dr clare hazel, premier foods (uk)

T2 and HT2 Toxins: Occurrence, Food Processing and Improved Analytical Methodology

Dr Clare Hazel, Premier Foods (UK)clare.m.hazel@premierfoods.co.uk

Dr Michelangelo Pascale, ISPA, CNR (Italy)michelangelo.pascale@ispa.cnr.it

Presentation Aims

To contribute data from two recently completed UK Government funded projects addressing:

1.Occurrence and fate of Fusariummycotoxins during commercial food processing – focus today on T2 and HT2 toxins

2.Development of improved methods for the determination of low levels of T2 and HT2 toxins in foodstuffs

Sixth Fusarium Forum 2009 9th-10th February 2009

Agenda

FSA projectGC-MS (Premier Foods, UK)HPLC-FD (ISPA-CNR, Italy)HPLC/MS-MS (ISPA-CNR, Italy)

ISPA-CNR ongoing researchDetermination of total T-2 and HT-2

toxins in cereals by enzymatic hydrolysis of T-2 toxin

Development of improved methods for the determination of low levels of T2 and HT2 toxins in foodstuffs

IntroductionSampling and analysisOccurrence and fate during processing

OatsWheatMaize

Conclusions

Occurrence and fate of Fusarium mycotoxins during commercial food processing –focus on T2 and HT2 toxins

Fusarium Mycotoxins in Cereal Food ChainFunding

UK GovernmentUK Cereal Industry and Food Manufacturers

ConsortiumUnique combination of government, food manufacturers and academic experts

Industrial PepsiCo (Quaker Oats)Premier Foods (formerly RHM)Smiths Flour MillersUnited BiscuitsR-Biopharm

DACSAMaizecorKelloggsCereal Partners

Academic KAS MycotoxinsCampden and Chorleywood Food Research AssociationHarper Adams University College

University of BristolCentral Science LaboratoryProf Ron Walker

Advisors Food Standards Agency (FSA)DEFRASNACMA

Home Grown Cereals AuthorityNational Association of British and Irish Millers

Overall Project Aim

UK recognised that:Fusarium mycotoxins do occur in UK and imported cerealsSignificant gaps in our knowledge e.g.• Fate in full scale commercial processing• Breakdown products, hidden metabolites and

potential toxicological significance

Project aim:To assist in the management of key mycotoxins in the cereal processing chain so as to best comply with current and future regulations and reduce the exposure of consumers to these contaminants

Focus on:Commonly occurring mycotoxins in UK cereals• Fusarium toxins

• Trichothecenes DON/NIV/T2/HT2, acetylated DON derivatives• Zearalenone (ZON)• Fumonisins (maize only)

Project Scope

Focus on:Production of milling intermediates frequently consumed in UK foodsCommercial scale

MAIZE WHEAT OATSMilling (Dry) Milling Milling

Breakfast cereals

Snacks

Bread, Cakes

Biscuits

Breakfast cereals

Oat breakfast cereals, biscuits etc

Approach

For all processes studied:Investigation conducted over multiple harvest years (2004-2007 harvests)Sampling points and sampling plans agreed at the outsetAnalytical requirements definedStudied same process on multiple occasions

Key question: Are all toxins present in the raw cereal accounted for at the end of processing?

= Process mass balance

Sampling and AnalysisSampling and Traceability

Critical that:

the samples taken are representative of the lot being processed

the raw material lot can be traced through the process

Sample points based on chemical and physical processes that may affect mycotoxinsSampling plans agreed with the UK FSA for each processPlans are as close as possible to EU sampling plan

Mycotoxin Analysis

ISO 17025 accredited method used for the determination of trichothecenesExtraction : Acetonitrile/water (84/16)Clean Up : Solid phaseDerivatisation carried out prior to determination by GC-MS multiple ion monitoring

FAPAS scores T2 and HT2: -0.1 to -1.3

Oats: Occurrence of T2 and HT2 and Fate During Milling

27 separate consignments of oats (from UK and Scandinavia (5 Finnish, 1 Swedish)) delivered to UK commercial plantFinished products and by-products taken for analysisFusarium mycotoxins analysed and mass balance across the process determined

