T2 and HT2 Toxins: Occurrence, Food Processing and Improved

Transkript

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]
Sixth Fusarium Forum 2009 9th-10th February 2009
Presentation Aims
To contribute data from two recently
completed UK Government funded
projects addressing:
1. Occurrence and fate of Fusarium
mycotoxins 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
Agenda
Occurrence and fate of
Fusarium mycotoxins during
commercial food processing –
focus on T2 and HT2 toxins
Introduction
Sampling and analysis
Occurrence and fate during processing
Oats
Wheat
Maize
Conclusions
Development of improved
methods for the determination
of low levels of T2 and HT2
toxins in foodstuffs
FSA project
GC-MS (Premier Foods, UK)
HPLC-FD (ISPA-CNR, Italy)
HPLC/MS-MS (ISPA-CNR, Italy)
ISPA-CNR ongoing research
Determination of total T-2 and HT-2
toxins in cereals by enzymatic hydrolysis
of T-2 toxin
Fusarium Mycotoxins in Cereal Food Chain
Consortium
Unique combination of government, food manufacturers
and academic experts
Industrial

Academic

Advisors

PepsiCo (Quaker Oats)
Premier Foods (formerly RHM)
Smiths Flour Millers
United Biscuits
R-Biopharm

KAS Mycotoxins
Campden and Chorleywood
Food Research Association
Harper Adams University College

Food Standards Agency (FSA)
DEFRA
SNACMA

DACSA
Maizecor
Kelloggs
Cereal Partners
University of Bristol
Central Science Laboratory
Prof Ron Walker
Home Grown Cereals
Authority
National Association of
British and Irish Millers
Funding

UK Government
UK Cereal
Industry and
Food
Manufacturers
Overall Project Aim
UK recognised that:
¾ Fusarium mycotoxins do occur in UK and imported
cereals
¾ Significant 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
Project Scope
Focus
on:
Commonly occurring mycotoxins in UK cereals
•
Fusarium toxins
•
•
•
Trichothecenes DON/NIV/T2/HT2, acetylated DON derivatives
Zearalenone (ZON)
Fumonisins (maize only)
Focus on:
Production of milling intermediates frequently consumed in
UK foods
Commercial scale
MAIZE
WHEAT
OATS

Milling (Dry)

Milling

Milling
¾
Breakfast cereals
¾
Bread, Cakes
¾
¾
Snacks
¾
Biscuits
Oat breakfast
cereals, biscuits etc
¾
Breakfast cereals
Approach

