Comparative Performance of Rapid Diagnostics for the Detection of T-2 and HT-2 Toxins in Oats
Abstract
:1. Introduction
2. Results and Discussion
2.1. Compliance/Non-Compliance in Relation to the EU Guidance Limits
2.2. Test Kit Performance Characteristics
3. Materials and Methods
3.1. Chemicals and Materials
3.2. Sample Collection and Preparation
3.3. LC-MS/MS Analysis of Oat Samples
3.3.1. Sample Extraction
3.3.2. LC-MS/MS Parameters
3.3.3. LC-MS/MS Validation and Method Performance
3.4. Test Kit Analysis of Oat Samples
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Sample ID | Sample Type | LC-MS/MS | Test Kit 1 | Test Kit 2 | Test Kit 3 | Test Kit 4 |
---|---|---|---|---|---|---|
(µg/kg) | ||||||
1 | Processed | 6.2 | 17.8 | 13.5 | 35.2 | 10.5 |
2 | Processed | 7.8 | 23.8 | 16.4 | 33.4 | 20.7 |
3 | Processed | 7.6 | 37.4 | 20.6 | 36.5 | 14.1 |
5 | Processed | 33.2 | 27.1 | 38.9 | 48.2 | 24.5 |
6 | Processed | 14.3 | 0 | 28.2 | 38.2 | 15.1 |
7 | Processed | 0.5 | 0 | 0 | 27.9 | 8.6 |
8 | Processed | 49.4 | 246.2 | 48.5 | 62.2 | 41.0 |
9 | Processed | 1.0 | 0 | 15.8 | 0 | 8.2 |
10 | Processed | 18.0 | 31.8 | 36.9 | 53.5 | 26.6 |
11 | Processed | 26.7 | 0 | 31.8 | 35.4 | 24.7 |
12 | Processed | 7.1 | 0 | 16.5 | 32.3 | 16.5 |
13 | Processed | 1.8 | 0 | 10.6 | 26.5 | 17.8 |
14 | Processed | 6.8 | 0 | 15.8 | 37.5 | 22.2 |
15 | Processed | 9.1 | 0 | 18 | 36.8 | 15.3 |
16 | Processed | 38.8 | 13.9 | 38.1 | 40.6 | 29.6 |
17 | Processed | 11.7 | 0 | 18.4 | 34.1 | 17.6 |
18 | Processed | 22.4 | 6.3 | 37.3 | 40.9 | 34.0 |
19 | Processed | 16.1 | 11.8 | 15.8 | 33.4 | 22.0 |
20 | Processed | 38.0 | 0 | 47.9 | 53 | 36.1 |
21 | Processed | 16.2 | 29.4 | 22.3 | 38.2 | 18.9 |
22 | Processed | 5.2 | 1.4 | 23.5 | 26.2 | 13.0 |
23 | Processed | 49.4 | 0.7 | 42.2 | 51.8 | 31.2 |
24 | Processed | 22.7 | 0 | 25.5 | 40.6 | 31.7 |
25 | Processed | 13.7 | 8.9 | 8.6 | 37.3 | 29.0 |
27 | Processed | 46.9 | 5.4 | 43 | 53 | 33.4 |
28 | Processed | 22.7 | 40.3 | 31.2 | 45.7 | 12.9 |
29 | Processed | 4.2 | 69.3 | 6.7 | 27 | 45.4 |
30 | Processed | 20.7 | 43 | 40.8 | 43.6 | 20.1 |
31 | Processed | 9.5 | 101.5 | 19.2 | 29.2 | 18.0 |
32 | Processed | 3.3 | 216.7 | 9.7 | 27.4 | 14.5 |
33 | Processed | 0.0 | 0 | 10.9 | 0 | 13.5 |
34 | Processed | 2.9 | 4.7 | 15.3 | 27.8 | 44.0 |
35 | Processed | 16.3 | 62.9 | 12.7 | 29.2 | 12.6 |
36 | Processed | 1.0 | 0.92 | 5.3 | 0 | 23.4 |
37 | Processed | 15.3 | 8.5 | 24.1 | 33 | 15.2 |
38 | Processed | 2.0 | 0 | 15.3 | 0 | 8.0 |
39 | Processed | 4.7 | 0.47 | 0 | 0 | 8.4 |
40 | Processed | 0.0 | 0 | 9.8 | 0 | 10.6 |
