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X. Niu, T. Emmanuel K., Z. Zhao and Z. Han, Anal. Methods, 2020, DOI: 10.1039/D0AY01787F.
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TECHNICAL NOTE
Kássio M. G. Lima et al.
ATR-FTIR spectroscopy with chemometric algorithms of multi-
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 Page 1 of 19 Analytical Methods

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A Reliable and Accurate UHPLC-MS/MS Method for
View Article Online
DOI: 10.1039/D0AY01787F
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Screening of Aspergillus, Penicillium and Alternaria
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9 mycotoxins in Orange, Grape and Apple Juices
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Analytical Methods Accepted Manuscript

13 Wenbo Guo1,‡, Junhua Yang1,‡, Xueke Niu1, Emmanuel K. Tangni2, Zhihui Zhao1,
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15 Zheng Han1,*
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1 Institute for Agro-food Standards and Testing Technology, Shanghai Key Laboratory of
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23 Protected Horticultural Technology, Shanghai Academy of Agricultural Sciences, Shanghai
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25 201403, China
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27 2 Organic Contaminants and Additives, Sciensano, Leuvensesteenweg 17, Tervuren 3080,
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29 Belgium; emmanuel.tangni@sciensano.be
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35 *Correspondence: hanzheng_ok@163.com
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37 ‡ These authors contributed equally to this work.
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 Analytical Methods Page 2 of 19

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4 Abstract DOI: 10.1039/D0AY01787F

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8 An ultra-high performance liquid chromatography–tandem mass spectrometry
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10 (UHPLC-MS/MS) method was developed for simultaneous determination of 15
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12 mycotoxins, including aflatoxins (B1, B2, G1, G2), ochratoxins (A, B, C), citrinin, patulin,

Analytical Methods Accepted Manuscript

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14 and the emerging Alternaria toxins (alternariol, alternariol monomethyl ether, altenuene,
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16 tentoxin, tenuazonic acid, altenusin) in orange, grape and apple juices. Different extraction
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18 approaches, sorbents, chromatographic columns and mobile phases were investigated for
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20 establishment of the optimal QuEChERS procedure and UHPLC-MS/MS conditions.
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22 Recoveries were in the range of 74-110%, the limits of detection (LODs) and limits of
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24 quantification (LOQs) ranged from 0.05 to 0.1 ng mL−1 and from 0.1 to 5.0 ng mL−1,
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26 respectively. Matrix effects were evaluated and matrix-matched calibration curves were
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28 used to compensate for matrix effects and achieve an accurate quantification. The
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30 correlation coefficients (R2) of the linearity were higher than 0.99 and the relative standard
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32 deviations (RSDs) of intra- and inter-day precision were under 13%. The method was
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34 subsequently applied to 22 fruit juice samples. The high frequencies (90.9%) of mycotoxins
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36 not only proved the reliability and sensitivity of the currently established method, but also
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38 demonstrated that fruit juices are susceptible to different mycotoxins, which need to be
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40 continuously monitored in the future.
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Keywords: Mycotoxins; Juices; Screening; UHPLC-MS/MS
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 Page 3 of 19 Analytical Methods

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3 1 Introduction View Article Online
DOI: 10.1039/D0AY01787F
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5 Fruit juices, especially orange, grape and apple juices, are favored by more and more
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7 consumers around the world due to its superior color, taste and nutrition1. However, as
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reported, these products are susceptible to various toxigenic fungi, i.e., Aspergillus spp.,
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10 Penicillium spp., and Alternaria spp, when they are not well processed or improperly
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12 stored2. It is noteworthy that these toxigenic fungi usually could produce different

Analytical Methods Accepted Manuscript

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14 mycotoxins3. As the most important secondary metabolites of Aspergillus spp., aflatoxin B1
15 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2), classified in
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17 group I as human carcinogens by the International Agency for Research on Cancer (IARC),
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19 have been found in apple juice and orange4, 5. Ochratoxin A (OTA), ochratoxin B (OTB)
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and ochratoxin C (OTC) could be produced by Aspergillus spp. and Penicillium spp., while
22 citrinin (CIT) and patulin (PAT) are produced by Penicillium spp., which are all frequently
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24 found in apple, orange or grape samples and their products6, 7. Alternaria toxins, the
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26 emerging mycotoxins produced by Alternaria spp, are frequently found in fruits and
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vegetables among which, the most important members include alternariol (AOH),
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29 alternariol monomethyl ether (AME), altenuene (ALT), altenusin (ALS), tentoxin (TEN),
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and tenuazonic acid (TeA) 10. Acute and chonic ingestion of mycotoxins could pose high
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33 potential risks to humans such as carcinogenesis, teratogenesis, nephrotoxicity,
34 hepatotoxicity, as well as reproductive and developmental toxicities 11-13. To minimize the
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36 risks of mycotoxins, the European Commission (EU), USA, Canada and China have
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38 established the maximum levels (MLs) of 50 μg kg-1 for PAT and 2 μg kg-1 for OTA in fruit
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juices, respectively.
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41 Analysis of mycotoxins in fruit juices is difficult due to their complex matrices
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43 consisting of various chemical components. Several analytical methods, i.e., countercurrent
44 14,
45 chromatography (CCC) high-performance liquid chromatography (HPLC) with
46 ultraviolet (UV) and fluorescence (FL) detector 15, 16, and high-performance liquid
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48 chromatography tandem mass spectrometry (HPLC-MS/MS) have been used for the
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detection of mycotoxins in fruit juices 17-19. The methodology of HPLC-MS/MS achieves
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its preferred status due to the high sensitivity, simple sample preparation and their
53 compatibility with almost the whole range of compound polarities. Nevertheless, most of
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55 the previous studies were only directed toward a single class of mycotoxins in fruits juices,
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57 as a consequence, the same sample needs to be analyzed multiple times to cover all relevant
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analytes. Therefore, within the field of mycotoxin analysis in fruit juices, a clear trend
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60 toward the establishment of multi-analytes method can be seen.
 Analytical Methods Page 4 of 19