T2 and HT2 results

Occurrence of T2 and HT2 in Oats at Intake in the UK, 2004-2007

Region No. of samples

Toxin Mycotoxin µg/kg Both present

<10 10-19

50-499

500-999 >1000 Max

UK/Eire 21T2 0 0 5 14 1 1 1610

21HT2 0 0 0 14 4 3 3570

Scandin-avian 6

T2 0 0 0 6 0 0 2216

HT2 0 0 0 4 2 0 730

Total 27T2 0 0 5 20 1 1 1610

27HT2 0 0 0 18 6 3 3570

•All consignments contained T2 and HT2 toxin. •HT2 typically 2-3 times higher than T2 toxin•Level in UK oats higher than in Scandinavian oats

44% of raw oats >500µg/kg T2 + HT2

Oat ProcessingSampleRaw Oats

Cleaning

Dehulling

Groat Husks/By-product

PelletKilning

Light Dark

Oat Flakes

Sample

Colour sorted

Sample

HT2 - Fate During Milling

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Sample

Raw oatOat flakePellet

• T2 and HT2 toxins show the same pattern• Dehulling, removal of husks results in a very large loss of both T2

and HT2• High level in the husk, and hence the pellet by-product• Lower levels in oat flakes, max combined = 120µg/kg

(µg/kg) Raw oats Oat flakes Oat pelletsT2 Mean 219 21 921

Range 25-1610 <10-65 71-6120HT2 Mean 581 25 2363

Range 81-3570 <10-55 306-23580

Oat Processingµg

Oat Summary

RAW OATST2 and HT2 co-occur in all samples (UK and Scandinavian origin)T2 + HT2 >500µg/kg in 44% of samples

PROCESSINGProcessing of oats highly effective in reducing the level of all trichothecenes in the oat flakes Reductions of >90% consistently achievedOat flakes maximum T2 + HT2=120µg/kgMycotoxins concentrated in the pellet by-products for animal feedAll toxins accounted for in process streams (mass balance)

Wheat: Occurrence of T2 and HT2 and Fate During Milling

60 consignments (2004-2007) Primarily UK originDestined for bread making (35 lots) and breakfast cereal production (25 lots)Finished products and by-products taken for analysisMass balance across the process determined

Occurrence of T2 and HT2 in Wheat at Intake in the UK, 2004-2007

Intake No. of samples

Toxin Mycotoxin µg/kg Both present

<10 10-19

50-499

500-999 >1000 Max

At wheatmills

35T2 33 2 0 0 0 0 12

1HT2 30 5 0 0 0 0 13

At breakfast cereal plant

25T2 24 1 0 0 0 0 13

1HT2 18 5 2 0 0 0 28

Low incidence of low levels of T2 and HT2 in wheat

Wheat MillingWHEAT

CLEANEDWHEAT

CONDITIONEDWHEAT

BREAKSIFTER

REDUCTIONSIFTER

REDUCTIONROLLS

SAMPLE 1

SAMPLE 3 SAMPLE 5

SAMPLE 4

SAMPLE 6BREAKFLOUR

WHEAT FLOUR

BRAN WHEATGERM

SAMPLE 7 SAMPLE 8

Preliminary cleaning

SCREENINGS− Large foreign objects− Metal, Stones, Wood

− Other cereals, seeds, weed seeds, straw, sticks

− Shriveled grain

SCREENINGS

BREAK ROLLSSAMPLE 2

T2 and HT2 and Fate During Dry Milling of Wheat

ns = no sample supplied.

52956636252466414451391336214666

<102917ns

<101414

<10<1026

<10102313

<101112

<10<10<10<10<10<10<10<10<10<10<10<10<10

28353624222977435756491841223662

<101311ns102123111334

<10123116

<10<10<10<10<10<1012

<10<10<10<10<10<10<10

BranGermWheatBranGermWheatHT2T2

Mycotoxin µg/kg

Both T2 and HT2 found to concentrate in germ and bran fractions not detected in white flour

