditions can provide mould
dard deviation ≤20 and 9 %). The developed method was growth and mycotoxin development. There have been many
applied to 15 commercial dried fruits: raisins, figs, apricots, investigations on the occurrence of mycotoxins in these prod-
plums, and dates purchased in local markets from Spain. ucts. Dried fruits, particularly dried vine fruit (raisins, sultanas
Among the mycotoxins studied, enniantins and aflatoxins and currants), and figs have been of considerable interest as
were the most predominant mycotoxins. various dried fruits are in great demand in the health food
markets (Trucksess and Scott 2008).
Keywords Mycotoxins . QuEChERS . Dried fruits . Only a few mycotoxins in dried fruits have been
LC/MS/MS regulated by law. The European Union (EU) has im-
posed vigorous regulations to limit the presence of
ochratoxin (OTA) in dried vine fruit (currants, raisins,
I. Azaiez : J. Mañes : M. Fernández-Franzón (*)
and sultanas; 10 μg kg−1) as well as aflatoxin (AF)B1 in
Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy,
University of Valencia, Av. Vicent Andrés Estellés s/n, dried fruits for human consumption or food ingredient
46100 Burjassot, Spain (2 μg kg−1) and total aflatoxins (4 μg kg−1 for the sum of
e-mail: [email protected] AFB1 +AFB2 +AFG1 +AFG2) (Commission of the European
F. Giusti : G. Sagratini
Communities 2006). Also, a relatively new EU regulation
Scuola di Scienze del Farmaco e dei Prodotti della Salute, (Commission of the European Communities 2010) was set
Università di Camerino, via S. Agostino 1, 62032 Camerino, Italy for the total aflatoxins in dried figs (10 μg kg−1).
 Food Anal. Methods

Nowadays, the general trend is the performance of multi- QuEChERS procedure has not been used in extraction of
mycotoxin methods which permit the analysis of a wide range mycotoxins from dried fruits. Finally, its application to the
of mycotoxins with varying physicochemical characteristics determination of the studied mycotoxin in real samples has
in a single run. Thus, the combination of liquid chromatogra- been accomplished successfully.
phy (LC) with tandem-mass spectrometry (MS/MS) has
proved to be a powerful tool for the simultaneous determina-
tion of different classes of mycotoxins (Capriotti et al. 2012; Materials and Methods
Tang et al. 2013), and it has become the selected technique in
multiresidue analysis. Reagents and Standards
The analysis of mycotoxins in dried fruits can be an ex-
tremely challenging task due to minute amounts usually pre- Acetonitrile and methanol were high performance liquid chro-
sents in the samples and the large quantities of co-extracted matography grade and supplied by Merck (Darmstadt,
compounds especially carbohydrates which can adversely Germany). Deionized water (<8 MΩ cm−1 resistivity) was
affect the method and instrument performance. purified using Milli-Q® SP Reagent water system plus from
The methods proposed for the determination of mycotoxins Millipore Corp. (Bedford, USA).
in dried fruits are mainly based on liquid chromatography Solid phases used were Sepra C18-E (50 μm, 65 A°)
fluorescence detector for aflatoxins and OTA (Karbancıoglu- endcapped silica based C18 from Phenomenex (Torrance,
Guler and Heperkan 2008; Reinhold and Reinhardt 2011; USA) and Sep-Pak®C18 cartridges and Classic CN cartridges
Zinedine et al. 2007). Most of these methods use solid–liquid from Waters Corporation (Massachusetts USA).
extraction and immunoaffinity column (IAC) for clean-up, but Analytical grade reagent formic acid, acetic acid (purity,
IAC is an expensive and complex purification system which >98 %), and ammonium formate were obtained from Panreac
leads to low recoveries for some mycotoxins, because of the Quimica S.A.U. (Barcelona, Spain). Chromatographic sol-
complexity of these matrices and its use is limited to a single vents and water were degassed for 20 min using a Branson
mycotoxin. 5200 ultrasonic bath (Branson Ultrasonic Corp., CT, USA).
Recently, an extraction procedure defined as “quick, easy, Polypropylen syringes and filters (25 mm/0.45 μm and
cheap, effective, rugged, and safe” (QuEChERS) has been 0.22 μm) were from Analysis Vínicos (España). Magnesium
developed mainly for the extraction of pesticides, but sulfate anhydrous 99.5 % powder (MgSO4) was purchased
it has also been used for other compounds (Anastassiades from Alfa Aesar® Gmbh & Co KG (Germany) and sodium
et al. 2003). chloride (NaCl) from Merck (Darmstadt, Germany).
The QuEChERS method has several advantages over most The standards of aflatoxin AF(B1, B2, G1, and G2), OTA,
traditional methods of analysis, such as its simplicity, small diacetoxyscirpenol (DAS), fumonisins (FB1 and FB2),
solvent usage; waste minimum steps, and effectiveness for beauvericin (BEA), and enniatins Enns (Enn A, Enn A1,
cleaning-up complex samples (Sospedra et al. 2010; Enn B, and Enn B1) were purchased from Sigma Aldrich
Rodríguez-Carrasco et al. 2012). Its inherent speed and sim- (Madrid, Spain). T-2 toxin (T-2) and HT-2 toxin (HT-2) stock
plicity are also important when analyses of large number of solutions (in acetonitrile) were acquired from Biopure
samples in short time flame are required. It includes two steps: Referenzsubstanzen GmBH (Tulln, Austria). Fumonisin B3
the first one is a partitioning step via salting-out extraction, (FB3) was supplied by the PROMEC unit (Program on
and the second one is a dispersive solid-phase extraction step Mycotoxins and Experimental Carcinogenesis, Tygerberg,
that involves further clean-up using combinations of MgSO4 South Africa). Stock standard solutions of all mycotoxins
and different sorbents, such as C18 or primary and secondary were made in methanol, except FB1, FB2, and FB3 that were
amine (PSA). QuEChERS based methods have been recently diluted in acetonitrile/water 50:50 (v/v). They were all kept in
reported for the extraction of different mycotoxins from vari- safety conditions at −20 °C. Purity checks of stock solutions
ety of food matrices such as cereal products (Cunha and are made by UV measurement since their molar absorptivities
Fernandes 2010; Ediage et al. 2011; Ferreira et al. 2012), eggs are known.
(Garrido Frenich et al. 2011), beer (Tamzura et al. 2011), From the individual stock standard solutions, a standard
spices (Yogendrarajah et al. 2013), and bee pollen mixture was prepared at the following concentrations: Enn B,
(Rodríguez-Carrasco et al. 2013). The main advantages of this Enn B1, Enn A, and Enn A1 and AFB1, AFB2, AFG1, and
extraction procedure were not only the low amount of organic AFG2 (0.5 μg mL−1); OTA and T-2 (2 μg mL−1); BEA,
solvent required, but also the fastness and the good recoveries FB1, FB2, and FB3; DAS; and HT-2 (5 μg mL−1). All
obtained for the tested mycotoxins (Ferreira et al. 2012). other working solutions for determine calibration curves,
The aim of the present study is to adapt, apply and evaluate ion suppression, recoveries, and limit of detection were
the QuEChERS procedure to the extraction of multiple my- prepared by diluting the standard mixture in methanol or
cotoxins in dried fruits. To the best of our knowledge, matrix extract.
Food Anal. Methods