For all processes studied:
Investigation
conducted over multiple harvest
years (2004-2007 harvests)
Sampling points and sampling plans agreed
at the outset
Analytical requirements defined
Studied 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 Analysis
Sampling
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 mycotoxins
¾ Sampling plans agreed with the UK FSA for each process
¾ Plans are as close as possible to EU sampling plan
¾
Mycotoxin
Analysis
ISO 17025 accredited method used for the determination of trichothecenes
Extraction : Acetonitrile/water (84/16)
Clean Up : Solid phase
Derivatisation 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
plant
¾ Finished products and by-products taken
for analysis
¾ Fusarium mycotoxins analysed and mass
balance across the process determined
¾
¾ T2
and HT2 results
Occurrence of T2 and HT2 in Oats at Intake in the UK, 2004-2007
Region
UK/Eire
Scandinavian
Total
No. of
samples
21
6
27
Toxin
Mycotoxin µg/kg
Both
present
<10
1019
2049
50499
T2
0
0
5
14
1
1
1610
HT2
0
0
0
14
4
3
3570
T2
0
0
0
6
0
0
221
HT2
0
0
0
4
2
0
730
T2
0
0
5
20
1
1
1610
HT2
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
500>1000 Max
999
21
6
27
44% of raw oats
>500µg/kg T2 + HT2
Oat Processing
Raw Oats
Sample
Cleaning
Dehulling
Sample
Sample
Groat
Husks/By-product
Sample
Kilning
Pellet
Colour sorted
Sample
Light
Oat Flakes
Dark
Sample
Sample
HT2 - Fate During Milling
3000
Raw oat
Oat flake
2500
Pellet
HT2 (µg/lg)
2000
1500
1000
500
0
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
• 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
T2
HT2
µg/kg
µg/kg
Oat Processing
(µg/kg)
Mean
Raw oats
219
Oat flakes
21
Oat pellets
921
Range
25-1610
<10-65
71-6120
Mean
581
25
2363
Range
81-3570
<10-55
306-23580
Oat Summary
RAW OATS
T2 and HT2 co-occur in all samples (UK and
Scandinavian origin)
T2 + HT2 >500µg/kg in 44% of samples
PROCESSING
Processing of oats highly effective in reducing the level
of all trichothecenes in the oat flakes
Reductions of >90% consistently achieved
Oat flakes maximum T2 + HT2=120µg/kg
Mycotoxins concentrated in the pellet by-products for
animal feed
All toxins accounted for in process streams (mass
balance)
Wheat
Wheat: Occurrence of T2 and HT2 and Fate
During Milling
60 consignments (2004-2007)
¾ Primarily UK origin
¾ Destined for bread making (35 lots) and
breakfast cereal production (25 lots)
for analysis
¾ Mass balance across the process
determined
¾
Occurrence of T2 and HT2 in Wheat at Intake in the UK, 2004-2007
Intake
No. of
Toxin
samples
Mycotoxin µg/kg
<10
At wheat
mills
At
breakfast
cereal
plant
T2
33
1019
2049
2
0
Both
present
50- 500>1000 Max
499 999
0
0
0
12
35
1
HT2
30
5
0
0
0
0
13
T2
24
1
0
0
0
0
13
25
1
HT2
18
5
2
0
Low incidence of low levels of T2 and HT2 in wheat
0
0
28
Wheat Milling
WHEAT
Preliminary cleaning
−
−
SCREENINGS
Large foreign objects
Metal, Stones, Wood
SILO
SCREENINGS
−
−
Other cereals, seeds, weed
seeds, straw, sticks
Shriveled grain
CLEANED
WHEAT
SAMPLE 1
Water
CONDITIONED
WHEAT
BREAK ROLLS
SAMPLE 2
BREAK
SIFTER
SAMPLE 3
SAMPLE 5
REDUCTION
ROLLS
REDUCTION
SIFTER
WHEAT
FLOUR
BREAK
FLOUR
SAMPLE 4
SAMPLE 6
BRAN
SAMPLE 7
WHEAT
GERM
SAMPLE 8
T2 and HT2 and Fate During Dry Milling of Wheat
Mycotoxin µg/kg
T2
HT2
Wheat
Germ
Bran
Wheat
Germ
Bran
<10
<10
<10
<10
<10
<10
12
<10
10
<10
<10
<10
<10
<10
<10
<10
<10
13
11
ns
10
21
23
11
13
34
<10
12
31
16
<10
34
28
35
36
24
22
29
77
43
57
56
49
18
41
22
36
62
<10
11
12
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
29
17
ns
<10
14
14
<10
<10
26
<10
10
23
13
<10
19
52
95
66
36
25
24
66
41
44
51
39
13
36
21
46
66
ns = no sample supplied.
Both T2 and HT2 found to concentrate in germ and bran fractions not
detected in white flour
Wheat Summary
CLEANED 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
¾
86 consignments (2004-2007)
¾ 56
from France, 30 from Argentina
Destined for snack product and breakfast
cereal manufacture
for analysis
¾ Mass balance across the process
determined
¾
Occurrence of T2 and HT2 in Maize at Intake in the UK, 2004-2007
Region
No. of
samples
French 1
Dent
26
French 2
Dent
30
French
Combined
55
Argentina
Flint
30
Toxins
Mycotoxin µg/kg
Both
present
<10
1019
2049
50499
500999
>1000
T2
13
9
2
2
0
0
107
HT2
10
6
7
3
0
0
149
T2
21
3
6
0
0
0
29
HT2
12
9
7
2
0
0
65
T2
34
12
8
2
0
0
107
HT2
22
15
14
5
0
0
149
T2
30
0
0
0
0
0
<10
HT2
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
Max
12
9
21
0
Dry Milling of Maize
Raw maize
Clean
Temper
Mill 1 : French (SW)
Mill 2 : French (Loire)
Mill 3 : Argentinean
De-germ
Mill
Range of products depending on final food product
Flour
Maize grits/flour
Flaking grits
Tortilla
Extruded snacks
Breakfast cereals
T2 and HT2 in Milling Streams from France
Whole
maize
Grits
(>500µ)
Flour
(<500µ)
Germ
Meal/bran
39%
0
17%
44%
100%
Range
<10-107
<10
<10-25
<10-23
11-93
HT2
Whole
maize
Grits
(>500µ)
Flour
(<500µ)
Germ
Meal/bran
70%
9%
22%
61%
100%
<10-149
<10-20
<10-30
<10-62
18-213
T2
Incidence
Incidence
Range
T2 and HT2 concentrated in the germ, meal and bran fractions
Lower in the grits and flour fractions
Maize Summary
MAIZE
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 food
fractions
•
Considerable increases in fractions intended for feed
T2 and HT2:Improved
Analytical Methodology
UK FSA Funded
Project 2007-2008
Improved Analytical Methodology
Aims
To 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)
Improved Methodology: Objectives

