Mycotoxin working

group NYS Department of Agriculture & Markets

Food Laboratory Division

AAFCO Check Sample results- Kits- 20146118.5 1200 1300 44 236.8 242.3

Lab # Method Total Aflatoxin DON Total Fumonisin Ocra A T-2 /HT-2 Zon

42 LC-(mycosep) R1 20.33 Vicam-1320

R2 23.06 Vicam-1520

297 Neogen R1 19.75 1

R2 20.25 1.1

4 Vicam R1 14 2000

R2 14 1900

957 Neogen R1 23.5 1 2 29.6 0.2

R2 14.7 0.8 1.5 25.9 0.2

660 Neogen R1 16.9 1 22.9 0.2

R2 17.2 1.4 24.4 0.4

202 r-Biopharm ridascreen R1 1.3 135.9

R2 1.3 136

3 Neogen R1 19.2 1100

R2 18.1 1100

27 Neogen R1 18.8 1100 1900 252.5

R2 17.1 1100 1900 299.6

964 Neogen R1 19 1125 1400 26.1 841.5 461

R2 19.1 1075 2100 27.3 811.8 493.4

227 Neogen R1 18 1300 2000 2376 318

R2 19 1200 2000 3480 494

33 Neogen R1 1300

R2 1300

35 r-Biopharm ridascreen R1 1524 2358 159.3

R2 1261 3265 184.8

202 r-Biopharm ridascreen R1 23.391 1.8 251.3

R2 22.068 2 282.1

958 Neogen R1 17.9 1.9

R2 18.2 2

959 Neogen R1 0.7

R2 0.1

Assigned Value

N= none detected KIT results 201461 Poultry feed

Reproducible results over all

Inconsistent reporting of

significant figures

T-2/HT-2 differences from

reference lab results-

AAFCO Check Sample results-KITS 201462 & 20146342.6 2700 1800 188.9 244.6 250.6

Lab # Method Total Aflatoxin DON Total Fumonisin Ocra A T-2 /HT-2 Zon

42 LC-(mycosep) R1 45 2400

R2 38 2050

297 Neogen R1 34.8 270

R2 35.9 280

4 Vicam R1 24 1.3

R2 22 1.3

957 Neogen R1 31.7 2400 1900 95.3 0.183

R2 38.1 2200 1600 44.9 0.195

660 Neogen R1 37.4 2478 39.7 302

R2 36.9 2756 34.1 293.1

202 r-Biopharm ridascreen R1 20.4 238.9 103.2

R2 20.4 235.9 104.1

3 Neogen R1 28.3 2300 203 255

R2 28 2100 203 213

27 Neogen R1 31.9 2500 1600 264

R2 24.4 2100 1900 284

964 Neogen R1 2300 2925 97.8 8418 383.1

R2 2000 2690 100.2 8659 391.2

227 Neogen R1 36 2200 3000 222 40100 168

R2 36 2900 3000 209 40760 151

33 Neogen R1 30.4 2400 2200 254

R2 33.4 2350 2450 227

35 r-Biopharm ridascreen R1 49.2 1812 2938 295.8

R2 54.328 3160 2833 176.9

202 r-Biopharm ridascreen R1 20.4 2.1 3

R2 20.4 2.1 3

958 Neogen R1 31.7 2.5 3 41 N

R2 31.8 2.4 3.1 41.8 N

959 Neogen R1 2400 3300 52.2

R2 2400 2200 69.3

2050 Neogen R1 37.4 1.6 2.43 86.1

R2 35.2 1.7 2.5 88.8

N= none detected KIT results 201463 Cattle feed

Assigned Value21.5 830 2100 85.7 46.9 183.9

Lab # Method Total Aflatoxin DON Total Fumonisin Ocra A T-2 /HT-2 Zon

42 LC-(mycosep) R1 19 3

R2 19 3

297 Neogen R1 23 800

R2 23.6 800

4 Vicam R1 15 1500

R2 14 1300

957 Neogen R1 25.1 600 2100 32.4 100

R2 18.4 600 1300 30.6 200

660 Neogen R1 19.1 900 23.9 246.9

R2 23.7 934.8 32.7 200.8

202 r-Biopharm ridascreen R1 11.1 N N

R2 11 N N

3 Neogen R1 15.4 1.6

R2 18.3 1.5