LC/MS/MS Analysis 2 h at room temperature to allow the mycotoxins absorption

into the matrix.
Detection and quantification was performed with 3200 In this study, in order to optimize the extraction procedure,
QTRAP® LC/MS/MS System (Applied Biosystems, different volumes of organic solvents as methanol and aceto-
Foster City, CA) with a Turbo V® ionspray ESI source nitrile (10; 22.5 and 32 mL) were assayed along with different
coupled to Agilent 1200 chromatograph (Agilent Tech- proportions of acetic acid (0; 0.5 and 1 %). Then, MgSO4
nologies, Palo Alto, CA, USA). The instrument data were (7.5 g) and NaCl (3 g) were added in order to obtain a
collected and processed using the Analyst® version 1.5.2 complete dispersion and humidification of the sample, which
software. will permit a better extraction of the mycotoxins. For clean-up
Chromatographic separation of analyte was performed step CN, C18 cartridge and C18 powder were proved. From
with a reversed-phase analytical column (Gemini ® C18 col- analytical point of view, the best extraction results were ob-
umn, 3-μm particle size, 150×2 mm, I.D.), equipped with a tained via the following steps:
C18 (4×2 mm, I.D.; 5 μm security guard cartridge) all from
Phenomenex, Madrid, Spain. The mobile phases were Step I Five grams of raisins were homogenized with
composed of two eluents both containing 5 mM ammo- 7.5 mL of water with acetic acid (1%) for 3 min with
nium formiate, the eluent A was water/formic acid 99:1 a vortex. The obtained mash was extracted with
(v/v) and the eluent B was methanol/formic acid 22.5 mL of acetonitrile for 3 min with a vortex.
99:1(v/v). The flow rate was 0.25 mL min−1. The gra- Step II Anhydrous MgSO4 (7.5 g) and NaCl (3 g) were
dient program started with 90 % A and 10 % B and added into the mixture and the homogenizates were
was kept 3 min, afterwards a linear gradient was ap- extracted under continuous mechanic shaking for 1 h
plied, reaching 70 % B after 1.5 min (holding time, in order to facilitate the extraction and partitioning of
3 min). Other linear gradients to 80 % B (6 min) and the mycotoxins compounds into the organic layer.
90 % B (14 min) were included. Finally, gradient Step III The extract was centrifuged for 10 min at 5,000 rpm,
switched back (5 min) to 90 % A. 5 °C and subsequently the supernatant layer was
MS/MS was performed in the selected reaction monitoring collected.
(SRM) mode using ESI in positive mode. For each compound, Step IV The eluate was evaporated to dryness under a nitro-
two characteristic product ions were monitored; the first and gen stream at 40 °C, using a multisample Turbovap
most abundant one was used for quantification, while the LV Evaporator (Zymark, Hoptkinton, USA) and
second one was used as a qualifier. was dissolved with 1 mL of 5 mM ammonium
For LC/MS/MS analysis, scheduled SRM was used formiate aqueous solution/methanol (50/50, v/v)
with a 120 s SRM detection window and 1 s of target acidified with 1 % of formic acid. Each sample
scan time. The applied parameters were: ion spray volt- was filtrated through a 0.22-μm PTFE filter prior
age, 5,500 V; source temperature, 450 °C; curtain gas, to the LC/MS/MS analysis.
20; ion source gas 1 (sheath gas), 50 psi; ion source gas
2 (drying gas), 55 psi. Nitrogen served as nebulizer and
collision gas.
Results and Discussion
Samples
Optimization of Chromatographic Conditions
Fifteen samples of dried fruits: plums (3), raisins (3), apricots
(3), figs (3), and dates were collected from different local The main aim of this study was to develop a single run
markets and supermarkets from Valencia. They were kept at multimethod for mycotoxin on dried fruits subject to cur-
−18 °C until analysis. rent and future EU legislation. The MS/MS method was
based on a previous reported LC/MS/MS method per-
Extraction and Clean-up Optimization formed in our laboratory, selecting those mycotoxins that
gave better sensitive in positive ionization (Rubert et al.
The extraction was performed by a modified version of 2012). The optimization of MS parameters was performed
QuEChERS procedure. In a 50-mL polypropylene centrifuge by flow injection analysis for each compound; entrance
tube, 5 g±(0.01 g) of homogenized sample were weighed. potential (EP) was set at 10 V, for all analytes. The
Sample recovery was investigated with 5 g of the blank dried quantification and qualification ion transitions of the re-
fruit samples using three different fortification levels: 50, 100, spective mycotoxins, the declustering potential, collision
and 200 μL of the standard mix. The spiked samples were energy, and collision cell exit potential programmed are
shaken for 3 min with a (mechanical mixing) vortex and left shown in Table 1, as well as the retention times.
 Food Anal. Methods

