Article Cite This: J. Agric. Food Chem. 2019, 67, 8035−8044
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Using Magnetic Multiwalled Carbon Nanotubes as Modified QuEChERS Adsorbent for Simultaneous Determination of Multiple Mycotoxins in Grains by UPLC-MS/MS Shuai Ma,†,‡ Meng Wang,‡ Tianyan You,† and Kun Wang*,†,§
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†
Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013, P.R. China ‡ Beijing Research Center for Agricultural Standards and Testing, Risk Assessment Laboratory for Agro-Products (Beijing), Ministry of Agriculture, Beijing Municipal Key Laboratory of Agriculture Environment Monitoring, No. 9 Middle Road of Shu Guang Hua Yuan, Haidian Dist., Beijing 100097, P.R. China § Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China S Supporting Information *
ABSTRACT: The simultaneous detection of multiple mycotoxins is important due to the increased toxic effects of combined mycotoxins in grains. In this research, a combination of modified QuEChERS with ultrahigh-performance liquid chromatography−tandem mass spectrometry (UPLC-MS/MS) was used for simultaneous detection of 20 mycotoxins in grains. A series of different types of magnetic (Fe3O4) nanoparticles modified with multiwalled carbon nanotubes (Fe3O4MWCNTs) were designed as modified QuEChERS adsorbents for facile and efficient purification and for target interferences removal in the matrices. When there is an external magnetic field, the proposed modified QuEChERS method uses a shorter pretreatment time compared with the traditional QuEChERS method, which makes it possible to conduct high-throughput analyses. To optimize the QuEChERS process, the extraction solvent and the type and amount of the Fe3O4-MWCNTs were investigated. Under optimal conditions, the method was validated and showed satisfactory linearity (r2 ≥ 0.9965), good recovery (73.5−112.9%), good precision (1.3−12.7%), and excellent sensitivity (ranging from 0.0021 to 5.4457 ng g−1), which indicates that this method can be used for detecting multiple mycotoxins in real samples. KEYWORDS: magnetic multiwalled carbon nanotube, QuEChERS, mycotoxins, UPLC-MS/MS
1. INTRODUCTION Mycotoxins are toxic secondary metabolites formed and released by many fungal species.1,2 It has been estimated that more than 25% of agricultural crop harvests worldwide are contaminated by various mycotoxins during their growth, harvesting, drying, and storage.3,4 The toxic effects of mycotoxins can be divided into teratogenic, carcinogenic, mutagenic, nephrotoxic, estrogenic, immunotoxic, and dermatoxic categories.5−7 Even though there are 300 to 400 mycotoxins that are commonly found, the most important mycotoxins are aflatoxins (AFs), ochratoxin, zearalenone, citrinin, patulin, trichothecenes (such as T-2 toxin), and deoxynivalenol (DON). Most of these mycotoxins are chemically stable and highly resistant even when cooked at high temperatures, so mycotoxins can easily enter the feed and food chain, resulting in a serious threat to human and animal health.8,9 Several methods have been reported for the analysis of mycotoxins, including electrochemical biosensors,10 optical biosensors based on specific antibodies or aptamers,11−13 enzyme-linked immunosorbent assays (ELISAs),14 and liquid chromatography−tandem mass spectrometry (LC-MS/ MS).15−19 Although the electrochemical biosensors and ELISA methods address the challenges associated with mycotoxin detection, they are only suitable for single © 2019 American Chemical Society
mycotoxin and cannot simultaneously detect multiple mycotoxins. However, multiple mycotoxins always exist in the same sample, leading to additive or even synergistic effects and increased toxicity.20 As a powerful analytical tool, LC-MS/ MS has frequently been used due to its high accuracy, specificity, and selectivity for the identification and quantification of multiple mycotoxins. Sample preparation is an essential step in the LC-MS/MS analysis of complex matrix effect samples, and solid-phase extraction (SPE) and QuEChERS are the most widely used pretreatment methods.21−24 SPE can provide good purification, but the procedure is usually costly, complicated, and timeconsuming, which limits the pretreatment speed when working with a large number of samples containing multiple mycotoxins. The traditional QuEChERS method is carried out using salts to extract and DSPE adsorbents, including primary secondary amine (PSA), graphitized carbon black (GCB), and C18, for purification.25−27 However, effective purification cannot be achieved by conventional DSPE adsorbents (PSA, GCB, and C18) in ultratrace simultaneous Received: Revised: Accepted: Published: 8035
January 4, 2019 June 19, 2019 June 24, 2019 June 24, 2019 DOI: 10.1021/acs.jafc.9b00090 J. Agric. Food Chem. 2019, 67, 8035−8044
Article
Journal of Agricultural and Food Chemistry
Figure 1. Comparison of recoveries for different extraction solvent.