Wheat SummaryCLEANED WHEAT

Low incidence of low levels of T2 and HT2 toxins over four harvest years

EFFECT OF MILLING

T2 and HT2 concentrate in germ and bran fractions

Lower in white flour

SUBSEQUENT PROCESSING

BREAD AND CAKE BAKING, BISCUIT and CRACKER PRODUCTION

Levels too low for meaningful study

Maize: Occurrence of T2 and HT2 and Fate During Milling

86 consignments (2004-2007) 56 from France, 30 from Argentina

Destined for snack product and breakfast cereal manufactureFinished products and by-products taken for analysisMass balance across the process determined

Occurrence of T2 and HT2 in Maize at Intake in the UK, 2004-2007

Region

No. of samples

Toxins Mycotoxin µg/kg Both present

<10 10-19

50-499

500-999 >1000 Max

French 1Dent

26T2 13 9 2 2 0 0 107

12HT2 10 6 7 3 0 0 149

French 2Dent

30T2 21 3 6 0 0 0 29

9HT2 12 9 7 2 0 0 65

French Combined 55

T2 34 12 8 2 0 0 10721

HT2 22 15 14 5 0 0 149

ArgentinaFlint

30T2 30 0 0 0 0 0 <10

0HT2 30 0 0 0 0 0 <10

Origin of the maize significant. Type of maize may be significant?

French maize, two samples >100µg/kg T2 + HT2

Dry Milling of MaizeRaw maize

Temper

De-germ

Range of products depending on final food product

Tortilla

Mill 1 : French (SW)

Maize grits/flour

Extruded snacks

Flaking grits

Breakfast cereals

Mill 2 : French (Loire)

Mill 3 : Argentinean

T2 and HT2 in Milling Streams from France

11-93<10-23<10-25<10<10-107Range

100%44%17%039%Incidence

Meal/branGerm Flour(<500µ)

Grits(>500µ)

Whole maizeT2

18-213<10-62<10-30<10-20<10-149Range

100%61%22%9%70%Incidence

Meal/branGerm Flour(<500µ)

Grits(>500µ)

Whole maizeHT2

T2 and HT2 concentrated in the germ, meal and bran fractions

Lower in the grits and flour fractions

Maize SummaryMAIZE

French: Moderate incidence of low levels of T2 and HT2 toxins over four harvest years, only 4% exceeded combined level of 100µg/kg (Dent maize)

No T2 or HT2 in Argentinean maize (Flint maize)

EFFECT OF MILLING

T2 and HT2 concentrate in germ and meal/bran fractions

Low incidence of low levels in flour and grits

SUBSEQUENT PROCESSING

SNACK and BREAKFAST CEREAL PRODUCTION

Levels too low for meaningful study

Conclusions

Data presented shows that over the four years of the project, T2 and HT2 toxins:

• Occur in different cereals at different incidences and levels

e.g. oats compared to wheat

• Different geographical origins of the same cereals can have a very different levels

• Oats: UK compared to Scandinavia

• Maize: France compared to Argentina (but may be dent vs. flint)

• Processing results in reduction of levels in the some human foodfractions

• Considerable increases in fractions intended for feed

T2 and HT2:Improved Analytical Methodology

UK FSA Funded Project 2007-2008

Improved Analytical Methodology

AimsTo develop analytical methods for T2 and HT2 toxins having a combined limit of quantification of 8 µg/kg (5 µg/kg T-2 and 3 µg/kg HT-2)

Validated for cereal and cereal based food stuffs (oats and processed cereal products: breakfast cereals, biscuits, pasta, snack products)

Assessment of T-2 and HT-2 Standard PurityDetermination of the Molar Extinction Coefficient of T-2 and HT-2 toxinsEvaluation of Immunoaffinity Columns for Sample Clean UpGC-MS Method Development

Raw Wheat, Barley, Maize, OatsProcessed Wheat, Maize, Oat Products

HPLC-FD Method DevelopmentRaw Wheat, Barley, Maize, OatsProcessed Wheat, Maize, Oat Products

HPLC-MS/MS Method DevelopmentRaw Wheat, Barley, Maize, OatsProcessed Wheat, Maize, Oat Products