Assessment of T-2 and HT-2 Standard Purity

Determination of the Molar Extinction Coefficient of T-2 and HT-2 toxins

Evaluation of Immunoaffinity Columns for Sample Clean Up

GC-MS Method Development
Raw Wheat, Barley, Maize, Oats
Processed Wheat, Maize, Oat Products

HPLC-FD Method Development

HPLC-MS/MS Method Development

In-house validation of developed methods
Improved Methodology: Phase 1
Objective
Outcome
To assess T2 and
HT2 standard purity
Commercially available standards were assessed by
GC-MS and LC-MS/MS
T-2 and HT-2 standard purity was shown to be
greater than 97%
To obtain a value for
the molar extinction
coefficient (ε) for
both T-2 and HT-2
toxins
To assess T-2 and
HT-2 toxin release
from immunoaffinity
columns (IACs) by
HPLC-MS/MS
ε (T-2 toxin) = 4022 ± 56 (L mol-1 cm-1)
ε (HT-2 toxin) = 4001 ± 48 (L mol-1 cm-1)
Absolute toxin amount released from the IACs < 200 pg
The contamination from the IACs will result < 0.2 µg/kg
The tested IACs are suitable for further method
development
Analytical Method Development Protocol
¾
¾
Based on existing methods improvement
Assessed on unprocessed cereals
¾
¾
¾
Assessed for use in processed food products
Method validation (using specially prepared
blank food test materials)
¾
¾
¾
¾
Limits of quantification determined for T2 and HT2
(based on signal/noise ratio of 10/1)
Precision/recovery
Linearity/range
Uncertainty of measurement determined
Methods documented and reviewed
T2 and HT2:Improved
GC-MS Developments
GC-MS Method - Current
Existing GC-MS end determination method
¾Acetonitrile/water
extraction
¾Solid phase (SPE) column clean up
¾TMS derivatives
Current LoQ = 10µg/kg per toxin
Based on most abundant ion response
Four ions monitored for each toxin
Abundance
T2TMS
Broad matrix applicability
7000
Toxin
T2
Ions (m/z)
122
185
290
HT2TMS
TIC: BWS01.D
6000
45.29
44.99
5000
350
4000
3000
HT2
122
185
275
2000
347
1000 44.73
44.80
Time-->
44.90
45.00
45.10
45.20
45.30
45.40
45.50
Improved GC-MS for the determination of
Improved
GC-MS
Method
T
-2 and HT
-2 toxins
T-2
HT-2

Assessment of IAC columns
Good clean up on raw cereal
and food products
Sensitivity limited by column
loading capacity