linearity, sensitivity, and precision and was then applied for further grain samples analysis. The excellent adsorption capacity and magnetic separation ability of Fe3O4-MWCNTs simplify the preparation of samples, and the proposed method is simple, rapid, cost-effective, and efficient.
determination of mycotoxins, especially for difficult matrices like grains, which leads to unsatisfying LODs and restricts the widespread application of the QuEChERS method.28 Moreover, high-speed centrifugation is essential to separate the DSPE adsorbent and sample solution after purification, which is time-consuming and not suitable for many samples. In recent years, magnetic separation has improved the purification procedure, and the combination of magnetic materials and QuEChERS has attracted much attention.29,30 With magnetic materials, the DSPE adsorbent and sample solution are magnetically separated within 3 s under an external magnetic field. Therefore, the key to improving the QuEChERS method is the design and selection of ideal purification materials to increase the purification efficiency and to shorten the pretreatment time. Multiwalled carbon nanotubes (MWCNTs) have special structures and characteristics, including a large and specific surface area, abundant π electrons, and hollow and layered structures, and MWCNTs are good DSPE adsorbents in QuEChERS to purification aconitines,31 antibiotics,32 resorcylic acid lactones,33 and pesticides.34,35 In this work, we introduce magnetic properties into MWCNTs by modifications with Fe 3O 4 nanoparticles (MNPs) and then characterized the Fe3O4-MWCNTs and further used as a DSPE adsorbent in a modified QuEChERS procedure. With the help of Fe3O4-MWCNTs, separation of extracts and adsorbents could be accomplished immediately with no need for large centrifugal devices, and the synthesis is simple and easy. Thanks to the advantages in performance, price, and operation, Fe3O4-MWCNTs could be used to achieve rapid and thorough analysis in a modified QuEChERS process. After coupling with ultrahigh-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/ MS), here we created a method for the simultaneous determination of 20 mycotoxins in grains. Parameters affecting the extraction and purification efficiency were systematically analyzed using rice as a representative sample. Based on the optimum conditions, the method was validated regarding the
2. EXPERIMENTAL SECTION 2.1. Sample Preparation. Briefly, 5.0 g of grains was added into 50 mL polypropylene centrifugation tubes. Acetonitrile/water (25 mL; 80:20, v/v, 1% acetic acid) was added, and the tube was vigorously shaken with a vortex mixer for 1 min. Then, after citrate buffer (4 g of MgSO4, 1 g of NaCl, 1 g of Na3Cit·2H2O, and 0.5 g of Na2Cit·1.5 H2O) were added, the mixture was shaken vigorously for 1 min followed by centrifugation at 8000 rpm for 5 min. Fe3O4MWCNTs (20 mg) and 1.0 mL of the purified supernatant were added to a 2.0 mL microcentrifuge. The mixture was mixed vigorously for 1 min and separated by a magnetic force created by a magnet. The solution was passed through a 0.22 μm PTFE membrane filter (Pall, MI, USA), and 5 μL of the final solution was analyzed by UPLC -MS/ MS. 2.2. UPLC-MS/MS Analysis. Using a Waters Acquity UPLC system (Milford, MA, USA) at 40 °C, 20 mycotoxins were isolated using an Acquity Cortecs UPLC C18 column (1.6 μm particle size, 2.1 mm × 100 mm, Waters). The mobile phase was composed of 5 mM NH4AC and Milli-Q water (A) and MeOH (B) at a flow rate of 0.3 