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OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control Service Agriculture Analytical Service Mycotoxin Analysis - Extensive Evaluation and New Methodology Tim Herrman Professor and Director Presented at the III Global Feed and Food Congress Cancun, Mexico April, 2010

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Page 1: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMISTTexas Feed and Fertilizer Control Service Agriculture Analytical Service

Mycotoxin Analysis - Extensive Evaluation and New Methodology

Tim HerrmanProfessor and Director

Presented at the III Global Feed and Food Congress

Cancun, MexicoApril, 2010

Page 2: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Page 3: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Page 4: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

1 2 3 4 7 8 9 10 11 12 13 14 15 SumTotal no. of elevators receiving corn 79 43 14 18 18 8 23 7 1 10 26 16 3 266.0Elevators with License 23 19 8 6 10 6 12 5 0 8 17 9 1 124.0% Elevators with License 29% 44% 57% 33% 56% 75% 52% 71% 0% 80% 65% 56% 33% 47.0%Elevators testing for aflatoxin 79 30 14 18 18 2 22 7 1 10 25 16 2 244.0% Elevators testing for aflatoxin 100% 70% 100% 100% 100% 25% 96% 100% 100% 100% 96% 100% 67% 92%Elevators not testing for aflatoxin 0 13 0 0 0 6 1 0 0 0 1 0 1 22.0% Elevators not testing for aflatoxin 0% 30% 0% 0% 0% 75% 4% 0% 0% 0% 4% 0% 33% 8%Elevators testing that were positive 31 31 13 16 16 2 19 5 0 7 19 15 0 174.0% Elevator testing positive 39% 72% 93% 89% 89% 25% 83% 71% 0% 70% 73% 94% 0% 65.0%Elevators correctly labeling 70 39 14 16 16 3 22 6 0 9 14 13 1 223.0% Elevators correctly labeling 89% 91% 100% 89% 89% 38% 96% 86% 0% 90% 54% 81% 33% 84.0%Elevators not correctly labeling 9 2 0 2 1 2 1 0 0 1 11 3 0 32.0% Elevators not correctly labeling 11% 5% 0% 11% 6% 25% 4% 0% 0% 10% 42% 19% 0% 12.0%Elevators unknown if correctly labeling 0 2 0 0 1 3 0 1 1 0 1 0 2 11%Elevators unknown if correctly labeling 0% 5% 0% 0% 6% 38% 0% 14% 100% 0% 4% 0% 67% 4.0%Elevators with completed records 39 40 14 12 16 3 23 7 1 9 7 10 1 182% Elevators with completed records 49% 93% 100% 67% 89% 38% 100% 100% 100% 90% 27% 63% 33% 69.0%Elevators with incompleted records 40 3 0 6 1 2 0 0 0 1 16 6 0 75% Elevators with incompleted records 51% 7% 0% 33% 6% 25% 0% 0% 0% 10% 61% 38% 0% 28.0%Elevators unknown if records are completed 0 0 0 0 1 3 0 0 0 0 3 0 2 9%Elevators unknown if records are completed 0% 0% 0% 0% 6% 38% 0% 0% 0% 0% 12% 0% 67% 3.0%Total Corn Received (bu) 73,140,006 60,775,891 5,506,997 4,860,917 5,376,160 8,703 7,082,752 1,276,618 5,991,222 1,850,176 12,226,127 6,657,662 0 184,753,231

Total Corn shipped (bu) 13,951,219 42,160,403 1,888,671 1,193,686 1,745,965 0 2,566,019 518,252 0 375,000 4,526,995 6,838,987 0 75,765,197

Adulterated corn shipped w/label (bu) 822,098 2,823,986 938,671 624,251 440,602 0 632,786 1,800 0 118,000 185,898 56,000 0 6,644,092

AREA

Page 5: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

SumTotal no. of e levators receiving corn 266E levators with License 124% Elevators with License 0.47E levators testing for aflatoxin 244% Elevators testing for aflatoxin 0.92E levators not testing for aflatoxin 22% Elevators not testing for aflatoxin 0.08E levators testing that were positive 174% Elevator testing positive 0.65E levators correctly labeling 223% Elevators correctly labeling 0.84E levators not correctly labeling 32% Elevators not correctly labeling 0.12E levators unknown if correctly labeling 11% Elevators unknown if correctly labeling 0.04E levators with completed records 182% Elevators with completed records 0.69E levators with incompleted records 75% Elevators with incompleted records 0.28E levators unknown if records are completed 9% Elevators unknown if records are completed 0.03Total Corn Received (bu) 184753231