41 | Processed | 1.2 | 0.89 | 8.5 | 0 | 6.8 |
42 | Processed | 3.8 | 0 | 14.4 | 28.6 | 16.4 |
43 | Processed | 0.2 | 3.5 | 0 | 0 | 8.5 |
45 | Processed | 42.2 | 42.5 | 40.2 | 43.2 | 29.5 |
48 | Processed | 3.0 | 0 | 1.2 | 0 | 18.0 |
50 | Processed | 0.0 | 80.9 | 0 | 0 | 6.9 |
53 | Unprocessed | 790.4 | 966 | 377.2 | 439.4 | 336.4 |
56 | Unprocessed | 1105.1 | 1166.5 | 690 | 864 | 758.2 |
57 | Unprocessed | 404.2 | 319.7 | 471.8 | 429.6 | 380.9 |
58 | Unprocessed | 895.3 | 690.5 | 442.4 | 476.2 | 448.0 |
59 | Unprocessed | 353.2 | 523 | 261.4 | 190 | 237.1 |
63 | Unprocessed | 488.8 | 686.5 | 395.2 | 420.2 | 509.4 |
68 | Unprocessed | 513.7 | 764.5 | 351.4 | 540.2 | 452.1 |
71 | Unprocessed | 839.6 | 1122.5 | 966.1 | >range | 641.8 |
72 | Unprocessed | 45.8 | 76.8 | 84.6 | 55.4 | 49.9 |
74 | Unprocessed | 506.7 | 608 | 424.3 | 542.6 | 381.1 |
76 | Unprocessed | 13.7 | 0 | 7.2 | 0 | 16.9 |
77 | Unprocessed | 578.0 | 610 | 394.3 | 449.8 | 383.4 |
80 | Unprocessed | 582.9 | 717 | 80.5 | 94 | 78.4 |
81 | Unprocessed | 482.3 | 656.5 | 561.5 | 628.6 | 484.7 |
83 | Unprocessed | 1024.8 | 694 | 506.8 | 619.5 | 429.7 |
85 | Unprocessed | 388.8 | 572.5 | 532.6 | 346.1 | 425.4 |
86 | Unprocessed | 387.3 | 355 | 303.8 | 307.1 | 247.8 |
88 | Unprocessed | 536.2 | 474.5 | 286.2 | 521 | 350.9 |
90 | Unprocessed | 1025.0 | 1354.5 | 986 | 1454.4 | 718.7 |
93 | Unprocessed | 395.0 | 217.5 | 283.5 | 368 | 437.7 |
96 | Unprocessed | 1080.0 | 1173.5 | 755.1 | 1020 | 913.0 |
100 | Unprocessed | 0.0 | 1129.3 | >range | >range | 5092.4 |
101 | Unprocessed | 55.4 | 21.1 | 42.1 | 37.1 | 28.4 |
108 | Unprocessed | 80.9 | 21.8 | 0 | 0 | 10.2 |
116 | Unprocessed | 8.2 | 1.2 | 0 | 0 | 28.6 |
117 | Unprocessed | 458.2 | 416 | 317.9 | 503.8 | 297.5 |
118 | Unprocessed | 417.8 | 487.5 | 270.3 | 481.4 | 337.7 |
130 | Unprocessed | 0.0 | 0 | 6.1 | 0 | 19.0 |
134 | Unprocessed | 3993.3 | 1477 | 1461.5 | >range | 2919.4 |
137 | Unprocessed | 503.1 | 863.5 | 360.5 | 496.4 | 302.2 |
142 | Unprocessed | 463.6 | 560.1 | 425.1 | 490 | 223.9 |
148 | Unprocessed | 162.7 | 111.7 | 116.6 | 129 | 62.4 |
150 | Unprocessed | 14.3 | 124.9 | 33.6 | 65.2 | 54.5 |
151 | Unprocessed | 66.8 | 134.8 | 133.1 | 105.8 | 75.0 |
154 | Unprocessed | 1114.6 | 1148 | 972.7 | 1497.9 | 985.5 |
155 | Unprocessed | 808.5 | 625.5 | 461.3 | 736.2 | 629.5 |
157 | Unprocessed | 768.0 | 1319 | 676.8 | >range | 694.8 |
164 | Unprocessed | 424.7 | 478 | 515.9 | 457.6 | 256.1 |
165 | Unprocessed | 9.4 | 36.1 | 46.3 | 50.2 | 28.5 |
167 | Unprocessed | 1427.7 | 487 | 62.1 | 172.8 | <range |
169 | Unprocessed | 288.4 | 268.6 | 281.4 | 227.3 | 181.3 |