mL min−1. The linear gradient eluting program was as follows: initial 5% B, 0.5 min 5% B, 7 min 90% B, 7.5 min 90% B, 7.6 min 5% B, 9 min 5% B. The subsequent rebalancing time was 1.4 min before the next injection, with a total running time of 9 min. The injection volume was 5 μL. The separated analytes were detected by an orthogonal Z-spray ionization (ESI) interface on a Waters XEVO TQS mass spectrometer. Based on the structural characteristics of the analyte, the positive ion and negative ion modes were applied. The parameters were as follows: source temperature of 150 °C, desolvation temperature of 500 °C, capillary voltage of 2.5 kV/−0.8 kV, conical gas flow rate of 150 L h−1, desolvation gas flow rate of 1000 L h−1, capillary voltage of 150 L h−1, and conical gas flow rate of 150 L h−1. The multiple reaction monitoring (MRM) mode was established for quantification and identification of the targeted analytes with the conditions shown in Table S1. MassLynx v4.1 and Targetlynx (Waters) were used for data analysis. 8036
DOI: 10.1021/acs.jafc.9b00090 J. Agric. Food Chem. 2019, 67, 8035−8044
Article
Journal of Agricultural and Food Chemistry
Figure 2. Comparison of recoveries for purification with different types of Fe3O4-MWCNTs
Figure 3. Comparison of recoveries for purification with different amount of Fe3O4-MWCNTs. 2.3. Method Evaluation. In accordance with the Commission Regulation 401/2006/EC37 and the European SANCO guidelines 12571/2013,38 verification was carried out to determine the performance of the proposed QuEChERS method, including the linear range, limit of detection (LOD), quantitative (LOQ), matrix effect (ME), accuracy (percent recovery), and precision (percent RSD). Mycotoxins were classified as group A (recovery experiments performed at 0.1 and 5 ng g−1), including AFB1, AFB2, AFG1, AFG2, OTB, and TEN; group B (recovery experiments at 1 and 50 ng g−1), including ALT, T-2, MPA, and ZEN; group C (recovery experiments at 5 and 250 ng g−1), including 3-ADON, 15-ADON, FUS-X, HT-2, PAT, and DAS; and group D (recovery between 10 and 500 ng g−1), including DON, TEA, NEO, and NIV. The linearity of the method was evaluated by adding blank samples at six concentration levels through the calibration curve of matrix matching where the peak area was used as the analyte response. The calibration curve was constructed by drawing the relationship between
peak area (Y) and analyte concentration (X), and the calibration curve equation and correlation coefficient (r2) of each mycotoxin were calculated. The LOD and LOQ were calculated as the concentrations corresponding to the signal-to-noise (S/N) ratio of 3 and 10, respectively. The recoveries and precision of the method were investigated through standard addition. The blank rice samples were added to three concentration levels (low, neutral, and high concentration with scalars corresponding to 2, 5, and 10 times the lowest concentration point in the linear range of the four groups, respectively) for three consecutive days. After mixing, the standard sample was placed at room temperature until the acetonitrile solvent evaporated. Then, the samples were extracted, purified, and analyzed by UPLC-MS/MS. The recovery rate (R%) was calculated by the following formula: R% = 100% × measured concentration/fortification level 8037
DOI: 10.1021/acs.jafc.9b00090 J. Agric. Food Chem. 2019, 67, 8035−8044
Article
Journal of Agricultural and Food Chemistry
Figure 4. Comparison of matrix effects for different DSPE sorbent materials.