Table 1 Optimized MS\MS

parameters for each mycotoxin Analyte Rt (min) Precursor ion (m/z) Product ions (m/z) DP (V) CE (V) CXP (V)

AFB1 8.4 313.0 [M+H]+ 241.1Q 46 39 4

128.0q 41 4
AFB2 8.4 315.0 [M+H]+ 287.0Q 81 33 6
259.0q 39 6
AFG1 8 329.0 [M+H]+ 243.0Q 76 39 6
200.0q 29 6
AFG2 7.9 331.1 [M+H] +
313.0Q 61 27 6
189.0q 39 4
BEA 16.7 801.2 [M+NH4]+ 784.1Q 116 27 10
244.1q 39 6
DAS 8.2 384.0 [M+NH4]+ 307.2Q 66 15 16
105.0q 63 12
ENN A 17.9 699.4 [M+NH4]+ 210.1Q 76 35 14
228.2q 59 16
ENN A1 17.1 685.4 [M+NH4]+ 210.2Q 66 37 8
214.2q 59 10
ENN B 15.4 657.3 [M+NH4]+ 196.1Q 51 39 8
214.0q 59 10
ENN B1 16.2 671.2 [M+NH4]+ 214.1Q 66 61 10
228.1q 57 12
FB1 8.8 722.4 [M+H]+ 334.3Q 101 51 20
352.3q 45 26
FB2 10.4 706.4 [M+H]+ 336.2Q 131 50 16
q
318.3 50 16
FB3 9.6 706.5 [M+H]+ 336.3Q 100 45 16
318.4q 45 16
HT-2 9.2 442.2 [M+NH4]+ 215.1Q 61 19 4
263.0q 19 8
OTA 11.2 404.0 [M+H]+ 102.0Q 91 27 6
Q quantification, q qualification, 239.0q 97 6
DP the potential difference, CE T-2 9.9 484.2 [M+NH4]+ 185.1Q 76 29 4
collision energy, CXP collision 215.1q 22 4
cell exit potential

Tuning experiments favored the choice of the ESI(+) mode provides the best sensitivities for all compounds. The addition of
since EU limits for AFB1, OTA on dried fruits were met. Even ammonium formiate to the aqueous mobile phase clearly en-
for HT-2, T-2, DAS, BEA, FBs, and Enns the ionization in hanced the sensitivity for T-2, HT2, DAS, and the other myco-
positive mode was successful, whereas FUS X, DON, and toxins detected under their ammonium adduct [M+NH4]+,
NIV gave no or very weak signals in the positive ion mode whereas formic acid in both mobile phases increased the overall
(Sulyok et al. 2006) (these mycotoxins were not included in sensitivity for the acidic compounds, i.e., FB1, FB2, and OTA
this study). The majority of mycotoxins have a higher tenden- (Desmarchelier et al. 2010). In negative mode, the formiate
cy to ionize in ESI+ forming protonated molecular ions. buffer is preferred to formic acid, which makes it impractical
Moreover, it has been observed that a limited ionization of to switch polarity during the same injection even when techni-
trichothecenes as protonated or deprotonated molecular ions cally possible in the MS. Therefore, it was decided to maintain
was obtained with ESI. This problem was overcome by mon- the positive mode in the multimethod for the entire run.
itoring ammonium adducts (Garrido Frenich et al. 2009;
Sulyok et al. 2010). Extraction and Clean-up Optimization
LC mobile phases commonly used for mycotoxins analysis
are usually water, acetonitrile and methanol, with or without Owing to the great chemical diversity of mycotoxins and the
addition of salts, acids, or bases. Methanol as organic solvent complexity of dried fruits matrices validated in this study, the
Food Anal. Methods