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3 The increasing need for multiple mycotoxins analysis in fruit juices has promoted the
View Article Online
DOI: 10.1039/D0AY01787F
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5 research on the efficient cleanup procedures considering the co-extractive interferences
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7 such as esters, sugars and pigments. Several sample clean-up techniques including
8 20, 21
solid-phase extraction (SPE) immunoaffinity column (IAC) and multifunctional
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10 purification column 22, have been used for mycotoxins purification in previous researches,
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12 which are frequently expensive and time- and solvent-consuming. Recently, a quick, easy,

Analytical Methods Accepted Manuscript

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14 cheap, effective, rugged and safe simple, rapid and effective method (QuEChERS), based
15 on a simple sample extraction using methanol, acetonitrile, water or their mixtures followed
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17 by the addition of salts and adsorbents, has been established and widely used for the
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19 determination of mycotoxins in crops and feed samples 23-25.
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The aim of this study is to develop and validate a modified QuEChERS-based
22 ultra-high performance liquid chromatography tandem mass spectrometry
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24 (UHPLC-MS/MS) method for simultaneous determination of the frequently found
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26 Aspergillus, Penicillium and Alternaria mycotoxins in fruit juices, to reveal the real
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contamination situations of multiple mycotoxins in these products.
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31 2 Materials and Methods
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33 2.1 Samples
34 A total of 22 fruit juice samples (9 orange juice samples, 7 grape juice samples and 6
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36 apple juice samples) were randomly collected from the local supermarkets in Shanghai. All
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38 samples were stored at 4 °C until analysis.
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2.2 Chemicals and reagents
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41 Acetonitrile and methanol (LC-MS grade) were purchased from Merck (Darmstadt,
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43 Germany). Formic acid (HPLC grade), ammonium acetate (HPLC grade), anhydrous
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45 magnesium sulfate (MgSO4, analytical grade), sodium chloride (NaCl, analytical grade),
46 primary-secondary amine (PSA), graphitized carbon black (GCB) and octadecylsilyl (C18)
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48 were purchased from ANPEL (Shanghai, China). Pectinase with the activity over than 50 U
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50 mg-1 was purchased from Titan (Shanghai, China). The pectinase was dissolved in water to
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prepare the pectinase solutions with the concentration of 20 mg mL-1. Ultrapure water was
53 prepared by a Millipore system (Millipore, Billerica, MA, USA).
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55 The analytical standards of AFB1 (2.03 μg mL-1), AFB2 (0.505 μg mL-1), AFG1 (2.01
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57 μg mL-1), AFG2 (0.508 μg mL-1), OTA (10.23 μg mL-1), OTB (10.1 μg mL-1), PAT (10 μg
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mL-1), AOH (100.3 μg mL-1), AME (102.3 μg mL-1), TEN (100.4 μg mL-1), TeA (101.1 μg
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60 mL-1), and CIT (100.3 μg mL-1) were purchased from Romer labs (Union, MO, USA).
 Page 5 of 19 Analytical Methods

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3 Solid portions of OTC (99.1%), ALT (98.0%) and ALS (99.4%) were purchased from
View Article Online
DOI: 10.1039/D0AY01787F
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5 AdipoGen (Liestal, Basel, Switzerland). The solid standards were dissolved in acetonitrile
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7 to prepare the stock solutions with the concentration of 100 μg mL-1. Subsequently, a mixed
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working solution with the concentrations of 100 ng mL-1 for AFB1 and AFG1, 25 ng mL-1
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10 for AFB2 and AFG2, and 1000 ng mL-1 for OTA, OTB, OTC, CIT, PAT, TEN, TeA, ALS,
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12 ALT, AOH and AME, was prepared. The working solution was stored at -20 °C in brown

Analytical Methods Accepted Manuscript

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14 glass vials.
15 2.3 UHPLC–MS/MS analysis
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17 UHPLC-MS/MS analysis was performed on a Waters ACQUITY UPLC system
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19 (Waters, Milford, MA, USA). Chromatographic separation was achieved on an Agilent
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Poroshell EC18 column (3.0 ×100 mm, 2.7 μm) with methanol (A) and 5 mmol L-1
22 ammonium acetate (B) as the mobile phase. The flow rate was 0.4 mL min-1 and the
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24 gradient elution procedure was applied as follows: initial 10% A; 0.5 min, 10% A; 1.5 min,
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26 50% A; 5.0 min, 90% A; 6.0 min, 90% A; 6.2 min, 10% A; 8.0 min, 10% A. The injection
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volume was 3 μL and the column temperature was 40 °C.
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29 AB SCIEX Triple Quad TM 5500 mass spectrometer (AB Sciex, Foster City, CA,
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31 USA) was used for MS/MS analysis in positive electrospray ionization mode (ESI+) and in
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33 negative electrospray ionization mode (ESI-). The parameters for MS/MS detection were
34 set as follows: ion spray voltage, 5500 V (ESI+) and 4500 V (ESI-); source temperature, 500
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36 °C (ESI+) and 450 °C (ESI-); ion source gas 1 (GS1), 50 Psi; ion source gas 2 (GS2), 50
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38 Psi; collision gas (CAD), 8 Psi. Multiple reaction monitoring (MRM) mode was used for
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quantification and confirmation of each mycotoxin with the parameters shown in Table 1.
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41 2.4 Sample preparation
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43 An aliquot (2 mL) of each homogenized fruit juice was transferred into a 50 mL
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45 centrifuge tube and 10 mL of acetonitrile/water (84/16, v/v) was used for extraction. For the
46 juices containing small suspended particles, the samples should be pre-added with 50 μL
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48 pectinase and kept in dark for 2 h at room temperature. The mixture was shaken at 180 rpm
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50 for 20 min and ultrasonicated for 20 min. Then, 2 g NaCl and 100 mg C18 were added to the
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slurry, severely shaken for 30 s immediately, and centrifuged at 5000×g for 5 min.
53 Subsequently, 6.0 mL of the supernatant was evaporated and the residues were re-dissolved
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55 with 1 mL of methanol/water containing 5 mmol L-1 ammonium acetate (20/80, v/v) and
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57 filtered through a 0.22 μm PTFE membrane filter for injection.
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2.5 Method validation
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 Analytical Methods Page 6 of 19