Table 1. Linear Ranges, Linear Equations, Correlation Coefficient (r2), Limits of Detection (LOD), and Limits of Quantification (LOQ) of the 20 Mycotoxins in Rice compound
linear range (ng·g−1)
TEA NIV DON FUS-X NEO 3-ADON 15-ADON AFG2 AFG1 OTB AFB2 MPA ALT AFB1 DAS HT-2 TEN T-2 PAT ZEN
10.0−500 10.0−500 10.0−500 5.0−200 10.0−500 5.0−200 5.0−200 0.1−5.0 0.1−5.0 0.1−5.0 0.1−5.0 1.0−50 1.0−50 0.1−5.0 5.00−200 5.00−200 0.1−5.0 1.0−50 5.0−200 1.0−50
linear equation Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
= = = = = = = = = = = = = = = = = = = =
1220.75X 282.164X 2529.97X 704.858X 24.7148X 5754.32X 3291.29X 1740.41X 6745.36X 2579.52X 1997.05X 703.185X 2290.77X 16857.2X 32.7148X 1676.39X 5026.54X 1764.57X 757.529X 242.115X
− 224.725 + 77.7928 + 1205.67 + 9.31571 + 18.5806 + 44.4749 + 307.446 − 60.847 + 223.685 + 384.11 − 45.089 + 428.533 + 230.471 − 2060.1 + 18.5806 − 57.1302 + 1321.84 − 100.925 − 148.623 − 105.823
The accuracy of these repetitive conditions was evaluated by the relative standard deviation (RSD, %), which is determined by intraday and interday measurements. The matrix effect was evaluated according to the following formula: Matrix effect (%) = (the slope of the calibration curve in the matrix − the slope of the calibration curve in the reagent solution)/the slope of the calibration curve in the reagent solution. 2.4. Statistics and Data Analysis. Experimental data are presented as the mean values ± SD. Mass instrument control, data acquisition, and the processing of TQ-S were performed using MassLynx 4.1 software. The statistical analyses and graphing were carried out with Microsoft Excel 2016 and SigmaPlot 12. Characterization of the Fe3O4-MWCNTs composites was carried out with Origin 8.0.
r2
LOD (ng·g−1)
LOQ (ng·g−1)
0.9982 0.9980 0.9999 0.9995 0.9965 0.9997 0.9994 0.9997 0.9993 0.9967 0.9997 0.9989 0.9991 0.9988 0.9979 0.9989 0.9990 0.9996 0.9994 0.9990
0.7071 0.5962 0.1187 0.0603 1.6337 0.0580 0.0999 0.0336 0.0032 0.0015 0.0074 0.0059 0.0052 0.0012 0.0823 0.0305 0.0006 0.0027 0.3144 0.0025
2.3570 1.9874 0.3957 0.2009 5.4457 0.1933 0.3329 0.1120 0.0106 0.0049 0.0245 0.0195 0.0173 0.0039 0.2742 0.1016 0.0021 0.0092 1.0482 0.0084
3. RESULTS AND DISCUSSION 3.1. Characterization of the Fe3O4-MWCNTs Composites. The size and morphology of the magnetic materials were investigated by TEM, X-ray diffraction (XRD), and FTIR spectroscopy analyses. The Fe3O4 nanoparticles gather during the reaction, and the TEM image of the magnetic materials (Figure S1) shows that the MWCNTs are surrounded by Fe3O4 nanoparticles. The average diameter of the Fe3O4 nanoparticles is mainly distributed from 100 to 200 nm. Figure S2 shows the XRD structure of MWCNTs and Fe3O4MWCNTs. Six typical diffraction peaks appeared at 2θ = 31.04, 35.58, 43.15, 53.78, 57.25, and 63.03°, corresponding to crystal planes (220), (311), (400), (422), (511), and (440), 8038