solvent composition applied for extraction was an important For the majority of mycotoxins, unsatisfactory recoveries were
parameter during the development of the multi-mycotoxin obtained when only acetonitrile was used as extraction solvent.
method. Optimization studies were performed with prune Low recoveries were also obtained with the mixture of acetonitrile/
samples spiked with Enns and aflatoxins (10 μg kg−1); T-2 water (55:45). It could be assumed that smaller solvent-sample
and OTA (40 μg kg−1); and BEA, fumonisins, DAS, and HT- ratios can result in a saturation of the extraction solvent when
2 (100 μg kg−1). samples with a large amount of solutes are extracted such as
Different mixtures of methanol, or acetonitrile and water with samples with a high content of sugar. The best recovery results
or without organic acids were evaluated. Several extractions with were obtained using the proportion acetonitrile/water (75:25).
methanol/water (80:20) mixtures were firstly carried out. Al- In order to improve the recoveries obtained, we optimized the
though mycotoxins extraction was satisfactory, increased amounts ratio sample/water, increasing the amount of water required to
of matrix components such as polysaccharides and brown pig- obtain a suitable homogenized dried fruit sample. “Wetting” prior
ments were also extracted (Ediage et al. 2011), which could result to the extraction is recommended in the Document SANCO/
in column spoilage (Knudsen et al. 2011) and polluted MS 12495/2011 whenever the matrix contains less than 40 % of
equipment causing MS damage. Consequently, acetronitrile was moisture. Therefore, water/solvent/salt amount was increased
considered to be the best choice for this type of matrix. and better recoveries were obtained with the high volumes tested.
Firstly a modification of QuEChERS method was applied Several types of agitations were put into practice in order to
by adding acid to the extraction solvents. Initially mixtures of improve extractions’ performances. Ultra-sonication, horizon-
acetonitrile/water without and with 0.5, and 1 % of acetic acid tal shaker, manual and vortex agitation were evaluated. The
were checked. The results are presented in Fig. 1. best results in terms of recoveries were obtained using vortex
Comparison of the results from each of the samples indi- agitation (data no shown).
cated that acidified (1 %) aqueous acetonitrile provided the Finally, a clean-up procedure for reducing co-extracted
best overall performance. Without acidification, FBs could not compounds and matrix interferences was evaluated. Original
be extracted, and low recoveries were obtained especially for QuEChERS usually used PSA but this step has been avoided
OTA, HT2, and T2. At low pH, the four carboxylic groups of as fumonisins are strongly retained because of the ionic
FBs are protonated leading to less interaction and binding to affinity between the primary and secondary ammine of the
the sample matrix so acid solvents are the best choice (Garrido PSA and the carboxylic groups of fumonisins (Desmarchelier
Frenich et al. 2011; Knudsen et al. 2011). et al. 2010). Conventional SPE cartridges (C18 and CN) and
Afterwards, extractions with four different volumes and pro- C18 powder were evaluated. The obtained results are present-
portions of acetonitrile/water mixture with 1 % of acetic acid were ed in Fig. 3. Lower recovery rates were obtained for most of
evaluated (volume of water was kept almost fixed (7.5/8 mL) and the mycotoxins tested, when using C18 powder clean-up. It
acetonitrile volume was varied). The results are shown in Fig. 2. was assumed that they were not well recovered for being

160

140

120
Recovery rates (%)

100

80

60
1% acid
40 0.5% acid
without acid
20

Fig. 1 Effect of the acetic acid addition on the recovery results obtained for the extraction of spiked prune samples
 Food Anal. Methods

120

Recovery rates (%) 100

80

60

40

20

0
OTA HT2 T-2 DAS FB2 FB3 FB1 AFB1 AFB2 AFG1 AFG2 ENNB ENNB1 ENNA1 ENNA BEA

a: v= 17.5ml; CH3CN:H2O(55:45) plus acid 1% b: v= 40ml; CH3CN:H2O(80:20) pus acid 1%

c: v= 30ml; CH3CN:H2O(75:25) plus acid 1% d: v= 30 ml CH3CN (100) plus acid 1%

Fig. 2 Effect of the solvents proportion and volume on the extraction recovery of mycotoxins in prunes

partially absorbed by C18 powder. In order to improve recov- Method Validation

ery rates, clean-up was carried out using solid-phase extract
(SPE) cartridges C18. The recovery rates of HT2, DAS, FBs, Method validation was performed in terms of linearity, intra-
AFB1 and AFG2 have improved while those corresponding to and interday repeatability, matrix effect, limit of detection
OTA, T2, Enns and BEA have decreased. To reduce the (LODs), limits of quantifications (LOQs) and recovery. Vali-
retention of mycotoxins with the solid phase, the cartridges dation parameters were studied on prune samples except for
were washed with 2 mL of methanol at the end of each elution. recoveries that were studied also on apricots, figs, raisins and
Recoveries data showed a slightly improvement for all myco- dates.
toxins. Similar recovery rates were obtained while using Results of the evaluation are summarized in Table 2. To
(SPE) cartridges of CN. It can be clearly observed that poorer compare the efficiency of each extraction procedure, absolute
results were obtained when clean-up procedure was applied. recoveries were determined by spiking blank extract samples
Therefore, no clean-up was used for further experiments. (standards added to blank samples of each matrix studied) at

140

120

100
Recovery rates (%)

80

60

40

20

0
OTA HT2 T-2 DAS FB2 FB3 FB1 AFB1 AFB2 AFG1 AFG2 ENNB ENNB1 ENNA1 ENNA BEA

clean-up with C18 powder clean-up with C18 cartridge

clean-up with CN cartridge clean-up with C18 cartridge, the eluting with methanol
without clean-up step