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3 The mycotoxins-free juices were used as the blank samples for method validation. The
View Article Online
DOI: 10.1039/D0AY01787F
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5 linearity was evaluated in neat solvent and in different fruit juices with the spiked
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7 mycotoxins at the concentrations of 0.1-20 ng mL-1 for AFB1, AFB2, AFG1, AFG2, 0.1-100
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ng mL-1 for OTA, OTB, OTC, TEN, TeA, ALT, and 1-200 ng mL-1 for AOH, AME, ALS,
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10 CIT, PAT, respectively. The calibration curves were constructed by plotting the responses
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12 versus analyte concentrations and the acceptable criteria of R2 was ≥0.99. The limits of

Analytical Methods Accepted Manuscript

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14 quantification (LOQs) were the lowest concentration point of the calibration curves for
15 different fruit juices, which were usual calculated with a theoretical signal to noise (S/N) of
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17 10. The limit of detection (LODs) were the lowest concentration that could be determined
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19 and calculated with a theoretical signal to noise (S/N) of 3. Matrix effect was calculated
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according to equation (1), in which, the slopematrix was the slope of matrix-matched
22 calibration curve prepared in the blank fruit juices, and slopesolvent was the slope of standard
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24 calibration curve prepared in the neat solvent. The positive and negative values were
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26 representative for signal suppression and signal enhancement, respectively.
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28 slopesolvent  slopematrix
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Matrix effect (%) =  100 (1)
30 slopesolvent
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32 Recoveries and precisions were evaluated in blank samples spiked with low,
33 intermediate, and high levels (1, 5, and 20 ng mL-1 for aflatoxins, and 5, 50 and 100 ng
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35 mL-1 for the others) in six replicates. Simultaneously, the relative standard deviations
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37 (RSDs) of low, intermediate, and high spiked levels in the same day and in five successive
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days were used for evaluation of the intra-day precision and inter-day precision,
40 respectively.
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44 3 Results and Discussion
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3.1 Optimization of the MS/MS conditions
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47 MS/MS parameters were optimized by continuous injection of each mycotoxin
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49 standard solution using syringe pump at a concentrations of 50-500 ng mL-1. Q1 scan and
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51 product ion scan were employed to obtain the precursor ions and product ions in ESI+ or
52 ESI-, respectively. The declustering potential (DP), entrance potential (EP), collision energy
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54 (CE) and collision cell exit potential (CXP) were optimized for quantification and
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56 confirmation transitions. The optimal parameters including the precursor ions, product ions,
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DP, EP, CE, CXP are shown in Table 1. Most mycotoxins showed a higher sensitivity and
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59 lower background signal interferences in ESI+ mode, except for PAT, ALS and TeA which
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got more satisfactory responses in ESI- mode (Figure 1).
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Page 7 of 19 Analytical Methods

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4 Table 1 Mass parameters for 15 mycotoxins.
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Retention time Precursor ions Product ions DP EP CE CXP
6 Mycotoxins
7 (min) (m/z) (m/z) (V) (V) (V) ( V)
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Analytical Methods Accepted Manuscript

9 AFB1 3.88 313.1 [M+H]+ 241.2*/269.0 84 10 50/43 10
10 AFB2 3.74 329.0 [M+H]+ 287.0*/259.0 52 9 34/38 10
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12 AFG1 3.54 329.0 [M+H]+ 243.0*/311.0 45 10 35/38 8
13 AFG2 3.41 331.0 [M+H]+ 285.0*/245.0 62 9 40/37 13
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15 OTA 4.23 404.0 [M+H]+ 358.0*/239.0 71 10 26/30 11
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OTB 3.66 370.0 [M+H]+ 205.0*/187.1 76 10 28/48 7
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18 OTC 5.82 432.0 [M+H]+ 358.1*/238.9 60 8 25/42 9
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20 CIT 3.86 251.0 [M+H]+ 233.0*/205.0 60 9 23/35 10
21 PAT 2.45 153.0 [M-H]- 109.0*/81.2 -57 10 -12/-15 7
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23 AOH 4.47 259.1 [M+H]+ 185.1*/213.2 92 8 43/36 9
24 AME 5.63 273.2 [M+H]+ 184.1*/230.2 80 8 50/42 10
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26 ALT 3.85 293.0 [M+H]+ 257.2*/239.1 95 9 20/29 11
27 TEN 4.71 415.0 [M+H]+ 312.2*/256.4 95 10 30/40 10
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29 TeA 2.61 196.1 [M-H]- 139.0*/119.9 -102 8 -30/-30 8
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ALS 2.96 288.8 [M-H]- 245.1*/230.1 -120 10 -8/-8 9
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32 * Primary product ion; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential
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 Analytical Methods Page 8 of 19