178 | Unprocessed | 532.2 | 490 | 348.9 | 438.6 | 359.0 |
179 | Unprocessed | 1095.1 | 1452 | 949.9 | 1271.4 | 796.6 |
180 | Unprocessed | 499.8 | 369 | 313 | 296.6 | 282.0 |
184 | Unprocessed | 846.3 | 344.5 | 378.6 | 383.8 | 346.6 |
189 | Unprocessed | 606.6 | 642 | 440 | 514.8 | 530.9 |
195 | Unprocessed | 2583.0 | 2384.5 | 2331.1 | 3218 | 2962.2 |
198 | Unprocessed | 1458.6 | 562.5 | 12.8 | 132 | <range |
201 | Unprocessed | 553.2 | Invalid | 396.9 | 517 | 317.0 |
205 | Unprocessed | 1383.5 | 832 | Invalid | 1011.2 | 905.9 |
216 | Unprocessed | 499.3 | 490.7 | 515 | 614.8 | 525.8 |
218 | Unprocessed | 345.0 | 489.6 | 288.4 | 303.9 | 321.0 |
223 | Unprocessed | 0.0 | 0 | 8 | 0 | 8.1 |
227 | Unprocessed | 2964.6 | 1284.5 | 477.2 | 1616 | 1299.4 |
229 | Unprocessed | 17.5 | 264.1 | 42.1 | 42.5 | 30.6 |
ERM | Processed | 160 | 148.8 | 126.6 | 163.1 | 109.7 |
LC-MS/MS | ||||
---|---|---|---|---|
Positive | Negative | Total | ||
Test Kit 1 | Positive | 7 | 2 | 9 |
Negative | 1 | 82 | 83 | |
Total | 8 | 84 | 92 | |
Cohen’s Kappa (K) | 0.81 | |||
Test Kit 2 | Positive | 1 | 0 | 1 |
Negative | 7 | 84 | 91 | |
Total | 8 | 84 | 92 | |
Cohen’s Kappa (K) | 0.21 | |||
Test Kit 3 | Positive | 6 | 0 | 6 |
Negative | 2 | 84 | 86 | |
Total | 8 | 84 | 92 | |
Cohen’s Kappa (K) | 0.85 | |||
Test Kit 4 | Positive | 2 | 0 | 2 |
Negative | 6 | 84 | 90 | |
Total | 8 | 84 | 92 | |
Cohen’s Kappa (K) | 0.38 |
Rapid Test Kit | Number of False Positives | False Positive Rate (%) | Number of FalseNegatives | False Negative Rate (%) |
---|---|---|---|---|
Test Kit 1 | 2 | 2.2 | 1 | 1.1 |
Test Kit 2 | 0 | 0 | 7 | 7.6 |
Test Kit 3 | 0 | 0 | 2 | 2.2 |
Test Kit 4 | 0 | 0 | 6 | 6.5 |
Rapid Test Kit | Number of False Positives | False Positive Rate (%) | Number of False Negatives | False Negative Rate (%) |
---|---|---|---|---|
Test Kit 1 | 11 | 12 | 3 | 3.3 |
Test Kit 2 | 4 | 4.3 | 13 | 14.1 |
Test Kit 3 | 7 | 7.6 | 7 | 7.6 |
Test Kit 4 | 2 | 2.2 | 11 | 12 |
Test Kit 1 | Test Kit 2 | Test Kit 3 | Test Kit 4 | |
---|---|---|---|---|
Matrix–oats | Yes | No | No | Yes |
Limit of detection | 50 ppb | 10 ppb | 50 ppb | 12 ppb |
Quantification range | 50–3000 ppb | 10–800 ppb | 50–500 ppb | 10–360 ppb |
Test time * | 5 min | 5 min | 5 min | 45 min |
Ease of use | Easy | Easy | Easy | Technical skills required |
Kit reagents supplied | All supplied | No extraction buffer | All supplied | All supplied |
Cost per analysis | GBP 7.20 | GBP 16.80 | GBP 7.40 | GBP 7.44 |
False negative rate (%) | 1.1 | 7.6 | 2.2 | 6.5 |
False positive rate (%) | 2.2 | 0 | 0 | 0 |