Total Corn shipped (bu) 75765197

Adulterated corn shipped w/label (bu) 6644092

Page 6: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Laws, Rules, and Policies

Texas Commercial Feed Control Act, Chapter 141

Texas Commercial Feed Rules, Chapter 61

Feed Industry Memorandum No. 5-12Distribution of Aflatoxin-Containing Whole Grain and Oilseed in Commercial Channels and Their Use in Mixed Feed

Page 7: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Texas Commercial Feed Control Act

§141.002. Commercial Feed.

(c) The following are not commercial feeds subject to this Chapter:(2) whole grain or whole seed not containing toxins or chemical

adulterants;

§141.148. Distribution of Adulterated Feed.

(a) A person commits an offense if the person distributes, conspires todistribute, or causes another person to distribute commercial feed:(1) that is of a composition, quantity, or quality that is below or isdifferent from that which it is represented to possess by its label;(2) that is moldy, sour, heated, or otherwise damaged, because ofwhich it is injurious to animals;(6) that contains or bears a poisonous or deleterious substance thatmay render it injurious to animals under ordinary conditions of use;(9) that is otherwise unfit for feeding to animals;

§141.051. Labeling of Commercial Feed.

Page 8: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Rules: SUBCHAPTER H. ADULTERANTS

§61.61 Poisonous or Deleterious Substances

(6) grain, oilseeds, processed grain and oilseed meals containing aflatoxin B1, B2, G1, G2 above 20 parts per billion (ppb) individually or total except that with proper labeling as approved by the Office of the Texas State Chemist as follows: <50 ppb may be distributed when destined for wildlife; <100 ppb may be distributed when destined for breeding cattle and breeding goats not used in production of milk for human consumption, breeding swine, mature poultry, and sheep; <200 ppb may be distributed when destined for finishing swine (more than 100 lbs. body weight); <300 ppb may be distributed when destined for finishing cattle in confinement; grain containing >300 to <500 ppb requires a blending permit issued by the Office of the Texas State Chemist; aflatoxin >500 ppb in grain and >300 ppb in oilseed, processed grain, and oilseed meal may not enter commerce and a record of disposition shall be submitted to the Office of the TexasState Chemist.

Page 9: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Rules: SUBCHAPTER H. ADULTERANTS

§61.61 Poisonous or Deleterious Substances

(7) grain, oilseeds, processed grain, and oilseed meal containing fumonisin above 5 parts per million (ppm) except that with proper labeling as approved by the Office of the Texas State Chemist and targeted for animal species as follows: <20 ppm for swine and catfish not to exceed 50% of diet; <30 ppm for breeding ruminants, breeding poultry and breeding mink not to exceed 50% of diet; <60 ppm for ruminants > 3 months old being raised for slaughter, and mink being raised for pelt production not to exceed 50% of diet; <100 ppm for poultry being raised for slaughter not to exceed 50% of diet; all other species or classes of livestock and pet animals <10 ppm not to exceed 50% of diet except equids and rabbits which should not exceed 5 ppm and 20% of diet; >100 ppm requires a blending permit issued by the Office of the Texas State Chemist.

Page 10: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMISTFeed Industry Memorandum 5-12

Page 11: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Page 12: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Proposed Changes to Rules and Policy and Need for Further Research

Eliminate Grain Elevator licensing exemptionTest and approve rapid mycotoxin test kits for aflatoxin levels in excess of 100 ppb to 500 ppbWork to align testing procedures and results among the grain industry, risk management agency (RMA), FGIS private entities, and OTSCContinue LC/MS/MS method development for other toxins