In-house validation of developed methods

Improved Methodology: Objectives

Improved Methodology: Phase 1

OutcomeObjective

Absolute toxin amount released from the IACs < 200 pgAbsolute toxin amount released from the IACs < 200 pgThe contamination from the IACs will result < 0.2 The contamination from the IACs will result < 0.2 µµg/kgg/kgThe tested IACs are suitable for further method The tested IACs are suitable for further method developmentdevelopment

To assess T-2 and HT-2 toxin releasefrom immunoaffinitycolumns (IACs) by HPLC-MS/MS

To obtain a value for the molar extinction coefficient (ε) for both T-2 and HT-2 toxins

Commercially available standards were assessed by GC-MS and LC-MS/MSTT--2 and HT2 and HT--2 standard purity was shown to be 2 standard purity was shown to be greater than 97%greater than 97%

To assess T2 and HT2 standard purity

ε (T-2 toxin) = 4022 ± 56 (L mol-1 cm-1)

ε (HT-2 toxin) = 4001 ± 48 (L mol-1 cm-1)

Analytical Method Development Protocol

Based on existing methods improvementAssessed on unprocessed cereals

Limits of quantification determined for T2 and HT2 (based on signal/noise ratio of 10/1)

Assessed for use in processed food productsMethod validation (using specially prepared blank food test materials)

Precision/recoveryLinearity/range

Uncertainty of measurement determinedMethods documented and reviewed

T2 and HT2:Improved Analytical Methodology GC-MS Developments

GC-MS Method - Current

Existing GC-MS end determination methodAcetonitrile/water extractionSolid phase (SPE) column clean upTMS derivatives

Current LoQ = 10µg/kg per toxinBased on most abundant ion responseFour ions monitored for each toxin

Broad matrix applicability

347122HT2

350122T2Ions (m/z)Toxin

44.80 44.90 45.00 45.10 45.20 45.30 45.40 45.50

Time-->

Abundance

TIC: BWS01.D

44.99 45.29

T2-TMS

HT2-TMS

Assessment of IAC columnsGood clean up on raw cereal and food productsSensitivity limited by column loading capacity

Extraction: Conventional 84/16, acetonitrile/waterAdditional sample extract loaded onto SPE columnPost clean up and derivatisation steps, concentrate extract further prior to GC-MS

Increase in sensitivity (LoQ based on = S/N 10/1)

Interferences and recovery assessed at all stages-all results corrected for recovery

Improved GC-MS MethodImproved GC-MS for the determination ofT-2 and HT-2 toxinsImproved GC-MS for the determinationdetermination ofofTT--2 and HT2 and HT--2 toxins2 toxins

ExtractionGround sample (20 g) with

acetonitrile/water (84/16 v/v, 100 mL)

Solid phase columnclean-up

TMS Derivatisation-hexanebackwash

GC-MS determination

Filtered extract (20mL, 4 g of matrix)

Wheat, Barley and Maize-Raw and Processed(for some matrices it proved difficult to find blank samples at the limits of detection being achieved, few barley samples tested were positive, raw oats contain high levels of T2 and HT2 toxin, processed oat products positive at these low levels)

Recovery (%)

LoQ (µg/kg)

Recovery (%)

LoQ (µg/kg)

<1<1 >80%>80%Increased SPE loading and decreased hexane volume

HT2-TMS Ion 185T2-TMS Ion 122CEREAL BLANK (wheat/maize)

GC-MS Method- Improved

Recovery (%)

LoQ (µg/kg)

Recovery (%)

LoQ (µg/kg)

<1<1 >80%>80%Increased SPE loading and decreased hexane volume

HT2-TMS Ion 185T2-TMS Ion 122CEREAL PROCESSED (wheat breakfast cereal/maize snack)

Improved GC-MS Method: Performance

Linear over range 0-20µg/kgLinearity/Range

At levels of 1.0-6.2 µg/kg, repeatability (RDSr) 4.4-27.7%Repeatability

Spiking at multiple levels in multiple matrices (unprocessed andprocessed cereals). All recoveries 82-99%

Recovery

Achieves combined LoQ of 2µg/kg (1µg/kg for T2 and HT2) in all matrices (where blanks available)