Recovery (ERM sample) (%) | 93 | 79 | 102 | 69 |
Analyte | Precursor Ion (m/z) | Product Ion (m/z) | Declustering Potential (DV) | Collision Energy (eV) | Collision Cell Exit Potential |
---|---|---|---|---|---|
Aflatoxin B1 | 313.061 313.061 | 285.1 241.1 | 121 121 | 33 53 | 14 14 |
Aflatoxin B2 | 315.074 315.074 | 287.2 259.1 | 141 141 | 37 41 | 14 14 |
Aflatoxin G1 | 329.055 329.055 | 243.2 311.1 | 131 131 | 37 31 | 18 16 |
Aflatoxin G2 | 331.057 331.057 | 313 245.2 | 106 106 | 35 41 | 16 14 |
Deoxynivalenol | 297.097 297.097 | 249.1 203.2 | 91 91 | 21 21 | 20 20 |
Fumonisin B1 | 722.316 722.316 | 704.3 334.4 | 1 1 | 41 53 | 38 10 |
Fumonisin B2 | 706.309 706.309 | 336.1 354.3 | 126 126 | 49 47 | 20 18 |
Ochratoxin A | 404.092 404.092 | 239 358.1 | 81 81 | 33 21 | 12 18 |
T-2 Toxin | 484.3 484.3 | 215.2 185.1 | 76 76 | 29 31 | 18 11 |
HT-2 Toxin | 442.257 442.257 | 263.102 215.102 | 71 71 | 19 19 | 14 22 |
Zearalenone | 317.1 317.1 | 175 131.1 | −100 −100 | −34 −42 | −13 −8 |
Time (min) | MPA (%) | MPB (%) |
---|---|---|
0 | 99 | 1 |
1.0 | 99 | 1 |
3.0 | 50 | 50 |
9.0 | 1 | 99 |
11.5 | 1 | 99 |
12.0 | 99 | 1 |
14.0 | 99 | 1 |
Mycotoxin | HL Spike (μg/kg) | LL Spike (μg/kg) |
---|---|---|
Aflatoxin B1 | 5 | 1 |
Aflatoxin B2 | 5 | 1 |
Aflatoxin G1 | 5 | 1 |
Aflatoxin G2 | 5 | 1 |
Fumonisin B1 | 200 | 40 |
Fumonisin B2 | 200 | 40 |
Deoxynivalenol | 200 | 40 |
Zearalenone | 200 | 40 |
Ochratoxin A | 10 | 2 |
T-2 toxin | 200 | 40 |
HT-2 toxin | 200 | 40 |
Analyte | Mean Conc. (µg/kg) | StdDev | RSD (%) | RA (%) | RE (%) | LOD (ppb) | LOQ (ppb) |
---|---|---|---|---|---|---|---|
Aflatoxin B1 | 4.7 | 0.2 | 5.1% | 93.7% | 94.0% | 0.1 | 0.5 |
Aflatoxin B2 | 5.1 | 0.3 | 6.0% | 102.5% | 96.8% | 0.2 | 0.7 |
Aflatoxin G1 | 2.9 | 0.3 | 10.6% | 57.9% | 88.4% | 0.1 | 0.4 |
Aflatoxin G2 | 3.3 | 0.3 | 9.2% | 65.9% | 91.3% | 0.1 | 0.5 |
Deoxynivalenol | 191.2 | 6.2 | 3.2% | 95.6% | 100.8% | 3.7 | 12.4 |
Fumonisin B1 | 153.2 | 12.5 | 8.2% | 76.6% | 83.5% | 4.2 | 13.8 |
Fumonisin B2 | 168.9 | 10.6 | 6.3% | 84.5% | 86.9% | 2.6 | 8.7 |
Ochratoxin A | 9.2 | 0.4 | 4.2% | 91.9% | 95.3% | 0.3 | 1.0 |
T-2 Toxin | 187.2 | 4.5 | 2.4% | 93.6% | 98.5% | 3.4 | 11.4 |
HT-2 Toxin | 188.1 | 4.8 | 2.6% | 94.1% | 97.7% | 4.1 | 13.6 |
Zearalenone | 186.1 | 6.3 | 3.4% | 93.1% | 99.5% | 1.8 | 6.1 |
Test Kit 1 | Test Kit 2 | Test Kit 3 | Test Kit 4 | |
---|---|---|---|---|
Sample size | 10 ± 0.1 g | 5 ± 0.1 g | 10 g | 5 g |