Fig. 3 Recovery rates obtained applying a clean-up step using C18 powder, C18 cartridge, CN cartridge, and without clean-up
Food Anal. Methods

Table 2 LOQ and LOD (micrograms per kilogram), average recoveries (percent), and repeatability (%RSD), of the selected mycotoxins

Mycotoxins LODs (μg/kg) LOQs (μg/kg) Recovery (%) Repeatability (%)

Dates Apricots Raisins Figs Prunes Intraday Interday

AFB1 0.08 0.2 89±3 84±2 62±8 83±3 60±4 9 12

AFB2 0.08 0.2 99±15 101±14 86±6 92±8 97±13 4 13
AFG1 0.16 0.5 83±4 78±1 70±8 96±12 63±20 5 10
AFG2 0.3 0.9 89±7 118±16 85±4 83±2 117±20 4 14
T-2 1.60 5 135±8 90±13 130±12 83±6 114±5 2 17
HT-2 5 15 107±4 85±4 92±10 101±11 101±7 3 20
DAS 15 45 94±4 93±4 96±15 81±7 109±16 4 20
Enn A 0.3 1 62±3 71±7 61±11 63±8 64±19 3 16
Enn A1 0.3 1 60±3 81±6 79±10 61±7 67±1 2 17
Enn B 0.3 1 64±5 86±4 78±6 68±6 87±1 4 20
Enn B1 0.3 1 60±4 81±7 71±7 66±8 77±2 2 20
FB1 5 15 62±2 72±9 64±1 64±4 84±1 3 12
FB2 5 15 73±3 100±15 69±2 84±3 88±5 6 13
FB3 5 15 62±2 81±9 76±3 107±5 91±2 5 9
OTA 3 9 87±3 90±12 75±4 81±1 105±5 4 18
BEA 7 20 43±4 49±5 42±8 45±6 41±1 3 20

Recovery and repeatability data was obtained with spiked sample at 5 μg kg−1 of aflatoxins and Enns; 20 μg kg−1 of T-2 and OTA; 50 μg kg−1 of BEA;
FB1, FB2, and FB3; DAS; and HT-2

three concentration levels: Enn B, Enn B1, Enn A and Enn lower spiked level). The low recovery does not prevent a reliable
A1, and AFB1, AFB2, AFG1, and AFG2 (20, 10, 5 μg kg-1); determination of these compounds in the real samples because of
T-2 and OTA (80, 40, 20 μg kg-1); BEA, FB1, FB2 and FB3, the other validation parameters, especially repeatability (Relative
DAS and HT-2 (200, 100, 50 μg kg-1). As can be observed in standard deviations (RSD) <20 %) and sensitivity (limits of
Table 2, the recoveries were higher than 60 % for all analytes, quantification (LOQs) <20 μg kg−1), were acceptable. Recover-
except for BEA, which yielded recoveries bellow 41 % (at the ies were higher on apricots and prunes than in dates, figs and

Table 3 Evaluation of matrix ef-

fect: comparison of the calibration Mycotoxins Slope R2 SSE (%)
curves slopes correlation coeffi-
cient (r2) and calculation of signal Solvent Matrix matched Solvent Matrix matched
suppression/enhancement SSE
(percent) for the selected Afla B1 4×106 2×106 0.9992 0.9981 50
6
mycotoxins Afla B2 3×10 1×106 0.9987 0.9979 33
Afla G1 2×106 1×106 0.9997 0.9983 50
Afla G2 2×106 424,159 0.9984 0.9934 21
T-2 85,880 68,453 0.9998 0.9933 80
HT2 59,262 47,490 0.9997 0.9911 80
DAS 203,231 74,318 0.9983 0.9959 37
Enn A 2×107 1×107 0.9981 0.9904 50
Enn A1 3×107 2×107 0.9987 0.9922 67
Enn B 4×107 5×107 0.9997 0.9931 125
Enn B1 9×106 8×106 0.9995 0.9904 89
FB1 841,347 989,609 0.9992 0.9973 118
FB2 1×106 2×106 0.9992 0.9995 200
FB3 696,155 1×106 0.9996 0.9976 144
OTA 185206 156125 0.9995 0.9904 84
BEA 2×107 3×107 0.9999 0.9861 150
 Food Anal. Methods

XIC of +MRM (48 pairs): 313,1/241,0 amu Expected RT: 8,5 ID: AFTB1_313_241q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Sp... Max. 8,1e4 cps. XIC of +MRM (48 pairs): 315,1/286,9 amu Expected RT: 8,3 ID: AFTB2_315_287Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo S... Max. 2,2e4 cps. XIC of +MRM (48 pairs): 329,0/243,1 amu Expected RT: 8,1 ID: AFTG1_329_243Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo S... Max. 1,5e4 cps.