Extraction: Conventional
84/16, acetonitrile/water
Additional sample extract
loaded onto SPE column
Post 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
Extraction
Extraction
Ground
Groundsample
sample(20
(20g)g)with
with
acetonitrile/water
acetonitrile/water(84/16
(84/16v/v,
v/v,100
100mL)
mL)
Filtered extract (20
mL, 4 g of matrix)
Solid
Solidphase
phasecolumn
column
clean-up
clean-up
TMS
TMSDerivatisation-hexane
Derivatisation-hexane
backwash
backwash
GC-MS determination
GC-MS Method- Improved
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)
T2-TMS Ion 122
CEREAL BLANK
(wheat/maize)
Increased SPE loading and
decreased hexane volume
HT2-TMS Ion 185
LoQ
(µg/kg)
Recovery
(%)
LoQ
(µg/kg)
Recovery
(%)
<1
>80%
<1
>80%
T2-TMS Ion 122
HT2-TMS Ion 185
CEREAL PROCESSED
(wheat breakfast
cereal/maize snack)
LoQ
(µg/kg)
Recovery
(%)
LoQ
(µg/kg)
Recovery
(%)
Increased SPE loading and
decreased hexane volume
<1
>80%
<1
>80%
Improved GC-MS Method: Performance
The optimised method with improved sensitivity has been validated on wholewheat
pasta, oat based breakfast cereal and cornflakes:
Sensitivity
Achieves combined LoQ of 2µg/kg (1µg/kg for T2 and HT2) in
all matrices (where blanks available)
Recovery
Spiking at multiple levels in multiple matrices (unprocessed and
processed cereals). All recoveries 82-99%
Repeatability
At levels of 1.0-6.2 µg/kg, repeatability (RDSr) 4.4-27.7%
Linearity/Range
Linear over range 0-20µg/kg
T2/HT2 Linearity
450000
R2 = 0.9999
400000
350000
area
300000
R2 = 0.9999
250000
T2
200000
HT2
150000
100000
50000
0
0
5
10
15
µg/kg
20
25
MAIZE BASED
BREAKFAST CEREAL
Spiked with T2 and HT2
Improved GC-MS Method: Performance
Uncertainty of measurement
Determined as defined in EC No 401/2006 - three matrices
LoD (µg/kg)
T2
0.3
HT2
0.3
Spike Levels
(µg/kg)
Uf (µg/kg)
T2
HT2
5
1
1
10
2
2
50
9
9
200
36
36
* Uf = maximum standard uncertainty
Uf = [ (LoD\2)2 + (α × C)2 ]1\2
T2 and HT2:Improved
HPLC-FD Developments
HPLC-FD Method - Current
Existing HPLC-FD method (Visconti et al., 2005)
Methanol/water + NaCl extraction
Immunoaffinity column clean up (extract dilution with water)
Pre-column derivatisation with 1-AN
Applicability: wheat, maize, barley
LoD (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
Improved HPLC-FD for the determination of
T2 and HT2 toxins in cereal grains
Refinements