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Analytical Methods Accepted Manuscript

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each mycotoxin.
 Page 9 of 19 Analytical Methods

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DOI: 10.1039/D0AY01787F
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5 3.2 Optimization of the UHPLC conditions
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To achieve a base-line separation and high sensitivity for multiple mycotoxins,
8 gradient elution program with different mobile phases (methanol, acetonitrile, water) and
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10 additives (formic acid, ammonium acetate), as well as various chromatographic columns
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12 with different stationary phases and sizes were thoroughly optimized. Compared to

Analytical Methods Accepted Manuscript

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acetonitrile, most targeted mycotoxins shown better sensitivity when methanol was selected,
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15 especially for AFB1 and OTA (Figure S1). To further enhance the signal responses, 5 mmol
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17 L-1 ammonium acetate and 0.1% formic acid were tested as the additives in the mobile
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19 phase. When 0.1% formic acid was applied, the sensitivity of ALS was poor, and PAT,
20 TeA, CIT could not even be detected, and therefore, 5 mmol L-1 ammonium acetate was
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22 used (Figure S2). Waters ACQUITY UPLC BEH C18 column (2.1 ×100 mm, 1.7 μm),
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24 Agilent Poroshell EC18 column (3.0 ×100 mm, 2.7 μm) and Thermo Hypersil GOLD
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column (2.1 ×100 mm, 1.9 μm) were compared. When Waters ACQUITY UPLC BEH C18
27 column (2.1 ×100 mm, 1.7 μm) or Thermo Hypersil GOLD column (2.1 ×100 mm, 1.9 μm)
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29 was selected, a tailing peak was observed for TeA. Satisfactory resolutions and symmetrical
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31 peaks were achieved when Agilent Poroshell EC18 column (3.0 ×100 mm, 2.7 μm) was
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used (Figure 1).
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36 3.3 Optimization of the QuEChERS method
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38 Various extraction solvents such as methanol, acetonitrile, methanol/water (80/20, v/v),
39 acetonitrile/water (84/16, v/v) and acetonitrile/water/acetic acid (84/15/1, v/v/v) were
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41 evaluated by the spiked samples (5 ng mL-1 for AFB1, AFG1, AFB2, AFG2, and 50 ng mL-1
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43 for the others) (Figure 2). When methanol or acetonitrile was used, poor recoveries,
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especially for AFB1, AFG1 and CIT (11-70%), were obtained due to the improper polarity
46 of these extraction solvents 26. When acetonitrile/water/acetic acid (84/15/1, v/v/v) was
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48 used, satisfactory recoveries (70-98%) were got for the majority of mycotoxins except for
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50 CIT (50-66%) and PAT (57-69%). When methanol/water (80/20, v/v) was used,
51 unsatisfactory recoveries (56-68%) were obtained for AOH and AME. Satisfactory
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53 recoveries in the range of 76-110% were achieved for all targeted mycotoxins when
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55 acetonitrile/water (84/16, v/v) was selected, and therefore was used for the subsequent
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experiments.
58 Several adsorbent materials, i.e., PSA, C18, GCB, MgSO4 and NaCl were investigated
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60 to remove the interferences and improve the purification efficiency. As shown in Figure 3,
although PSA and GCB were frequently used to remove sugar, pigment and organic acids
 Analytical Methods Page 10 of 19

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3 in previous studies 27, 28, unsatisfactory recoveries (23-61%) were obtained for ALS, OTA,
View Article Online
DOI: 10.1039/D0AY01787F
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5 OTB, CIT, AOH, AME and ALT in this study, which could be caused by the ionic affinity
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7 and π–π interactions 29. In addition, because of the strong chelate effects with Mg2+ 30, low
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recoveries (36-58%) were obtained for CIT and TeA when MgSO4 was used. Finally, 2 g
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10 NaCl and 100 mg C18 were used for the clean-up step, resulting in the satisfactory
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12 recoveries of 76–106%.

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58 Figure 2 Extraction efficiencies of different solvents for various mycotoxins in spiked orange juice (A), grape
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 Page 11 of 19 Analytical Methods

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47 Figure 3 Purification efficiencies of different adsorbent materials for various mycotoxins in spiked orange
48 juice (A), grape juice (B) and apple juice (C) samples. The spiked concentrations were 5 ng mL-1 for AFB1,
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52 3.4 Method validation
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54 As shown in Table 2, the correlation coefficients (R2) of the calibration curves
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constructed in neat solvent, orange juice, grape juice and apple juice were greater than 0.99.
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57 High sensitivity was obtained with the LODs and LOQs in the range of 0.05-1 ng mL-1 and
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59 0.1-5 ng mL-1, respectively. Recoveries and precisions for the targeted mycotoxins under
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different spiked levels are listed in Table 3. In details, the recoveries ranged from 76% to
 Analytical Methods Page 12 of 19

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5 juice, respectively. The intra- and inter-day RSDs were in the range of 0.5-1% and 1-13%
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7 for various fruit juices, respectively. The results of recovery and precision tests were highly
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consistent to the criteria regulated in European Commission Regulation (EC) No. 401/2006
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10 and Commission Decision 2002/657/EC, which indicated the acceptable accuracy and
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12 reproducibility of the proposed method.