8,43 8,25 8,00

2,2e4 1,5e4
8,0e4
2,1e4 1,4e4
7,5e4 2,0e4
1,3e4
7,0e4 1,9e4

1,8e4 1,2e4
6,5e4
1,7e4
6,0e4 1,1e4

AFB1 AFB2
1,6e4

5,5e4

5,0e4
1,5e4

1,4e4

1,3e4
1,0e4

9000,0
AFG1

In t e n s i ty , c p s

In t e n s i ty , c p s
In t e n s i ty , c p s

4,5e4 1,2e4 8000,0

1,1e4
4,0e4
7000,0
1,0e4
3,5e4
9000,0 6000,0
3,0e4 8000,0
5000,0
7000,0
2,5e4
6000,0 4000,0
2,0e4
5000,0
3000,0
1,5e4 4000,0

3000,0 2000,0
1,0e4
2000,0
5000,0 1000,0
1000,0

0,0 0,0 0,0

6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 10,8 11,0 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6 9,8 10,0 10,2
Time, min Time, min Time, min

XIC of +MRM (48 pairs): 331,1/313,1 amu Expected RT: 7,8 ID: AFTG2_331_313Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo S... Max. 1,8e4 cps. XIC of +MRM (48 pairs): 442,2/262,8 amu Expected RT: 9,3 ID: HT2_442_263Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spra... Max. 7,3e4 cps. XIC of +MRM (48 pairs): 484,3/215,1 amu Expected RT: 10,0 ID: T-2_484_215Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spra... Max. 1,0e5 cps.

7,84 9,19 9,89

7,3e4 1,04e5
1,8e4
7,0e4 1,00e5
1,7e4
9,50e4
6,5e4
1,6e4 9,00e4

1,5e4 6,0e4 8,50e4

1,4e4 8,00e4
5,5e4

1,3e4

1,2e4
AFG2 5,0e4
HT-2
7,50e4

7,00e4
T-2
4,5e4 6,50e4
1,1e4
In t e n s i ty , c p s

In t e n s i ty , c p s
In t e n s i ty , c p s

6,00e4
4,0e4
1,0e4
5,50e4

9000,0 3,5e4 5,00e4

8000,0 4,50e4
3,0e4
7000,0 4,00e4
2,5e4 3,50e4
6000,0
3,00e4
2,0e4
5000,0
2,50e4
4000,0 1,5e4
2,00e4

3000,0 1,50e4
1,0e4

2000,0 1,00e4
5000,0
1000,0 5000,00

0,0 0,00
0,0 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 8,8 9,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4 11,6
6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6 Time, min Time, min
Time, min

XIC of +MRM (48 pairs): 404,3/239,0 amu Expected RT: 11,2 ID: OTA_404_239Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spr... Max. 2,6e4 cps. XIC of +MRM (48 pairs): 384,0/307,2 amu Expected RT: 8,3 ID: DAS_384_307Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spr... Max. 8364,5 cps. XIC of +MRM (48 pairs): 722,2/334,2 amu Expected RT: 8,8 ID: FB1_722_334Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spra... Max. 3,2e4 cps.

11,19 8,26 8,73

2,6e4 8364
3,2e4
8000
2,4e4
3,0e4
7500
2,2e4 2,8e4
7000
2,6e4
2,0e4 6500

1,8e4 OTA 6000

5500
DAS 2,4e4

2,2e4
FB1
1,6e4
2,0e4
5000
In t e n s i ty , c p s

In t e n s i ty , c p s

In t e n s i ty , c p s
1,4e4 1,8e4
4500

1,6e4
1,2e4 4000

1,4e4
3500
1,0e4
1,2e4
3000
8000,0
2500 1,0e4

6000,0 8000,0
2000

4000,0 1500 6000,0

1000 4000,0
2000,0
500 2000,0

0,0
9,8 10,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4 11,6 11,8 12,0 12,2 12,4 12,6 12,8 13,0 13,2 0 0,0
Time, min 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6 9,8 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4
Time, min Time, min

XICof +MRM(48 pairs): 706,2/336,3 amu Expected RT: 10,4 ID: FB2_706_336Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spr... Max. 2,4e4 cps. XIC of +MRM(48 pairs): 699,4/210,1 amu Expected RT: 17,1 ID: ENA_699_210Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spr... Max. 9,5e4 cps. XIC of +MRM(48 pairs): 685,4/210,2 amu Expected RT: 17,3 ID: ENA1_685_210Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo S... Max. 1,2e5 cps.

9,58 17,84 17,10

2,4e4 9,5e4 1,19e5
1,15e5
2,3e4 9,0e4
1,10e5
2,2e4
8,5e4 1,05e5
2,1e4
8,0e4 1,00e5
2,0e4

1,9e4
1,8e4
FB3 7,5e4

7,0e4
9,50e4

9,00e4

8,50e4
1,7e4
6,5e4 8,00e4
1,6e4 10,36
1,5e4 6,0e4
ENNA 7,50e4

7,00e4
ENNA1
In t e n s i ty , c p s
In t e n s ity , c p s

1,4e4
In t e n s ity , c p s

5,5e4
1,3e4 6,50e4

FB2
5,0e4
1,2e4 6,00e4
4,5e4 5,50e4
1,1e4
1,0e4 4,0e4 5,00e4

9000,0 4,50e4
3,5e4
8000,0 4,00e4
3,0e4
7000,0 3,50e4

6000,0 2,5e4 3,00e4

5000,0 2,0e4 2,50e4

4000,0 2,00e4
1,5e4
3000,0 1,50e4
1,0e4
2000,0 1,00e4
1000,0 5000,0 5000,00
0,0 0,0 0,00
8,4 8,6 8,8 9,0 9,2 9,4 9,6 9,8 10,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4 11,6 11,8 15,6 15,8 16,0 16,2 16,4 16,6 16,8 17,0 17,2 17,4 17,6 17,8 18,0 18,2 18,4 18,6 18,8 19,0 19,2 15,0 15,2 15,4 15,6 15,8 16,0 16,2 16,4 16,6 16,8 17,0 17,2 17,4 17,6 17,8 18,0 18,2 18,4 18,6 18,8 19,0 19,2 19,4 19,6
Time, min Time, min Time, min