DOI: 10.1021/acs.jafc.9b00090 J. Agric. Food Chem. 2019, 67, 8035−8044
Article
Journal of Agricultural and Food Chemistry
Table 2. Validation Parameters: Recoveries and Interday and Intraday Precisionsa at Three Concentrations (2 LOQ, 5 LOQ, and 10 LOQ) recovery (%) and RSD (%) compound TEA NIV DON FUS-X NEO 3-ADON 15-ADON AFG2 AFG1 OTB AFB2 MPA ALT AFB1 DAS HT-2 TEN T-2 PAT ZEN
2 LOQ 83.5 97.5 101.3 100.0 100 100.5 104.3 80.6 79.6 112.9 82.5 91.7 100 105.3 102.1 100.8 91.3 105.7 97.6 81.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
intraday precision RSD (%)
5 LOQ
8.8 6.1 0.9 4.3 6.2 1.8 5.7 1.2 10.5 5.5 9.3 3.5 7.2 7.6 7.3 4.5 5.9 3.9 2.7 8.1
106.3 101.3 101.25 92.3 91.3 93.8 91.2 82.9 84.8 73.5 84.1 89.7 78.9 89.2 89.7 98.8 91.3 95.0 90.0 77.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.0 1.8 1.7 3.9 5.8 1.8 1.9 7.4 5.5 8.8 9.2 4.0 1.9 10.2 4.0 1.8 1.9 3.7 2.2 6.2
10 LOQ 106.5 98.5 97.0 93.6 91.2 95.9 93.3 79.8 103.5 79.1 80.3 89.9 83.2 76.5 89.3 102.5 91.5 99.0 96.3 80.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
interday precision RSD (%)
2 LOQ
5 LOQ
10 LOQ
2 LOQ
5 LOQ
10 LOQ
9.8 3.7 11.5 6.2 4.9 8.3 6.4 5.7 3.4 10.0 10.1 10.37 3.5 10.8 2.6 4.5 4.4 12.7 4.9 7.6
8.6 1.9 4.1 5.6 3.2 7.6 3.3 2.5 8.4 9.7 8.2 7.8 6.1 4.2 3.3 5.7 6.1 7.4 4.4 8.7
1.8 3.9 8.5 0.9 7.8 4.7 6.1 7.2 8.5 2.6 7.5 5.6 5.4 1.3 1.2 6.8 9.6 3.1 8.4 6.2
12.8 10.6 9.3 10.7 7. 5 7. 8 5. 5 4.4 10.9 9.6 6.3 3.9 7.1 6.5 9.4 10.2 7.7 9.8 2.2 4.7
8.3 4.4 7.5 5.9 8.2 6.2 6.3 1.5 4.7 5.4 10.6 4.4 4.7 7.3 7.2 0.9 4.8 7.2 4.3 6.4
8.3 5.8 4.7 3.75 6.5 7.6 6.4 5.0 7.5 2.9 5.8 2.6 4.3 2.9 6.4 5.8 5.4 2.9 7.1 5.5
0.7 0.7 2.9 1.6 2.4 1.5 0.8 5.6 0.7 1.7 5.8 0.9 3.3 4.8 3.4 0.7 2.3 2.9 0.5 9.9
a
Relative standard deviation, RSD.
Table 3. Recoveries of Mycotoxins in 5 Grains at a Concentration of 2 LOQ compound TEA NIV DON FUS-X NEO 3-ADON 15-ADON AFG2 AFG1 OTB AFB2 MPA ALT AFB1 DAS HT-2 TEN T-2 PAT ZEN
rice 83.5 97.5 101.3 100 100 100.5 104.3 80.6 79.6 112.9 82.5 91.7 100 105.3 102.1 100.8 91.3 105.7 97.6 81.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
millet 8.8 6.1 0.9 4.3 6.2 1.8 5.7 1.2 10.5 5.5 9.3 3.5 7.2 7.6 7.3 4.5 5.9 3.9 2.7 8.1
76.7 87.3 90.0 94.9 85.7 82.9 82.5 79.9 82.4 100.6 85.6 80.5 90.0 107.7 100.0 73.2 102.6 89.7 71.8 79.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
wheat
9.4 5.2 4.1 8.2 0.8 4.8 5.3 11.7 4.9 7.6 8.3 2.2 7.5 6.5 2.6 1.9 9.1 2.1 3.9 5.5
81.0 82.4 105.0 97.4 88.1 87.8 92.5 85.2 78.7 74.5 86.9 79.6 92.5 81.6 95.0 92.7 109.5 100.0 89.7 73.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
9.0 1.7 7.2 3.9 3.7 4.8 5.5 12.5 5.8 10.1 7.5 6.4 7.7 9.3 5.1 1.8 8.3 0.5 7.1 8.9
corn 90.5 79.6 87.5 94.9 90.5 82.9 82.5 82.7 86.3 106.8 90.1 83.6 105.0 87.8 77.5 78.3 84.6 87.2 84.6 87.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