Sensitivity

T2/HT2 Linearity

R2 = 0.9999

100000

150000

200000

250000

300000

350000

400000

450000

0 5 10 15 20 25

µg/kg

MAIZE BASED BREAKFAST CEREALSpiked with T2 and HT2

The optimised method with improved sensitivity has been validated on wholewheat pasta, oat based breakfast cereal and cornflakes:

Uncertainty of measurementDetermined as defined in EC No 401/2006 - three matrices

Improved GC-MS Method: Performance

* Uf = maximum standard uncertainty Uf = [ (LoD\2)2 + (α × C)2 ]1\2

3636200

1150.30.3

HT2T2HT2T2

Uf (µg/kg)Spike Levels (µg/kg)

LoD (µg/kg)

T2 and HT2:Improved Analytical Methodology HPLC-FD Developments

HPLC-FD Method - Current

Existing HPLC-FD method (Visconti et al., 2005)Methanol/water + NaCl extractionImmunoaffinity column clean up (extract dilution with water)Pre-column derivatisation with 1-ANApplicability: wheat, maize, barleyLoD (signal/noise ratio of 3/1) = 5 µg/kg (T2) and 3 µg/kg (HT2)

Column: Phenyl-Hexyl Luna® (150 x 4.6 mm, 5 µm)

Mobile phase: gradient CH3CN:H2O (70:30 to 85:15)

Flow rate: 1 mL/min

Detection: fluorescence (λex.=381 nm, λem.=470 nm)

Derivatisation: pre-column with 1-anthroylnitrile

Derivatisation with 1-ANDerivatisation Derivatisation withwith 11--ANAN

IAC clean-upIAC IAC cleanclean--upup

Extractionsample (50 g) with methanol:water (9:1

v/v, 100 mL) + NaCl (1 g)

Extractionsample (50 g) with methanol:water (9:1

v/v, 100 mL) + NaCl (1 g)

Filtration (Wathman No. 4)Dilution 1:5 (v/v) with:PBS or 4% NaClFiltration (Wathman GF/A)

HPLC/FD HPLC/FD determinationdetermination

Improved HPLC-FD for the determination ofT2 and HT2 toxins in cereal grains

Improved HPLC-FD for the determinationdetermination ofofT2 and HT2 toxins in cereal T2 and HT2 toxins in cereal grainsgrains

Refinements

Additional sample extractloaded onto IAC20 mL = 2 g matrix equivalent

Different dilution solutionPBS (wheat, barley, oats) 4% NaCl (maize)

Use of different commercial immunoaffinity columns

2 4 6 8 10 12 14 16 18 20 22

blank wheat

9,8 10,0 10,2 10,4 10,6 10,8 11,0 11,2 11,4 11,6 11,8 12,0 12,2 12,4 12,6

17,5 18,0 18,5 19,0 19,5 20,0 20,5 21,0 21,5 22,0 22,5 23,0 23,5

S/N= 11

S/N= 12

2 4 6 8 10 12 14 16 18 20 22

spiked wheat

T2-(1AN)(4.3 µg/kg)

HT2-(1AN)2

(2.5 µg/kg)

T2-(1AN)

HT2-(1AN)2

Chromatographic profiles(Rhône IAC/20 mL =2 g matrix equivalent on column)

LoQ T-2 = 4 µg/kg

LoQ HT-2 = 2.5 µg/kg

Wheat spiked with T2 and HT2 at 5 µg/kg and 3 µg/kg,

respectively

Chromatographic profilesChromatographic profilesChromatographic profilesHPLC-FD chromatograms of a naturally contaminated maize sample by using water (A) and a 4% NaCl solution (B) as dilution solvent.

dilution with 4% NaClsolution

dilution with PBSdilution with PBS

Sodium chloride is necessary to precipitate the interfering compounds (e.g. proteins) that provided turbidity of solutions after dilution and that co-eluted with HT2-(1-

AN)2 derivative.