XIC of +MRM(48 pairs): 657,3/196,1 amu Expected RT: 15,3 ID: ENB_657_196Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo Spr... Max. 4,3e5 cps. XIC of +MRM(48 pairs): 671,2/214,1 amu Expected RT: 16,2 ID: ENB1_671_214Q from Sample 12 (D 200-500) of 06-13-2013 ines.wiff (Turbo S... Max. 4,8e4 cps.
XIC of +MRM(48 pairs): 801,2/244,1 amu Expected RT: 16,6 ID: BEA_801_244q from Sample 165 (F BLANK ) of 06-13-2013 ines.wiff (Turbo Spr... Max. 3,1e4 cps.

15,32 16,24
4,3e5 4,8e4 16,73
3,1e4
4,2e5 4,6e4
3,0e4
4,0e5 4,4e4

4,2e4 2,8e4
3,8e5

3,6e5 4,0e4
2,6e4
3,8e4
3,4e5
3,6e4 2,4e4

ENNB1
3,2e5

3,0e5

2,8e5
ENNB 3,4e4

3,2e4

3,0e4
2,2e4

2,0e4
BEA
2,6e5
2,8e4
In t e n s ity , c p s
In t e n s ity , c p s

In t e n s i ty , c p s

1,8e4
2,4e5 2,6e4
2,2e5 2,4e4 1,6e4

2,0e5 2,2e4
1,4e4
1,8e5 2,0e4

1,8e4 1,2e4
1,6e5
1,6e4
1,4e5 1,0e4
1,4e4
1,2e5
1,2e4 8000,0
1,0e5
1,0e4
8,0e4 6000,0
8000,0
6,0e4 6000,0 4000,0
4,0e4 4000,0
2000,0
2,0e4 2000,0

0,0 0,0 0,0

13,0 13,2 13,4 13,6 13,8 14,0 14,2 14,4 14,6 14,8 15,0 15,2 15,4 15,6 15,8 16,0 16,2 16,4 16,6 16,8 17,0 17,2 17,4 14,6 14,8 15,0 15,2 15,4 15,6 15,8 16,0 16,2 16,4 16,6 16,8 17,0 17,2 17,4 17,6 17,8 18,0 18,2 18,4 18,6 14,8 15,0 15,2 15,4 15,6 15,8 16,0 16,2 16,4 16,6 16,8 17,0 17,2 17,4 17,6 17,8 18,0 18,2
Time, min Time, min Time, min

Fig. 4 LC/ESI (+)/MS/MS chromatogram of a spiked blank plum sample (20 mg kg−1 of AFs and ENNs; 80 μg kg−1 of OTA and T-2 and 200 μg kg−1
of DAS, HT-2, and FBs and BEA)
Food Anal. Methods

raisins. These differences come out because the recovery of to evaluate the matrix effects. The signal suppression/
these mycotoxins strongly depends on the matrix type, and enhancement (SSE) was calculated according to Eq. 1 defined
acetonitrile extract is efficient only for some matrixes. More- by Sulyok et al. 2006 for each mycotoxin (see Table 3).
over, analysis methods for a wide range of compounds
usually require a compromise because the selected extraction SSE ¼ ðslope matrix‐matched calibration= ð1Þ
and clean-up conditions could not be the optimum for all the slope standard calibration in solventÞ  100
mycotoxins.
For repeatability and intermediate precision studies of the To evaluate this parameter, a commercial sample of prunes
LC/MS/MS procedure, five replicate determinations on the was used as being representative of the complexity of dried
same day and on five different days of a standard solution fruits.
were carried out. RSDs ranged from 2 to 9 % for run-to-run Significant signal suppression (SSE, 21–89 %) was ob-
precision and from 9 to 20 % for the day-to-day precision. The served for most mycotoxins whereas five of the mycotoxins
limit of detection (LODs) and LOQs for each mycotoxin were showed a significant signal enhancement (SSE, 118–200 %).
obtained from the signal-to-noise ratio. The LOD achieved for
this method range from 0.08 to 15 μg/kg and the LOQ range Analysis of Commercially Available Samples
from 0.2 to 45 μg/kg. The levels of LOQ are lower than the
maximum residue limits set by EU for the studied mycotoxins. A total of 15 domestic and imported dried fruits (three of each
The results obtained demonstrated a good linearity for all the fruit) available in Spanish markets were analyzed using our
mycotoxins within the tested interval, with correlation coeffi- developed method. The results are summarized in Table 4. In
cients higher than 0.998 for the entire target compounds, as none of the dried plum samples tested were any of the target
shown in Table 3. mycotoxins detected and they were not included in Table 4. In
Figure 4 shows a LC/ESI (+)/MS/MS chromatograms of a the rest of the analyzed samples, AFG2, OTA, HT2, Enns (B,
spiked blank plum sample. B1, and A1) and BEA were detected. Out of the 15 analyzed
Matrix effect depends on the analyte as well as the matrix, samples, 8 were found to be contaminated with at least one
which makes it necessary to determinate ion suppression/ mycotoxin. Enn B (seven positive samples) and Enn A1 (four
enhacement for every matrix–mycotoxin combination. Matrix positive samples) were the most commonly detected myco-
compounds could interfere the quantitative analysis of the toxins. The highest level of mycotoxins was detected in figs
compounds at trace levels, as well as greatly affect the method (227.7 μg kg−1 of Enn B).
accuracy and reproducibility. In the majority of the published Very little literature is available on emerging Fusarium
multi-mycotoxin methods that employ ESI, significant matrix mycotoxins in fruits although Fusarium strains (Fusarium
effects, have been observed (Desmarchelier et al. 2010; ramigenum, Fusarium solani, Fusarium proliferatum, and
Yogendrarajah et al. 2013). Table 3 shows the calibration Fusarium oxysporum) were isolated from dried fruits in sev-
curves obtained using six concentration levels, between eral investigations (Heperkan et al. 2012; Moretti et al. 2010).
LOQ and 100 times LOQ. The slope of the standard addition The results obtained in the present study are comparable to
plot was compared with the slope of standard calibration plot those obtained by Tolosa et al. (2013) in which the incidence