coix seed 8.5 1.2 6.4 7.1 5.4 3.3 2.0 9.8 7.3 6.5 11.9 5.0 7.3 8.2 2.8 3.3 6.8 2.7 4.3 3.1
77.1 90.1 90.0 94.9 92.9 87.8 85.0 83.3 91.6 96.8 84.3 85.2 95.0 78.7 102.5 75.9 113.1 103.3 79.5 71.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
8.7 3.9 1.7 1.4 4.2 5.5 6.1 7.7 6.3 8.9 8.5 5.9 6.2 10.8 9.3 5.0 4.4 3.6 0.8 2.3
oxygen-containing functional groups. The peaks at 582 cm−1 in the Fe3O4-MWCNTs were assigned to the FeO modes.40 This suggests the formation of the Fe3O4-MWCNTs nanocomposites and the interaction between Fe3O4 MNPs and MWCNTs. 3.2. Optimization of the QuEChERS Procedure. Improving the QuEChERS treatment of grain samples is the main objective of this study. Therefore, the parameters that affect the extraction and purification efficiency, including the extraction solution, and the selection and dosage of magnetic adsorbents were systematically analyzed in the rice samples. During the optimization of the sample preparation, the external
respectively. Peaks are consistent with the database in the JCPDS file (PDF no. 65/3107) and indicate a cubic shape of the Fe3O4. The peak near 2θ = 25.80° was attributed to the MWCNTs. Fe3O4 and MWCNTs peaks coexist in the synthesized Fe3O4-MWCNTs, indicating the successful synthesis of magnetic MWCNTs.39 The FT-IR spectrum of the Fe3O4-MWCNTs is shown in Figure S3. The peaks at 3420, 1735, and 1620 cm−1 correspond to the stretch vibrations of −OH, −CO, and −COOH, respectively, while the peaks at 1381 and 1173 cm−1 can be ascribed to the bending vibration of OH and the stretching vibration of CO, respectively. These data indicate that the acid-treated MWCNTs have many 8039
DOI: 10.1021/acs.jafc.9b00090 J. Agric. Food Chem. 2019, 67, 8035−8044
Article
Journal of Agricultural and Food Chemistry Table 4. Concentration of Mycotoxins in Grain Samplesa sample
TEA
ALT
AFG2
15-ADON
3-ADON
FUS-X
DAS
NEO
TEN
DON
NIV
ZEN
corn corn corn wheat wheat wheat wheat wheat wheat wheat rice rice rice rice rice
14.29 ND ND ND ND ND ND 10.97 12.76 ND ND ND ND ND ND
3.68 ND 4.18 6.28 5.28 4.40 6.13 3.60 8.00 3.80 4.63 2.85 5.40 7.98 5.40
1.06 1.07 0.75 0.80 0.65 0.27 0.55 0.15 0.50 0.47 0.56 0.36 0.48 0.44 0.33
27.00 26.13 ND ND ND ND 5.25 ND ND 9.13 ND ND ND ND 5.88
8.17 39.54 ND ND ND ND ND ND ND 13.82 ND ND ND ND 6.97
34.03 31.94 17.48 32.87 33.68 8.45 8.10 8.22 15.63 9.95 8.68 ND 13.43 10.76 7.87
ND 5.25 11.15 ND 11.15 ND ND ND ND 6.37 ND ND ND ND ND
ND 18.75 ND 12.50 ND ND ND ND ND ND 10.63 ND 12.50 ND 15.00
116.13 75.18 104.51 75.00 107.33 25.63 29.83 22.70 74.28 29.53 42.38 12.17 33.69 24.48 23.09
236.52 830.15 23.04 ND 31.62 ND 48.53 42.65 12.25 105.15 ND 121.81 22.30 ND 107.35
ND 96.64 ND ND ND ND ND ND ND ND ND ND ND ND ND
137.64 16.36 ND ND ND ND ND ND ND ND ND ND ND ND 26.77
In units of nanograms per gram (ng g−1). ND, not detected.