Analytical Methods Accepted Manuscript

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14 In general, matrix effect is considered tolerable when the extents of the signal
15 suppression/enhancement are lower than 20% 31. As shown in figure 4, matrix effect of
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17 apple juice was relatively lower compared to the other two matrices with the values no
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19 more than 20% for most mycotoxins except for ALS (-22%), AME (-30.1%) and AFB2
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(22%). For grape juice, ten (66.7%) mycotoxins showed strong matrix effects in the range
22 of-82-65%. With regard to orange juice, strong matrix effects were observed for all
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24 mycotoxins with the values ranged from -69% to 81%. Apparently, matrix effects still
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26 remained high for most of mycotoxins in this three matrices although a QuEChERS
27
cleanup step had been applied to remove interferes, i.e., sugars, pigments and organic acids.
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29 To achieve an accurate quantification, matrix-matched calibration curves for different fruit
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31 juices were used.
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Figure 4 Matrix effects of different mycotoxins in apple, grape and orange juices.
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3.5 Real sample analysis
 Page 13 of 19 Analytical Methods

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3 The developed method was applied to 22 fruit juice samples randomly collected from
View Article Online
DOI: 10.1039/D0AY01787F
4
5 the supermarkets in Shanghai, and the contaminations of mycotoxins are listed in Table 4.
6
7 The most frequently detected contaminant was TeA with the incidences (concentration
8
ranges) of 88.9% (0.3-4.1 ng mL-1), 71.4% (1.8-20.3 ng mL-1) and 83.3% (0.4-9.1 ng mL-1)
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10 in orange juice, grape juice and apple juice samples, respectively. PAT, OTA, OTB and
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12 AOH were also detected with the concentrations in the range of 0.9-8.6 ng mL-1, 0.6-1.7 ng

Analytical Methods Accepted Manuscript

13
14 mL-1, 0.9-1.9 ng mL-1 and 1.8-20.3 ng mL-1, respectively. ALT was found only in one apple
15 juice sample with the concentration of 0.6 ng mL-1. The contamination levels of Alternaria
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17 toxins were similar to the previous study, in which, large amounts of TeA and AOH were
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detected in fruit juices with the incidences of 50-100% 32-34. Generally, the survey results
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20
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clearly suggested that fruit juices are easily contaminated by Aspergillus, Penicillium and
22 Alternaria mycotoxins, and more attention should be paid on these mycotoxins to reduce
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24 the health risk to consumers.
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26
27
4 Conclusion
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29 In this study, a simple, rapid and accurate UHPLC-MS/MS strategy based on the
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31 modified QuEChERS approach for simultaneous determination of 15 mycotoxins in various
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33 fruit juices was proposed. The method was carefully validated by determining the linearity,
34 recovery, precision, matrix effect and successfully applied in real fruit juice samples. The
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36 fruit juices were proved to be easily contaminated by different mycotoxins, i.e., PAT, OTA,
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38 TeA, which could pose potential health risks. Thus, continuous monitoring of multiple
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mycotoxins by the current method is considered to be significant to confirm the current
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41 contamination and further ensure the safe consumption of fruit juices.
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Analytical Methods Page 14 of 19

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4 Table 2 Linearity and sensitivity of the targeted mycotoxins in various fruit juices
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Orange juice Grape juice Apple juice
6
7 Calibration Linear range R2 LOD LOQ Calibration curve Linear R2 LOD LOQ Calibration curve Linear R2 LOD LOQ
8 Toxins