Extraction | Add extraction powder. Add 50 mL distilled or deionized water. Shake vigorously for 3 min or blend for 1 min. Filter or centrifuge for 30 s at 2000× g. | Add 25 mL extraction buffer. Vortex for 2 min. Filter (≤5 min). | Add extraction powder. Add 37 mL distilled or deionized water. Shake for 1 min 30 s. Filter. | Add 25 mL of ready-to-use extraction buffer. Shake the sample for 10 min. Centrifuge for 10 min at 3000× g |
Analysis | Mix 100 μL extract with 1500 μL sample diluent. Insert a test strip into the cartridge. Insert the cartridge into the reader. The barcode is read. Enter a sample ID. Add 400 μL sample extract. | Transfer 100 μL of filtered extract to the LFD sample well. Develop for 5 min. Insert the LFD into the reader. | Mix 600 μL of running buffer with 100 μL of sample extract. Transfer 150 μL to a tube and add the test strip. Develop for 5 min. Insert the test strip into the reader. | Dilute the supernatant 1:1 with methanol/distilled water (70/30; v/v). To 50 μL of standard or sample in separate duplicate wells, add 50 μL conjugate and 50 μL antibody. Mix and incubate at room temperature for 30 min. Wash 3 times with 250 μL wash buffer. Add 100 μL of substrate/chromogen to each well, mix and incubate for 15 min at room temperature in the dark. Add 100 μL of the stop solution to each well. Measure the absorbance at 450 nm. |
Results | Results are displayed on the reader screen. Calibration cartridges are provided by the manufacturer. | Results are displayed on the reader screen. Automatic calibration through the barcode and lot ID of the strips. | Results are displayed on the reader screen. Automatic calibration through the barcode and lot ID of the strips. | Evaluation of results through software (absorbance values of the samples interpolated against those of the calibration curve). Calibration curve performed by the analyst using standards provided. |
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© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Meneely, J.; Greer, B.; Kolawole, O.; He, Q.; Elliott, C. Comparative Performance of Rapid Diagnostics for the Detection of T-2 and HT-2 Toxins in Oats. Molecules 2023, 28, 6657. https://doi.org/10.3390/molecules28186657
Meneely J, Greer B, Kolawole O, He Q, Elliott C. Comparative Performance of Rapid Diagnostics for the Detection of T-2 and HT-2 Toxins in Oats. Molecules. 2023; 28(18):6657. https://doi.org/10.3390/molecules28186657
Chicago/Turabian StyleMeneely, Julie, Brett Greer, Oluwatobi Kolawole, Qiqi He, and Christopher Elliott. 2023. "Comparative Performance of Rapid Diagnostics for the Detection of T-2 and HT-2 Toxins in Oats" Molecules 28, no. 18: 6657. https://doi.org/10.3390/molecules28186657