Page 13: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

New Methodology

Conventional LC/Fluorescence MethodTime consuming, Error-prone cleanup

and derivatization steps involved

Modern LC/MS/MS methodSelective, Sensitive, High throughput

Upgrade

Wei Lee, Lead PI and AuthorSusie Dai, Co-PI, Author, CMS Team Leader

Page 14: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Waters Quattro Premier (MS-MS) with Acquity UPLC

Page 15: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Electrospray Ionization (ESI)

Triple Quadrupole System(Tri-Q)

LC/ESI/MS/MS

Page 16: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Sample Preparation and AnalysisExtraction Filtration

Solid Phase Extraction

Derivatization by NDA

Addtion ofInternal Standard

Adjust pH of the extract

LC/Fluorescence LC/MS/MS

Ground Corn Samples

Page 17: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

MRM transitions of fumonisins and isotope labeled internal standard. Total analysis time: 5 min (Submitted to JAOAC Int.,2009)

LC/MS/MS Analysis of Fumonisins

Page 18: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Analyte

Nominal concentration

(µg g-1)Recovery

(%)

Accuracy (%)

Intra-day RSD (%)

(n=5)

Inter-day RSD (%)

(n=3)FB1 0.2 95 -5 7.2 4.8

0.5 93 -7 7.9 5.51.0 95 -5 3.5 3.0

FB2 0.2 107 7 4.3 3.60.5 108 8 3.1 3.11.0 105 5 1.8 2.3

FB3 0.1 108 8 3.1 4.00.25 103 3 2.4 4.30.50 98 -2 2.7 2.3

FB1 0.2 97 -3 7.5 6.20.5 98 -2 6.8 5.81.0 93 -7 7.1 5.5

FB2 0.2 105 5 5.7 4.80.5 107 7 4.2 3.61.0 104 4 2.8 4.0

FB3 0.1 94 -6 4.4 5.40.25 96 -4 2.4 2.10.50 96 -4 2.3 3.1

Validation of LC/MS/MS Method

Food

Feed

Page 19: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Validation of LC/MS/MS MethodSample

IDAnalyte HPLC/Fluorescence

measurement (µg g-1)LC/MS/MS measurement

(µg g-1)RSD (%)for LC/MS/MS

measurements (N=5)

1 FB1 1.40 1.49 2.0FB2 0.35 0.37 6.6FB3 0.27 0.26 7.7

2 FB1 4.10 3.98 3.1FB2 1.17 1.25 3.7FB3 0.69 0.65 3.5

3 FB1 9.30 8.82 3.6FB2 2.67 2.86 3.2FB3 1.38 1.50 5.0

4 FB1 44.60 45.59 2.4FB2 13.07 13.36 6.6FB3 6.43 5.95 7.4

Comparison of LC/MS/MS method with conventional HPLC/Fluorescence method. (Submitted to JAOAC Int.,2009)

Page 20: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Aspects HPLC/Fluorescence HPLC/MS/MS

Sample preparation

10 h 2.5 h

Instrumentation 6 h 2 h

Protocol 2 days Half day

Which method is better?

Note: Time was calculated based on analysis of a set of 24 regulatory samples.

Page 21: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Summary

A fast, robust, and reliable method, for simultaneous detection of the fumonisin B1, B2 and B3 in the corn-based matrix was developed and validated.

A Standard Operating Procedure (SOP) for fumonisin analysis was established and applied of regulatory samples.

Page 22: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Furthermore ……Preliminary results of the development of Aflatoxin analysis

Multiple reaction monitoring (MRM) of Aflatoxin B1, B2, G1 and G2

Page 23: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

Manager, Quality Assurance

Dr. Jim E. Balthrop

Manager, Laboratory Operations

Sara M. Williams

Analysts in CMS group:

Ted A. OplingerCindy K. McCormick Linda G. Menefee

Lab Methods Development Acknowledgement

Page 24: Mycotoxin Analysis - Extensive Evaluation and New Methodologyotscweb.tamu.edu/OTSC-Present/2010/IFIF-tjh.pdf · OFFICE OF THE TEXAS STATE CHEMIST Texas Feed and Fertilizer Control

OFFICE OF THE TEXAS STATE CHEMIST

OFFICE OF THE TEXAS STATE CHEMIST445 Agronomy Road College Station, TX 77840

(979) 845 1121 http://otsc.tamu.edu