RSDr(%)

Recovery (%)

RSDr(%)

Recovery (%)

>85.4>82.2 <3.1<3.6wheat/maize/oats/barley

HT2 toxinT2 toxin

CEREAL

HPLC-FD Method - ImprovedLimit of quantification (LoQ) of the method, recovery and repeatability (RSDr) for raw cerealsraw cereals spiked with T2 and HT2 toxins at 100 µg/kg

LoQ (µg/kg)signal to noise ratio of 10:1

6.0 - 6.62.0 - 2.64.0wheat/maize/oats/barley

T2+HT2HT2 toxinT2 toxin

CEREAL

1.9855.288maize based snack-fried

2.5973.890oats based breakfast cereal

5.1963.592wheat based breakfast cereal

4.5984.787cornflakes

3.7992.794wholewheat pasta

3.0874.085pasta

(%)Recovery

(%)RSDr

(%)Recovery

(%)Matrix

HT-2 toxinT-2 toxin

Assessment for use in processed cereal productsAssessment for use in processed cereal products

*RSDr, relative standard deviation (n=3)

Recovery experiments were performed at level of 100 µg/kg T2 toxin and 100 µg/kg HT2 toxin (triplicate experiments).

For maize based snack-extruded and wheat based biscuits interfering peaks at the retention time of HT-2 derivative were observed

The optimized method with improved sensitivity has been validated on wholewheat pasta, oat based breakfast cereal and cornflakes:

wholewheat pasta cornflakes oat based breakfast cereal

A single batch of three uncontaminated (‘blank’) test materials were used for in-house validation aimed to determine precision, recovery at low (40 µg/kg, 10 × LoQ) and high levels (200 µg/kg, 50 × LoQ), selectivity and LoQ.

In-house validation of HPLC-FD improved method

Limit of quantification (LoQ) of the HPLC-FD method, recovery andrepeatability (RSDr) for wholewheat pasta, cornflakes, oat based breakfast wholewheat pasta, cornflakes, oat based breakfast cerealcereal spiked with T2 and HT2 toxins at 40 µg/kg and 200 µg/kg.

RSDr(%)

Recovery (%)

RSDr(%)

Recovery (%)

>95.8>83.3 <5.9<6.6wholewheat pastacornflakesoat based breakfast cereal

HT2 toxinT2 toxin

Processed cereal product

6.4 - 6.92.5 - 2.73.9 - 4.3wholewheat pastacornflakesoat based breakfast cereal

36362008840

0.81.3

HT2T2HT2T2

Uf *(µg/kg)Spike Levels

(µg/kg)LoD (µg/kg)

HPLC-FD Method - Improved

T2 and HT2: Improved Analytical Methodology HPLC-MS/MS Developments

Improved HPLC-MS/MS for thedetermination of T2 and HT2 toxinsImproved HPLC-MS/MS for thedeterminationdetermination ofof T2 and HT2 toxinsT2 and HT2 toxins

Solid phase columnclean-up (Oasis® HLB)Solid phase column

clean-up (Oasis® HLB)

HPLC-MS/MS determinationHPLC-MS/MS determination

Filtered extract(5mL, 1 g of matrix)

Development of sample extractionand clean up procedure

Matrix Effect InvestigationEvaluation of the influence of co-elutingmatrix compounds on ionisation of analytes

Optimisation of MS/MS parameters(triple quadrupole)

APCI ion sourcePositive ionisation

Modifier: 5 mM ammonium acetate

Based on most abundant ion response, five ions monitored for each toxin (MRM mode)

Analyte Precursor Ion Q1 (m/z) Q3 (m/z) 323.2 263.3 245.2 215.1

HT-2 [HT-2+NH4]+ 442.1

365.1 305.2 245.2 215.1

T-2 [T-2+NH4]+ 484.2

Comparison of calibration curves obtained by dissolving standard T2/HT2 toxins in: ------ HPLC mobile phase; ------ Matrix extract purified on Oasis HLB column

100000

200000

300000

400000

500000

600000

700000

0 2.5 5 7.5 10 12.5

ng inj

mobile phasew heat

Matrix Effect

Matrix assisted calibration is necessary for reliable quantitative analyses

100000

150000

200000

250000

0 2.5 5 7.5 10 12.5ng inj

mobile phase

w heat

HT2 toxinHT2 toxinT2 toxinT2 toxin

13C24 T2 toxin and 13C24 HT2 toxin are available starting from 2008 (Biopure, Austria)