a
added to the extraction solvents to improve the extraction efficiency.36,41 Wang’s study showed that the recovery of some mycotoxins by citric acid to extraction mixture is higher than that of formic acid and acetic acid, especially for PAT, indicating that the strong chelating effect of citric acid with heavy metals can protect mycotoxins.17 The addition of 1% acetic acid to the extraction solvent can improve the recovery of AFs, TEN, DON, HT-2, and T-2.21,25 Consequently, we optimized the acid addition by using solutions with 0%, 1%, and 3% acetic acid and 10 M L−1 citric acid. The ACN/water (80:20, v/v containing 1% acetic acid) had a generally good recovery rate for the target mycotoxins. As shown in Figure 1, the extraction efficiency for the 20 mycotoxins assayed was satisfactory (range: 73.5−112.9%). This result is consistent with previous studies on cereals and food matrices. When the acetic acid content was increased to 3%, more impurities were extracted.17,21 However, the addition of citric acid did not
Table 5. Comparison with Similar Works adsorbent material
linear ranges (ng g−1)
LOQs (ng g−1)
types of mycotoxins
ref
m-MWCNTs MWCNTs MWCNTs Fe3O4-MWCNTs
3−1500 0.1−500 10−500 0.1−500
0.3−1.5 0.02−0.10 0.05−2.90 0.0006−1.6337
4 4 6 20
18 36 44 this work
standard method of matrix matching was used to calculate the recovery rate. 3.2.1. Optimization of the Extraction Solution. The extraction solution, which is crucial for effective extraction during the QuEChERS procedure, was evaluated. In previous reports, ACN and ACN/water have frequently been used as extraction solutions for mycotoxin analysis, and acid is also
Figure 5. Comparison of recoveries for purification with Fe3O4-MWCNTs, PSA, C18, and GCB. 8040
DOI: 10.1021/acs.jafc.9b00090 J. Agric. Food Chem. 2019, 67, 8035−8044
Article
Journal of Agricultural and Food Chemistry
thus, the matrix effects could be ignored. Thus, it is important to select the excellent DSPE materials and matrix-matched calibration to get a reliable quantification in mycotoxins analysis when there are considerable matrix effects in grains. 3.4. Method Validation. We tested and verified the proposed method using optimized conditions by evaluating the recovery, linear range, correlation coefficients, precision, limits of detection (LODs), and limits of quantification (LOQs). The proposed method had satisfactory linearities for all target analytes with the correlation coefficients (r2) higher than 0.9965 for rice, 0.9910 for millet, 0.9918 for wheat, 0.9927 for corn, and 0.9961 for coix seed (Tables 1, S2, and S3). The LODs and LOQs for the 20 mycotoxins ranged from 0.0006 to 1.6337 and from 0.0021 to 5.4457 ng g−1, respectively. Using matrix-matched calibration standards, we evaluated the recovery at different concentrations (Tables 2 and 3). The recovery rate was 73.5−112.9% for rice, 71.8−107.7% for millet, 73.3−109.5% for wheat, 77.5−106.8% for corn, and 71.6−113.1% for coix seed. All relative standard deviations (RSDs) of grains were less than 12.5%, indicating good precision. Through intra- and interday accuracy testing, we can determine the method reproducibility. We calculated the intra- and interday RSD by adding three concentrations of mycotoxins to rice samples. Five parallel extractions of a sample solution over a day gave the intraday RSDs. The interday RSDs were determined by extracting sample solutions that had been independently prepared for a continuous 3 days. The results showed that the intraday and interday RSD were less than 12.7% and 12.8%, respectively (Table 2), indicating that the method reproducibility was acceptable. 