Analytical Methods Accepted Manuscript

curve (ng mL-1) (ng (ng range (ng (ng range (ng (ng
9
10 mL-1) mL-1) (ng mL-1) mL-1) mL-1) (ng mL-1) mL-1) mL-1)
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12 AFB1 y=47241x+44492 0.1-20 0.999 0.05 0.1 y=90487x+166365 0.1-20 0.998 0.05 0.1 y=104308x+370924 0.1-20 0.995 0.05 0.1
13 AFB2 y=33784x+26866 0.1-20 0.999 0.05 0.1 y=58751x-11428 0.1-20 0.999 0.05 0.1 y=63746x+31309 0.1-20 0.999 0.05 0.1
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15 AFG1 y=34945x+45758 0.1-20 0.999 0.05 0.1 y=58882x-1007 0.1-20 0.998 0.05 0.1 y=80781x+282853 0.1-20 0.996 0.05 0.1
16
AFG2 y=16777x+21471 0.2-20 0.995 0.1 0.2 y=25671x+17397 0.5-20 0.999 0.2 0.5 y=38073x+97378 0.5-20 0.998 0.1 0.5
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18 OTA y=15971x-5495 0.2-100 0.994 0.1 0.2 y=39106x+2935 0.1-100 0.999 0.05 0.1 y=22077x+3923 0.2-100 0.999 0.1 0.2
19
OTB y=72777x-19847 0.1-100 0.998 0.05 0.1 y=98527x+43432 0.2-100 0.999 0.05 0.2 y=98100x+161076 0.1-100 0.998 0.05 0.1
20
21 OTC y=295033x+418760 0.1-100 0.995 0.05 0.1 y=342025x+500237 0.1-100 0.995 0.05 0.1 y=418412x+87927 0.5-100 0.998 0.2 0.5
22
23 TEN y=38691x+8978 0.1-100 0.999 0.05 0.1 y=54419x+15763 0.1-100 0.999 0.05 0.1 y=54912x+18649 0.1-100 0.997 0.05 0.1
24 TeA Y=10244x+7391 0.5-100 0.996 0.2 0.5 y=15240+258359 0.1-100 0.998 0.05 0.1 y=17268x+41813 0.1-100 0.999 0.05 0.1
25
26 ALT y=99013+27697 0.1-100 0.998 0.05 0.1 Y=129116+150107 0.2-100 0.999 0.05 0.2 y=130969x+171906 0.1-100 0.999 0.05 0.1
27 AOH y=12735x+6118 1.0-200 0.993 0.5 1.0 y=12770x+1413 0.5-200 0.999 0.2 0.5 y=19321x+12142 1.0-200 0.999 0.5 1.0
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29 AME y=1829x-1567 0.5-200 0.997 0.2 0.5 y=1586x+1117 0.5-200 0.996 0.2 0.5 y=1407x+5006 0.5-200 0.994 0.2 0.5
30
ALS y=5351x-19308 0.5-200 0.994 0.2 0.5 y=6417x-54993 1.0-200 0.991 0.05 1.0 y=4305x-7285 1.0-200 0.997 0.5 1.0
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32 CIT y=94144x+20650 0.2-200 0.999 0.1 0.2 y=171792x+14334 0.5-200 0.996 0.1 0.5 y=418412x+87927 0.5-200 0.999 0.1 0.5
33
PAT y=8328x+70959 5.0-200 0.991 1.0 5.0 y=7433x+6535 1.0-200 0.999 0.5 1.0 y=10322x+26305 0.5-200 0.998 0.2 0.5
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4 Table 3 Recoveries and precisions of the 15 mycotoxins in various fruit juices (n=6).
5 Toxins Spiked Orange juice Grape juice Apple juice
6
7 levels Recovery Intra-day Inter-day Recovery Intra-day Inter-day Recovery Intra-day Inter-day
8 (ng mL-1)

Analytical Methods Accepted Manuscript

(Mean±SD, %) precision precision (Mean±SD, %) precision precision (Mean±SD, %) precision precision
9
10
(RSD, %) (RSD, %) (RSD, %) (RSD, %) (RSD, %) (RSD, %)
11 AFB1 1 87.3±2.1 4.6 5.7 79.9±0.9 2.5 4.8 85.6±1.7 3.4 5.7
12
5 98.7±5.8 1.9 3.4 94.3±2.1 0.9 3.6 94.3±2.6 1.5 3.8
13
14 20 83.5±1.4 2.4 2.5 84.6±3.7 3.1 4.9 94.5±6.7 4.0 2.9
15 AFB2 1 93.4±6.5 6.2 11.9 97.5±4.6 2.2 5.7 90.4±3.2 3.5 5.2
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17 5 83.4±4.9 3.5 2.8 87.3±2.4 0.8 1.2 95.7±5.3 2.7 9.0
18 20 102.3±3.5 2.6 10.4 85.6±1.8 3.4 2.5 89.3±4.6 5.6 3.4
19
20 AFG1 1 85.6±5.5 5.7 5.4 80.5±1.7 5.8 6.1 90.8±3.5 0.9 2.9
21 5 84.5±6.2 1.9 3.2 95.6±2.8 3.6 7.0 95.6±2.7 1.8 6.7
22
23 20 90.6±3.6 3.3 4.5 100.4±7.5 1.6 1.2 89.9±6.6 0.8 1.2
24 AFG2 1 79.8±4.5 2.1 1.8 79.6±3.3 2.5 3.0 98.7±1.3 1.4 3.5
25
5 84.3±1.8 1.2 5.6 86.5±8.3 3.5 2.9 100.9±8.8 5.7 4.6
26
27 20 89.7±7.7 2.8 3.4 89.6±4.5 4.6 7.8 97.6±2.3 2.2 10.3
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OTA 5 91.2±2.4 4.5 8.9 85.1±2.1 1.8 5.6 90.4±4.5 1.9 5.7
29
30 50 101.3±5.4 5.6 10.8 93.1±1.7 3.4 6.7 85.6±0.9 0.6 1.2
31 100 80.9±3.6 7.7 5.8 90.8±2.1 4.6 2.8 93.0±1.8 1.5 3.5
32
33 OTB 5 84.5±2.8 1.4 2.9 100.5±2.7 2.2 4.6 79.9±3.6 3.6 4.0
34 50 80.1±3.3 3.5 4.4 87.6±3.4 1.7 5.6 87.5±2.1 4.6 5.9
35
36 100 89.6±1.4 2.6 2.8 85.9±4.8 4.3 4.8 93.1±6.7 4.9 2.3
37 OTC 5 90.2±3.5 3.5 10.8 93.5±6.1 1.0 2.7 95.6±2.4 5.1 6.7
38
39 50 79.8±2.8 5.6 8.9 96.8±3.4 2.6 4.2 90.5±4.7 2.6 3.4
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Analytical Methods Page 16 of 19

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4 100 88.6±4.8 2.7 5.9 100.1±3.9 3.1 5.6 96.0±3.9 3.1 3.0
5 CIT 5 80.5±6.7 6.4 12.9 75.3±4.5 6.7 11.2 80.4±2.9 7.8 12.7
6
7 50 76.1±7.3 3.4 3.2 80.1±2.7 4.5 8.9 74.3±4.6 4.8 5.7
8