27 Neogen R1 15.3 600 1500 296.4

R2 12.7 600 1700 307.5

964 Neogen R1 24.5 800 1900 64.9 233 210

R2 16.2 900 2100 75.2 227 320

227 Neogen R1 15 700 2100 195 95 478

R2 24 600 3000 161 117.8 296

33 Neogen R1 18.6 900 1700

R2 17.6 700 2100

35 r-Biopharm ridascreen R1 23.384 579 1991 317.5

R2 25.01 774 2972 224.7

202 r-Biopharm ridascreen R1 500 3100

R2 600 3000

958 Neogen R1 29.4 1.1 9.2 28.6 N

R2 21 N 3.7 32.6 N

959 Neogen R1 1 1.9 42.7

R2 1 2.7 38.6

N= none detected KIT results 201462 Swine feed

Assigned Value

AAFCO check sample results- LC-MS/MS 201461

Assigned Value 18.5 1.6 0.5 0.5 1200 1300 400 200 44 236.8 242.3

Lab # Method AB1 AB2 AG1 AG2 DON FB1 FB2 FB3 Ocra A T-2 Zon

1 LC-MS/MS R1 15.63 N N N 733.1 1676 468.6 N N 95.98 301.1

R2 17.73 1.03 N N 709.9 1538 450.2 N 51.23 77.75 281.7

2033 LC-MS/MS R1 19.21 2.09 N N 1511 525 36 217.5 251.3

R2 19.22 2.28 N N 1248 486 39.21 199.3 240.6

918 LC-MS/MS R1 21.9 1.88 0.5 N 1210 160 N 41.7 131 190

R2 20.6 1.81 1.1 N 1420 240 N 40.5 120 167

218 LC-MS/MS R1 21.7 2.32 N N 49.38 206.5 286.5

R2 20.82 2.34 1.54 N 49.77 205.2 276.5

10 LC-MS/MS R1 28.6 4.1 N N 1312

R2 26.5 3.2 N N 995

2060 LC-MS/MS R1 31.024 2.609 1.27 0.091 461.3 2449 279.7 164.7 27.757 177.5 315.9

R2 30.292 2.006 1.543 0.112 642.5 2831 274.4 150.7 30.97 133.1 323.2

553 LC-MS/MS R1 N N N N 993.2 284.5 164.1 47.96 44.81

R2 N N N N 869.1 251.5 212 51.28 42.18

2052 LC-MS/MS R1 695 1052 296 150 149

R2 686 820 185 91 135

LC-MS/MS results 201461 Poultry feedN= none detected

Recoveries for each

analyte vary from

assigned value

Individual lab results

reproducible overall

Inconsistent T-2/HT-2

results

AAFCO check sample results- LC-MS/MS 2014612 & 63

Assigned value 20 1.5 0.5 0.5 830 1500 400 200 85.7 46.9 183.9

Lab # Method AB1 AB2 AG1 AG2 DON FB1 FB2 FB3 Ocra A T-2 Zon

1 LC-MS/MS R1 18.6 1.43 N N 594 1701 489.5 63.44 34.1 248.2

R2 20.26 1.23 0.93 N 488 1727 485.2 60.27 33.09 240.6

2033 LC-MS/MS R1 25.2 1.7 1.03 N 634 1623 762.5 42.25 23.92 135

R2 34.6 0.9 2.7 N 598 1465 650 43.55 29.58 150.8

918 LC-MS/MS R1 28.3 1.47 0.7 N 1040 96.9 34 138

R2 29.2 2.47 0.6 N 920 89.5 30 181

218 LC-MS/MS R1 24.1 2.1 1.1 N 83 45 212

R2 22.7 2 N N 87 43 208

10 LC-MS/MS R1 16.8 3 N N

R2 15.5 N N N

2060 LC-MS/MS R1 34.2 2.5 N N 468.2 2032 413.2 181.8 80.7 40.1 157.6

R2 32.8 3 N N 440.3 2458 439.1 180.9 77.1 42.3 169

553 LC-MS/MS R1 N N N N 650.7 1175 372.4 170.4 70.31 45.42 54.81

R2 N N N N 565.4 2348 490.7 305.6 75.89 36.54 58.97

2052 LC-MS/MS R1 15.8 NDG N 523 1259 434 169 43.1 174

R2 16 NDG N 550 1167 364 156 48.6 170

N= none detected LC-MS/MS results 201462 Swine feed Assigned value 40.2 1.7 0.7 0.5 2700 1400 270 140 188.9 244.6 250.6