Matrix assisted calibration can be avoided using Internal Standards

RSDr* (%)

Recovery* (%)

RSDr* (%)

Recovery* (%)

>72>70 <9<7wheat/maize/oats/barley

HT2 toxinT2 toxin

CEREAL

HPLC-MS/MS Method - ImprovedLimit of quantification (LoQ) of the method, recovery and repeatability (RSDr) for raw cerealraw cereal spiked with T2 and HT2 toxins

3.6 - 6.61.9 - 5.11.4 - 2.5wheat/maize/oats/barley

CEREAL

* Spiking range 10-500 µg/kg, n= 3 replicates

ExtractionGround sample (50 g) with methanol:water

(9:1 v/v, 100 mL, 1 g Na Cl)

ExtractionGround sample (50 g) with methanol:water

(9:1 v/v, 100 mL, 1 g Na Cl)

Immunoaffinity columnclean-up

HPLC-MS/MS determinationHPLC-MS/MS determination

Filtration(dilution with water)

HPLC-MS/MS Method – Improving sensitivity

HT2HT2

0.80.80.50.50.30.3IAC IAC (100 mg inj)(100 mg inj)

6.16.13.63.62.52.5SPE SPE (20 mg inj)(20 mg inj)

Combined Combined LoQLoQT2T2LoQ (LoQ (µµg/kg)g/kg)

Limit of quantification (LoQ), recovery and repeatability (RSDr)for wholewheat pasta, cornflakes, oat based breakfast cerealwholewheat pasta, cornflakes, oat based breakfast cereal spiked with T-2 and HT-2 toxins at 40 µg/kg and 200 µg/kg.

RSDr(%)

Recovery (%)RSDr (%)Recovery

>98>89 <7.5<7.5wholewheat pastacornflakesoat based breakfast cereal

HT2 toxinT2 toxin

6.0 - 7.94.5 - 5.91.5 - 3.1wholewheat pastacornflakesoat based breakfast cereal

In-House Validation of HPLC-MS/MS improved method

88401.61.0

36362008840

1.50.53636200

36362008840

2.00.7

HT2T2HT2T2

Uf *(µg/kg)Spike Levels

(µg/kg)LoD (µg/kg)

HPLC-MS/MS Method - Improved

oat based breakfast cereal

cornflakes

wholewheatpasta

Conclusions – Improved Analytical Methodology

Three improved methods (HPLC-FD, GC-MS, LC-MS/MS) for the determination of T-2 and HT-2 in cereals and cereal based products have been developed

Recovery and repeatability values fulfill criteria established by the Commission (Regulation EC No. 401/2006) for the acceptance of T2 and HT2 analytical methods

All the methods achieved LoQs of 8 µg/kg for the combined toxins (5 µg/kg T-2 and 3 µg/kg HT-2), or less, and all have been fully validated in multiple matrices (raw wheat, maize, barley and oats and several derived products)

Methods applicable for use by analytical laboratories for due diligence checks or for enforcement purpose

Three SOPs of the developed methods have been written in ISO format.

UK Food Standards Agency for financial support

All project participants for their support and enthusiasm

Acknowledgements

Lattanzio et al., J. Food Prot. (submitted)

Fusarium Forum • Brussels • 9-10 February, 2009

Determination of total T-2 and HT-2 toxins in cereals by enzymatic hydrolysis of T-2 toxin

Lattanzio M.T.V., Solfrizzo M., Pascale M., Visconti A.Institute of Sciences of Food Production (ISPA), CNR - Bari, Italy

The selective deacetylation of T-2 toxin at C4 position to give HT-2 has been observed during cereals extraction with phosphate buffer (PSB).

This conversion is due to some carboxylesterase activity of cereals.

Carboxyl esterase activities toward pesticide and xenobiotic esters have recently found in maize and sorghum (Gershater et al, 2006, Phytochemistry, 67:2561).

enzymes

OHOCOCH3

3 2 2(CH ) CHCH OCO

HT-2 toxin

916 10

(carboxylesterases)

PBS (shaking, 1 h, r.t.)