Analytical Methods Accepted Manuscript

100 78.1±5.4 1.3 3.5 77.8±6.8 5.0 6.9 78.7±4.1 5.9 8.6
9
10 PAT 5 99.2±4.5 5.6 10.8 89.1±3.9 4.3 5.6 95.8±5.0 8.9 11.7
11 50 89.5±2.1 4.5 3.9 97.3±8.7 3.6 2.9 97.3±3.1 6.8 7.3
12
100 90.3±5.6 2.6 4.4 95.6±10.1 3.7 8.5 98.2±1.9 3.2 2.9
13
14 AOH 5 98.5±0.9 1.1 1.8 90.4±3.6 2.3 2.6 89.2±7.8 5.3 4.0
15
50 100.7±2.8 2.3 5.7 97.6±1.5 1.1 3.7 92.3±10.1 1.9 3.6
16
17 100 83.2±3.5 3.3 4.1 98.2±5.5 0.9 2.3 94.5±2.7 2.7 6.1
18 AME 5 90.7±5.6 2.1 3.8 88.9±1.2 3.3 5.0 85.5±1.8 3.6 5.3
19
20 50 81.4±3.3 0.9 2.5 91.4±3.5 5.6 6.1 95.7±4.3 2.8 1.9
21 100 96.8±4.1 1.6 4.7 87.7±4.6 2.1 6.7 99.8±2.8 1.2 4.5
22
23 ALT 5 98.8±5.7 2.1 6.7 100.8±2.4 1.0 3.4 94.3±1.2 0.5 1.7
24 50 80.6±3.4 4.6 8.9 98.3±5.4 1.9 2.6 100.5±5.6 1.7 2.6
25
26 100 106.5±6.7 2.8 10.4 100.4±5.8 3.0 3.4 95.8±3.8 1.2 6.9
27 TEN 5 84.7±5.6 7.9 4.7 90.1±2.5 4.7 5.6 100.1±3.3 3.2 5.0
28
50 89.9±3.2 4.2 5.3 95.6±1.6 2.2 6.5 96.7±7.8 3.4 2.8
29
30 100 80.1±1.1 1.9 2.6 93.4±3.7 1.9 4.3 90.8±2.4 2.1 3.1
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TeA 5 86.5±2.2 3.5 5.7 89.7±4.6 2.7 5.4 87.5±3.9 4.8 6.7
32
33 50 90.4±0.5 1.3 2.2 86.9±7.9 4.8 7.3 88.6±1.8 3.0 5.4
34 100 95.4±3.5 0.9 3.7 84.3±1.8 5.1 6.9 90.8±6.8 2.6 4.7
35
36 ALS 5 108.7±6.9 2.6 8.7 98.3±10.1 5.7 12.1 99.3±5.8 5.7 10.2
37 50 96.4±6.1 5.1 6.4 94.3±5.7 3.6 5.1 109.7±11.1 2.5 4.5
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39 100 108.2±3.8 3.5 8.9 97.5±3.6 2.4 8.7 96.5±7.9 4.8 6.8
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 Page 17 of 19 Analytical Methods

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2 Table 4 Occurrence of the mycotoxins in various fruit juices.
3 Juice variety No. TeA (ng mL-1) Other mycotoxins (ng mL-1) View Article Online
DOI: 10.1039/D0AY01787F
4 Orange juice 1 2.7 －
5
6 2 1.7 －
7 3 4.1 AOH (<LOQ)
8 4 － －
9 5 2.7 －
10
6 <LOQ －
11
12 7 3.4 －

Analytical Methods Accepted Manuscript

13 8 0.3 －
14 9 0.8 －
15
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Grape juice 10 3.7 OTB (0.9)

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11 1.8 PAT (0.9), OTB (1.9)
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12 20.3 OTA (0.6)
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19 13 6.1 OTA (1.7), AOH (0.4)
20 14 － PAT (8.6), AOH (1.1)
21 15 18.3 PAT (16)
22 16 － －
23 Apple juice 17 9.1 －
24
25 18 <LOQ －
26 19 0.5 ALT (1.6)
27 20 0.3 －
28 21 0.6 PAT (<LOQ)
29 22 － －
30
31 －: not detected.
32 Author Contributions
33
34 Wenbo Guo and Junhua Yang performed the experiments; Xueke Niu contributed to
35
36 the sample preparation, Wenbo Guo wrote the manuscript; Emmanuel K. Tangni and
37
Zhihui Zhao reviewed the manuscript; Zheng Han conceived and designed the experiments.
38
39
40
41 Funding
42
43 This work was supported by the Shanghai Agriculture Applied Technology
44 Development Program, China [Grant No. 2019-02-08-00-12-F01148], the National Natural
45
46 Science Foundation of China (No. 31972178), and the Science and Technology Innovation
47
48 Action Plan Project of Shanghai Municipal Commission of Science and Technology [No.
49
50
17391901200].
51
52
53 Conflicts of interest
54
55 The authors declare no conflict of interest.
56
57
References
58
59 1. S. S. Chen and N. X. Qiu, Beverage Industry, 2005, 8, 43-46.
60
 Analytical Methods Page 18 of 19

1
2
3 2. G. S. Shephard and H. F. Vismer, Elsevier Inc., 2008, 335-349. View Article Online
DOI: 10.1039/D0AY01787F
4
5 3. E. M. Embaby, N. M. Awni, M. M. Abdelgalil and H. I. Elgendy,
6 International Journal of Agricultural Technology, 2015, 11, 693-712.
7
4. M. A. Abdel-Sater, A. A. Zohri and I. M. A., Journal of Food Science and
8
9 Technology, 2001, 38, 407-411.
10 5. M. L. Fernández-Cruz, M. L. Mansilla and J. L. Tadeo, Journal of Advanced
11
12
Research, 2010, 1, 113-122.
6. D. Spadaro, A. Garibaldi and M. L. Gullino, Food Additives & Contaminants

Analytical Methods Accepted Manuscript

13
14 Part B, 2008, 1, 134-139.
15
7. A. S. Akdeniz, S. Ozden and B. Alpertunga, Food Additives & Contaminants
Published on 27 October 2020. Downloaded by Goteborgs Universitet on 12/1/2020 9:16:42 AM.