3.5. Determination of Mycotoxins in Food Samples. The optimized QuEChERS method was used to determine the natural occurrence of mycotoxins in grain samples randomly collected from China’s local markets (Table 4 and Figure S4). Among the 15 samples, in total, 12 mycotoxins were detectable, (TEA, NIV, DON, FUS-X, NEO, 3-ADON, 15ADON, AFG2, ALT, DAS, TEN, and ZEN). All of the samples were found to contain AFG2 (0.15−1.07 ng g−1) and TEN (12.17−116.13 ng g−1), respectively. ALT and FUS-X were detected in 14 samples with concentrations in the range of 2.85−8.00 ng g−1 and 7.87−34.03 ng g−1, respectively. DON was detected in 11 samples (12.25−830.15 ng g−1). 15-ADON (5.25−27.00 ng g−1) and NEO (10.63−18.75 ng g−1) were detected in 5 samples. 3-ADON and DAS were detected in 4 samples with concentrations in the range of 6.97−39.54 ng g−1 and 5.25−11.15 ng g−1, respectively. TEA and ZEN were detected in 3 samples with concentrations in the range of 10.97−14.29 ng g−1 and 16.36−137.64 ng g−1, respectively. NIV was detected in 1 sample at a concentration of 96.64 ng g−1. The above results demonstrated that AFG2, TEN, ALT, FUS-X, and DON are common contaminants in grains. In addition, the proposed method provides a universal extraction and purification pretreatment method using modified QuEChERS, thus simplifying the analysis of multiple mycotoxins. 3.6. Comparison with Similar Works and Commercial DSPE Adsorbent. MWCNTs are one type of new nanomaterials and have been used as adsorbents to remove the interference compounds of matrixes in the QuEChERS method. For the evaluation of the current method, it was compared with similar works from the viewpoint of the adsorbent material used, linear ranges, and LOQs.18,36,44 As
improve the recovery rate for the most target mycotoxins. Therefore, ACN/water (80:20, v/v containing 1% acetic acid) was selected as the extraction solution. 3.2.2. Optimization of the Type of MWCNT. Considering the abundance of starch, protein, fat, and mineral substances in grains, these compounds will interfere with the detection of mycotoxins by mass spectrometry. Therefore, solid-phase dispersion was used as the extraction method, and several types of magnetic MWCNTs materials, including Fe3O4MWCNTs, Fe3O4-MWCNTs-OH, Fe3O4-MWCNTs-COOH, and Fe3O4-MWCNTs-2, were selected as the DSPE adsorbent materials to remove interference. Fe3O4-MWCNTs-2 is one type of MWCNT, which is obtained from MWCNTs-2 (O.D., 8−15 nm; length, ∼50 μm; purity greater than 98%) modified with Fe3O4 nanoparticles. As shown in Figure 2, satisfactory recovery rates were observed with Fe3O4-MWCNTs as the adsorbent for all 20 mycotoxins, but the Fe3O4-MWCNTs-OH and Fe3O4-MWCNTs-COOH had strong adsorption for and, thus, low recovery of TEA, TEN, ZEN, and AFs. This may be due to the interaction of hydroxyl and carboxy groups on the surface of MWCNTs-OH and MWCNTs-COOH with the amino and carbonyl groups of the target analyte molecules (TEA, TEN, ZEN, and AFs), which leads to the low recovery.42 Therefore, Fe3O4-MWCNTs adsorbent were selected as the DSPE adsorbent materials. 3.2.3. Optimization of the Amount of Fe3O4-MWCNTs. The amount of Fe3O4-MWCNTs added may directly affect the extraction efficiency of the target mycotoxins. Thus, amounts of 10, 20, and 30 mg of Fe3O4-MWCNTs were investigated (Figure 3). The extraction efficiency of all the mycotoxins increased as the amount of Fe3O4-MWCNTs increased from 10 to 20 mg of Fe3O4-MWCNTs. However, with 30 mg of Fe3O4-MWCNTs, the recovery of many of the target mycotoxins decreased to