Lab # Method AB1 AB2 AG1 AG2 DON FB1 FB2 FB3 Ocra A T-2 Zon

1 LC-MS/MS R1 29.8 1.6 N N 1688 1770 382 211 180 285

R2 26.9 1.4 N N 1661 1768 403 210 171 309

2033 LC-MS/MS R1 31.8 2.9 N N 2100 1720 780 180 118 147.5

R2 37.7 2.6 N N 2030 1650 680 190 107 148

918 LC-MS/MS R1 45.2 2.79 0.8 N 2100 228 126 118

R2 45.8 2.29 1 N 2370 218 119 141

218 LC-MS/MS R1

R2

10 LC-MS/MS R1

R2

2060 LC-MS/MS R1 41 2.4 N N 1147 1758 515.8 298.5 155.4 N 359.2

R2 35 1.7 N N 1751 1692 479.7 276.9 170.7 N 353.4

553 LC-MS/MS R1 4.31 N N N 1858 2139 509.9 301.8 206.1 25.72 79.03

R2 6.67 N N N 1591 3307 723.4 428.6 241 29.34 71.35

2052 LC-MS/MS R1 1271 1679 419.2 197.4 205.4

R2 1199 1605 376.4 173.2 135.2

555 LC-MS/MS R1 N N

R2 N N

N= none detected LC-MS/MS results 201463 Cattle feed

Reference Lab Method Overview

Aflatoxins; Modified AOAC 994.08 method

84/16 ACN/water extraction

Solid phase cleanup followed by dilutions

Analysis using HPLC post. coll. derv.

Deoxynivalenol; JOAOAC Vol. 88 No.4 MacDonald, S Et. Al.

84/16 ACN/water extraction

Solid phase cleanup then immunoaffinity cleanup

Analysis using HPLC-UV

Ocra A; AOAC #2000.03

3/1 MEOH/Water extraction

Samples diluted with PBS then ran through R-Biopharm

Rhone immunoaffinity cleanup

Analysis using HPLC-FD

T-2 & HT-2; J Ag & Food Chem. Vol. 42 Croteau, S, Et. Al.

84/16 ACN/water extraction

Solid phase cleanup using two different columns

followed by derivitization

Internal standard is added along with a Sodium

Bicarbonate solution

Analysis using GC

Zearalenone; JOAOAC Vol. 88 No.6 MacDonald, S Et. Al.

84/16 ACN/water extraction

Samples diluted with PBS then ran through R-

Biopharm Rhone immunoaffinity cleanup

Analysis using HPLC-FD

Fumonisin B1, B2, B3; Modified AOAC #2001.04 method

3/1 MEOH/Water extraction

Samples diluted with PBS then ran through R-

Biopharm Rhone immunoaffinity cleanup

Derivitization followed by dilution using a Phosphate

buffer

Analysis using HPLC-FD

Romer Labs Mycotoxin Method by LC-MS/MS

Extraction method for AFLA’s, DON, ZON, OCRA A, HT-2& T-2 (and others)

84/16 ACN/water for 90 minutes followed by filtering

A portion of filtered extract is concentrated then reconstituted

Analysis using LC-MS/MS

Fumonisin extraction

50/50 ACN/Water for 60 minutes followed by filtering

A portion of filtered extract is concentrated and reconstituted

Analysis using LC-MS/MS

Using internal standards

Correcting for recoveries

Methods received via Email for LC-MS/MS

Lab #918 Extraction method for AFLA’s, DON, FUM, ZON, OCRA A, & T-2

84/16 ACN/Water extraction for 90 minutes followed by filtering

For DON, FUM, ZON, OCRA A, & T-2 samples are diluted and ran on LC-MS/MS

Aflatoxins go through a further cleanup with Romer Myco-Sep Column and analyzed by LC-MS/MS

Having issues with Fumonisin recoveries

Suggestion: Fumonisins need a extract solution of 50/50 ACN/water

Using external standards

Lab #553 Extraction method for AFLA’s, DON, ZON, OCRA A, Mycophenolic acid & T-2

84/16 ACN/water extraction for 20 minutes followed by centrifugation

Solid phase cleanup using SAX-PSA followed by dilutions

Analyzed by LC-MS/MS

Fumonisin extraction

25/75 MEOH/water for 20 minutes followed by filtering

Analyzed by LC-MS/MS

Using external standards

Looking for extractions methods from other labs using LC-MS/MS

Matrix effects

area count

LC/MS/MS ESI

10 ppb 10 ppb % difference

no matrix in matrix

aflatoxin B1 2944 2000 32

aflatoxin B2 3630 2316 36

aflatoxin G1 2001 1199 40

aflatoxin G2 1076 525 51

Conclusions

Looking for more labs using LC-MS/MS methods to send there extraction method

84/16 ACN/water seems to be the magic ratio for AFLA’s, DON, ZON, OCRA A, & T-2

Fumonisins need a different extraction ratio (50/50-75/25)

Possibility of splitting AAFCO check sample methods up (Kit vs. LC)?

Reliably of reference lab results?

Is reference lab giving T-2 & HT-2 results?

Internal standards vs. external standards seem to be comparable

Committees guidance (feelings) on correcting for recoveries

Questions?

THANKS!!