16
17 Part B, 2013, 6, 265-269.
18 8. F. Huang, Y. Fu, D. Nie, J. E. Stewart, T. L. Peever and H. Li, Fungal Biology,
19
20 2015, 119, 320-330.
21 9. S. Fraeyman, S. Croubels, M. Devreese and G. Antonissen, Toxins, 2017, 9,
22 228.
23
24 10. V. Ostry, World Mycotoxin Journal, 2008, 1, 175-188.
25 11. S. Pal, N. Singh and K. M. Ansari, Toxicology Research, 2017, 6, 764-771.
26
12. S. Marin, A. J. Ramos, G. Canosancho and V. Sanchis, Food and Chemical
27
28 Toxicology, 2013, 60, 218-237.
29 13. M. Meena, S. Gupta, P. Swapnil, A. Zehra, M. K. Dubey and R. S. Upadhyay,
30
31
Frontiers in Microbiology, 2017, 8, 1451-1451.
32 14. C. Fan, X. Cao, M. Liu and W. Wang, Journal of Chromatography A, 2016, 1436,
33 133-140.
34
35 15. N. A. Alhazmi, Saudi Journal of Biological Sciences, 2010, 17, 353-359.
36 16. P. M. Scott, G. A. Lawrence and B. P. Y. Lau, Mycotoxin Research, 2006, 22,
37 142-147.
38
39 17. M. Wang, N. Jiang, H. Xian, D. Wei, L. Shi and X. Feng, Journal of
40 Chromatography A, 2016, 1429, 22-29.
41
18. C. Juan, J. Manes, G. Font and A. Juangarcia, Lwt - Food Science and
42
43 Technology, 2017, 86, 344-351.
44 19. Puntscher, Hannes, Kuett, Mary-Liis, Skrinjar, Philipp, Mikula, Podlech,
45
46
Joachim and Froehlich, Analytical & Bioanalytical Chemistry, 2018, 410,
47 4481-4494.
48 20. W. Guo, L. Wu, K. Fan, D. Nie, W. He, J. Yang, Z. Zhao and Z. Han, Scientific
49
50
reports, 2017, 7, 14484.
51 21. Z. Zhang, X. Hu, Q. Zhang and P. Li, Journal of Chromatography B, 2016, 1021,
52 145-152.
53
54 22. Y. Wei, M. Feng, Z. Zhu, J. He, J. Shen, Z. He, X. Qin and L. Zhou, Chinese
55 Journal of Chromatography, 2017, 35, 891.
56 23. A. L. Capriotti, C. Cavaliere, S. Piovesana, R. Samperi and A. Lagana,
57
58 Journal of Chromatography A, 2012, 1268, 84-90.
59 24. T. Pihlström, G. Blomkvist, P. Friman, U. Pagard and B. G. Österdahl,
60
Analytical & Bioanalytical Chemistry, 2007, 389, 1773-1789.
 Page 19 of 19 Analytical Methods

1
2
3 25. Malachova, Alexandra, Stranska, Milena, Vaclavikova, Marta, DOI: Elliott,
View Article Online
10.1039/D0AY01787F
4
5 Christopher, T. and Black, Analytical & Bioanalytical Chemistry, 2018, 410,
6 807-825.
7
26. A. Rahmani, S. Jinap, F. Soleimany, A. Khatib and C. P. Tan, European Food
8
9 Research & Technology, 2011, 232, 723-735.
10 27. I. Azaiez, F. Giusti, G. Sagratini, J. Manes and M. Fernandezfranzon, Food
11
12
Analytical Methods, 2014, 7, 935-945.
28. M. V. Barbieri, C. Postigo, N. Guillem-Argiles, L. S. Monllor-Alcaraz and M.

Analytical Methods Accepted Manuscript

13
14 L. d. Alda, Science of the Total Environment, 2019, 653 958-967.
15
29. I. R. Pizzutti, A. De Kok, J. Scholten, L. W. Righi, C. D. Cardoso, G. N.
Published on 27 October 2020. Downloaded by Goteborgs Universitet on 12/1/2020 9:16:42 AM.

16
17 Rohers and R. C. D. Silva, Talanta, 2014, 129, 352-363.
18 30. N. H. Aziz and L. A. A. Moussa, Food Control, 2002, 13, 0-288.
19
20 31. K. Wang, K. Lin, X. Huang and M. Chen, Journal of Agricultural and Food
21 Chemistry, 2017, 65, 5064-5073.
22 32. S. Hussain, M. R. Asi, M. Iqbal, N. Khalid and A. Ario, Toxins, 2020, 12, 52.
23
24 33. B. P. Y. Lau, P. M. Scott, D. A. Lewis, S. R. Kanhere, C. Cléroux and V. A.
25 Roscoe, Journal of Chromatography A, 2003, 998, 119-131.
26
34. P. P. Carballo Dionisia, Tolosa Josefa, Font G, Berrada Houda, Ferrer E
27
28 Revista de toxicología, 2018, 35, 2-6.
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60