T2 and HT2 Toxins: Occurrence, Food Processing and Improved Analytical Methodology
Dr Clare Hazel, Premier Foods (UK)[email protected]
Dr Michelangelo Pascale, ISPA, CNR (Italy)[email protected]
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
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
20-49
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
Sample
Sample
Sample
Colour sorted
Sample
Sample
HT2 - Fate During Milling
0
500
1000
1500
2000
2500
3000
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
HT2
(µg/
lg)
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
/kg
µg/k
g
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
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
20-49
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
SILO
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
Water
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
<1019
<101112
<10<10<10<10<10<10<10<10<10<10<10<10<10
28353624222977435756491841223662
<101311ns102123111334
<10123116
<1034
<10<10<10<10<10<1012
<1010
<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
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
20-49
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
Clean
Temper
De-germ
Range of products depending on final food product
Mill
Flour
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
185
185
275
290
347122HT2
350122T2Ions (m/z)Toxin
44.80 44.90 45.00 45.10 45.20 45.30 45.40 45.50
1000
2000
3000
4000
5000
6000
7000
Time-->
Abundance
TIC: BWS01.D
44.73
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)
ExtractionGround sample (20 g) with
acetonitrile/water (84/16 v/v, 100 mL)
Solid phase columnclean-up
Solid phase columnclean-up
TMS Derivatisation-hexanebackwash
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
R2 = 0.9999
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
0 5 10 15 20 25
µg/kg
area
T2HT2
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
9950
2210
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
5
10
15
20
25
30
35
40
45
50
2 4 6 8 10 12 14 16 18 20 22
blank wheat
7,0
8,0
9,0
10,0
11,0
12,0
13,0
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
7,0
8,0
9,0
10,0
11,0
12,0
13,0
14,0
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
5
10
15
20
25
30
35
40
45
50
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)
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 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
RSDr*
(%)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
LoQ (µg/kg)signal to noise ratio of 10:1
6.4 - 6.92.5 - 2.73.9 - 4.3wholewheat pastacornflakesoat based breakfast cereal
T2+HT2HT2 toxinT2 toxin
Processed cereal product
36362008840
0.81.3
HT2T2HT2T2
Uf *(µg/kg)Spike Levels
(µg/kg)LoD (µg/kg)
Uncertainty of measurementDetermined as defined in EC No 401/2006 - three matrices
HPLC-FD Method - Improved
* Uf = maximum standard uncertainty Uf = [ (LoD\2)2 + (α × C)2 ]1\2
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
ExtractionGround sample (10 g) with
acetonitrile/water (84/16 v/v, 50 mL)
ExtractionGround sample (10 g) with
acetonitrile/water (84/16 v/v, 50 mL)
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
185.3
365.1 305.2 245.2 215.1
T-2 [T-2+NH4]+ 484.2
185.3
Comparison of calibration curves obtained by dissolving standard T2/HT2 toxins in: ------ HPLC mobile phase; ------ Matrix extract purified on Oasis HLB column
WHEAT
0
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
WHEAT
0
50000
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
LoQ (µg/kg)signal to noise ratio of 10:1
3.6 - 6.61.9 - 5.11.4 - 2.5wheat/maize/oats/barley
T2+HT2HT2 toxinT2 toxin
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
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
Processed cereal product
LoQ (µg/kg)signal to noise ratio of 10:1
6.0 - 7.94.5 - 5.91.5 - 3.1wholewheat pastacornflakesoat based breakfast cereal
T2+HT2HT2 toxinT2 toxin
Processed cereal product
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)
Uncertainty of measurementDetermined as defined in EC No 401/2006 - three matrices
HPLC-MS/MS Method - Improved
* Uf = maximum standard uncertainty Uf = [ (LoD\2)2 + (α × C)2 ]1\2
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
O
O
H3C
H
H
CH2
OH
H
CH3
H
OHOCOCH3
3 2 2(CH ) CHCH OCO
HT-2 toxin
916 10
87
6
11
1
15
12
13
2
3
45
14
(carboxylesterases)
PBS (shaking, 1 h, r.t.)
O
O
H3C
H
H
CH2
OH
H
CH3
H
O COCH3OCOCH3
(CH3)2CHCH2OCO
T-2 toxin
916 10
87
6
11
1
15
12
13
2
3
45
14
Lattanzio et al., J. Food Prot. (submitted)
T2
HT-2WHEATWHEAT6000
4000
3000
2000
1000
5000
0
ng
300 60 12090time (min)
80
60
40
20
100
0
%
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.
T2
HT-26000
4000
3000
2000
1000
5000
0
ng
80
60
40
20
100
0
%
300 60 12090time (min)
MAIZEMAIZE
T2
HT-26000
4000
3000
2000
1000
5000
0
ng
300 60 12090time (min)
80
60
40
20
100
0
%
OATOATT2
HT-26000
4000
3000
2000
1000
5000
0
ng
300 60 12090time (min)
80
60
40
20
100
0
%
BARLEYBARLEY
conversion yield: 100% conversion yield: 89%
conversion yield: 42% conversion yield: 35%
Lattanzio et al., J. Food Prot. (submitted)
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
40
80
120
160
200
0 min 30 min 60min 90 min 120 min 180 min
µg/k
g
control
+ 20 mg maize proteins
+ 30 mg maize proteins
WHEATWHEAT
0.0
40.0
80.0
120.0
160.0
200.0
0 min 30 min 60min 90 min 120 min 180 min
µg/k
g
control
+ 30 mg maize proteins
BARLEYBARLEY
0
40
80
120
160
200
0 min 30 min 60min 90 min 120 min 180 min
µg/k
g
control
+ 30 mg maize proteins
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
Lattanzio et al., J. Food Prot. (submitted)
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:
Lattanzio et al., J. Food Prot. (submitted)
RecoveriesRecoveries and and repeatabilityrepeatability (LC(LC--MS/MS/MSMS) )
Spiking level, T2 + HT2 (µg/kg)
Recovery, % (RSDr, %)*
HT2
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
Lattanzio et al., J. Food Prot. (submitted)
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.
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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