Table 4 Mycotoxins detected in

analyzed samples Samples Mycotoxin concentration (μg kg−1)

AFG2 OTA HT2 Enn B Enn B1 Enn A1 BEA

Dates D1
D2 18.5
D3 53.1 2.2
Raisins R1 21.4
R2 4.3 4.9 10.2
R3
Figs F1 227.7 39.9 8.1 18.2
F2 150.3 30 6.9 131.8
F3
Apricots A1 43.6
A2
A3 53.3 2.7
 Food Anal. Methods

of Enn A, A1, B, and B1) and BEA in different dried fruits (13 Ediage EN, Di Mavungu JD, Monbaliu S, Peteghem VC, De Saeger S
(2011) A validated multianalyte LC-MS\MS method for quantifica-
samples) were assayed and 52.7 % of the samples were found
tion of 25 mycotoxins in casava flour, peanut cake and maize
to be contaminated with at least one of the four Enns with a samples. J Agric Food Chem 59:5173–5180
mean ranging from 0.025 to 0.666 mg kg−1 for Enns and Fernández-Cruz ML, Mansilla ML, Tadeo JL (2010) Mycotoxins in fruits
0.006 mg kg−1 for BEA. and their processed products: analysis, occurrence and health impli-
cations. JAR 1(2):113–122
Ferreira I, Fernandes JO, Cunha SC (2012) Optimization and validation
of a method based in a QuEChERS procedure and gas chromatog-
Conclusions raphy–mass spectrometry for the determination of multi-mycotoxins
in popcorn. Food Control 27(1):188–193
Garrido Frenich A, Vidal Martínez JL, Romero-González R, Aguilera-
The liquid chromatography coupled to mass spectrometry/
Luiz M (2009) Simple and high-throughput method for the
mass (LC/MS/MS) analysis allowed to identify unambiguous- multimycotoxin analysis in cereals and related foods by ultra-high
ly and quantify the 16 studied mycotoxins. The optimized performance liquid chromatography/tandem mass spectrometry.
QuEChERS extraction procedure was proved to be a fast Food Chem 117:705–712
Garrido Frenich AG, Romero-González R, Gómez-Pérez ML, Vidal JL
and efficient extraction method for the analysis of mycotoxins
(2011) Multi-mycotoxin analysis in eggs using a QuEChERS-based
in dried vine fruits, apricots, figs and plums. The results are so extraction procedure and ultra-high-pressure liquid chromatography
far satisfactory and a fully validated extraction and analysis coupled to triple quadrupole mass spectrometry. J Chromatogr A
method have been set in this study. 1218(28):4349–4356
Heperkan D, Karbancioglu Güüler F, Oktay HI (2012) Mycoflora and
natural occurrence of aflatoxin, cyclopiazonic acid, fumonisin and
Acknowledgments This work was supported by the Spanish Ministry
ochratoxin A in dried figs. Food Addit Contam A 29:277–286
of Science and Innovation (AGL2010-17024/ALI).
Karbancıoglu-Guler F, Heperkan D (2008) Natural occurrence of ochra-
toxin A in dried figs. Anal Chim Acta 617:32–36
Conflict of Interest Ines Azaiez has no conflict of interest. Federica Knudsen PB, Mogensen JM, Larsen TO, Nielsen KF (2011) Occurrence
Giusti has no conflict of interest. Gianni Sagratini has no conflict of of fumonisins B(2) and B(4) in retail raisins. J Agric Food Chem
interest. Jordi Mañes has no conflict of interest. Mónica Fernández has 59(2):772–776
no conflict of interest. This article does not contain any studies with Köppen R, Koch M, Siegel D, Merkel S, Maul R, Nehls I (2010)
human or animal subjects. Determination of mycotoxins in foods: current state of analytical
methods and limitations. Appl Microbiol Biotechnol 86:1595–1612
Moretti A, Ferracane L, Somma S, Ricci V, Mulèè G, Susca A, Ritieni A,
Logrieco AF (2010) Identification, mycotoxin risk and pathogenic-
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