O COCH3OCOCH3

(CH3)2CHCH2OCO

T-2 toxin

916 10

HT-2WHEATWHEAT6000

300 60 12090time (min)

Time course of natural conversion of T-2 into HT-2 in maize, wheat, oats and barley spiked with T-2 (5µg) and extracted with PBS.

HT-26000

300 60 12090time (min)

MAIZEMAIZE

HT-26000

300 60 12090time (min)

OATOATT2

HT-26000

300 60 12090time (min)

BARLEYBARLEY

conversion yield: 100% conversion yield: 89%

conversion yield: 42% conversion yield: 35%

T-2 decrease in wheat, oats and barley spiked with 200 mg/kg T-2 and extracted with PBS in presence or absence (control) of maize protein extract.

The complete conversion of T2 into HT2 in wheat, oats and barley can be obtained carrying out the extraction with phosphate buffer in presence of maize protein extract.

0 min 30 min 60min 90 min 120 min 180 min

control

+ 20 mg maize proteins

WHEATWHEAT

control

BARLEYBARLEY

control

OATSOATS

The extraction with phosphate buffer in presence of maize proteins can allow an accurate estimation of the sum of T-2 and HT-2 in cereals

Determination of total T-2 and HT-2 in cerealsDeterminationDetermination ofof total Ttotal T--2 and HT2 and HT--2 in 2 in cerealscereals

LCLC--MS/MSMS/MSHPLCHPLC--FD FD ((pprere--columncolumn derivatisation derivatisation withwith 11--AN)AN)

ImmunoaffinityImmunoaffinity columncolumncleanclean--upup

Extraction

Filtration

Phosphate buffer (pH 7.4) and maize protein extract

Antibody Antibody –– basedbased methodsmethodsELISAELISAFluorescenceFluorescence polarizationpolarizationStrip test Strip test

Perspectives of application:

RecoveriesRecoveries and and repeatabilityrepeatability (LC(LC--MS/MS/MSMS) )

Spiking level, T2 + HT2 (µg/kg)

Recovery, % (RSDr, %)*

Maize 20 95 (4.0)40 96 (6.9)

100 97 (0.2)200 94 (1.6)400 72 (2.1)

Wheat 20 68 (5.7)40 69 (2.4)

100 79 (5.6)200 84 (7.3)400 67 (3.3)

Oats 20 87 (3.4)40 73 (9.4)

100 68 (5.6)200 66 (8.1)400 61 (1.0)

* RSDr, relative standard deviation (n = 3).

LimitLimit ofofquantificationquantification

((signalsignal--toto--noisenoise ratio ratio ofof1:10)1:10)

maize 1.6 µg/kgwheat 1.3 µg/kgoats 1.3 µg/kg

EU Legislation (Commission Regulation (EC) No 1881/2006)Maximum limits will be established in unprocessed cereals and cereal products for the sum of T-2 and HT-2 toxin.

“… given the rapid in vivo conversion of T-2 to HT-2 the toxic effects of T-2 toxin and its metabolite HT-2 toxin could not be differentiated”.A group of PMTDI (provisional tolerable daily intake) of 60 ng/kg bw per day has been established for T-2 and HT-2 toxins alone or in combination.

JOINT FAO/WHO EXPERT COMMITTEE JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVESON FOOD ADDITIVES

The concept of determining the total content of T-2 and HT-2 in cereal samples, for both official control purposes and risk assessment studies, is definitely in line with incoming legislation requirements.

Conclusions – ISPAISPA--CNR CNR activitiesactivities

A method for the determination of the sum of T2 and HT2 toxins in cereals by enzymatic hydrolysis of T2 toxin has been developed.

The method has been validated in maize, wheat and oats with LoQ = 1.5 µg/kg for the combined toxins (T2+HT2).

Recovery and repeatability values fulfill criteria established by the Commission (Regulation EC No. 401/2006) for the acceptance of T2 and HT2 analytical methods.

The method uses PBS as extraction solvent, avoiding the use of organic solvents.

The method uses LC-MS/MS or HPLC-FD detection and is quite promising for application in rapid antibody-based methods.

The concept of determining the total content of T2 and HT2 in cereal samples is definitely in line with incoming legislation requirements that is considering the sum of T-2 and HT-2 toxins for maximum permitted levels.

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