Additional sample extract
loaded onto IAC
20 mL = 2 g matrix equivalent

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

Extraction
Extraction
sample
sample(50
(50g)g)with
withmethanol:water
methanol:water(9:1
(9:1
v/v,
100
mL)
+
NaCl
(1
g)
v/v, 100 mL) + NaCl (1 g)
Filtration (Wathman No. 4)
Dilution 1:5 (v/v) with:
PBS or 4% NaCl
Filtration (Wathman GF/A)
IAC
-up
IACclean
clean-up
Use of different commercial
immunoaffinity columns
Derivatisation
-AN
Derivatisationwith
with11-AN
HPLC/FD determination
Chromatographic profiles
(Rhône
(Rhône IAC/20
IAC/20 mL
mL =2
=2 g
g matrix
matrix equivalent
equivalent on
on column)
column)
50
Wheat spiked with T2 and
HT2 at 5 µg/kg and 3 µg/kg,
respectively
blank wheat
45
40
35
13,0
12,0
30
S/N= 12
T2-(1AN)
11,0
25
10,0
20
9,0
8,0
15
7,0
10
9,8
5
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
14,0
2
50
4
6
8
10
12
14
16
18
20
22
spiked wheat
45
HT2-(1AN)2
13,0
S/N= 11
12,0
11,0
10,0
40
9,0
35
8,0
30
T2-(1AN)
25
(4.3 µg/kg)
HT2-(1AN)2
7,0
17,5
18,0
18,5
19,0
19,5
20,0
20,5
21,0
21,5
22,0
22,5
23,0
(2.5 µg/kg)
20
LoQ T-2 = 4 µg/kg
15
LoQ HT-2 = 2.5 µg/kg
10
5
2
4
6
8
10
12
14
16
18
20
22
23,5
Chromatographic profiles
HPLC-FD chromatograms of a naturally contaminated maize sample by
using water (A) and a 4% NaCl solution (B) as dilution solvent.
dilution
dilutionwith
withPBS
PBS
dilution
dilutionwith
with4%
4%NaCl
NaCl
solution
solution
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-(1AN)2 derivative.
HPLC-FD Method - Improved
Limit of quantification (LoQ) of the method, recovery and repeatability
(RSDr) for raw cereals spiked with T2 and HT2 toxins at 100 µg/kg
LoQ (µg/kg)
signal to noise ratio of 10:1
CEREAL
wheat/maize/oats/barley
T2 toxin
HT2 toxin
T2+HT2
4.0
2.0 - 2.6
6.0 - 6.6
T2 toxin
CEREAL
HT2 toxin
Recovery
(%)
RSDr
(%)
Recovery
(%)
RSDr
(%)
>82.2
<3.6
>85.4
<3.1
Assessment for use in processed cereal products
Recovery experiments were performed at level of 100 µg/kg T2 toxin and 100 µg/kg
HT2 toxin (triplicate experiments).
T-2 toxin
HT-2 toxin
Matrix
Recovery
(%)
RSDr*
(%)
Recovery
(%)
RSDr*
(%)
pasta
85
4.0
87
3.0
wholewheat pasta
94
2.7
99
3.7
cornflakes
87
4.7
98
4.5
wheat based breakfast cereal
92
3.5
96
5.1
oats based breakfast cereal
90
3.8
97
2.5
maize based snack-fried
88
5.2
85
1.9
*RSDr, relative standard deviation (n=3)
For maize based snack-extruded and wheat based biscuits interfering peaks at the
retention time of HT-2 derivative were observed
In-house validation of HPLC-FD improved method
The optimized method with improved sensitivity has been validated on
wholewheat pasta, oat based breakfast cereal and cornflakes:
cornflakes
wholewheat pasta
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.
Limit of quantification (LoQ) of the HPLC-FD method, recovery and
repeatability (RSDr) for wholewheat pasta, cornflakes, oat based breakfast
cereal spiked with T2 and HT2 toxins at 40 µg/kg and 200 µg/kg.
LoQ (µg/kg)
Processed cereal product
wholewheat pasta
cornflakes
T2 toxin
HT2 toxin
T2+HT2
3.9 - 4.3
2.5 - 2.7
6.4 - 6.9
T2 toxin
wholewheat pasta
cornflakes
HT2 toxin
Recovery
(%)
RSDr
(%)
Recovery
(%)
RSDr
(%)
>83.3
<6.6
>95.8
<5.9
HPLC-FD Method - Improved
LoD (µg/kg)
T2
1.3
Uf *
(µg/kg)
Spike Levels
(µg/kg)
HT2
0.8
T2
HT2
40
8
8
200
36
36
Uf = [ (LoD\2)2 + (α × C)2 ]1\2
T2 and HT2: Improved
HPLC-MS/MS Developments
Improved HPLC-MS/MS for the
determination of T2 and HT2 toxins
Optimisation of MS/MS parameters
(triple quadrupole)
Development of sample extraction
and clean up procedure
Extraction
Extraction
Ground
Groundsample
sample(10
(10g)g)with
with
acetonitrile/water
acetonitrile/water(84/16
(84/16v/v,
v/v,50
50mL)
mL)
Matrix Effect Investigation
Filtered extract
(5mL, 1 g of matrix)
Evaluation of the influence of co-eluting
matrix compounds on ionisation of
analytes
Based on most abundant ion response, five ions
monitored for each toxin (MRM mode)
Analyte
Precursor Ion
Q1 (m/z)
Q3 (m/z)
Solid
Solidphase
phasecolumn
column
®
clean-up
clean-up(Oasis
(Oasis®HLB)
HLB)
323.2
263.3
HT-2
[HT-2+NH4]
+
442.1
245.2
215.1
185.3
365.1
HPLC-MS/MS
HPLC-MS/MSdetermination
determination
305.2
T-2
[T-2+NH4]
+
484.2
245.2
215.1
185.3
APCI ion source
Positive ionisation
Modifier: 5 mM
ammonium acetate
Matrix Effect
Comparison of calibration curves obtained by dissolving standard T2/HT2 toxins
in: --- HPLC mobile phase; --- Matrix extract purified on Oasis HLB column
WHEAT
WHEAT
700000
250000
mobile phase
600000
mobile phase
w heat
w heat
200000
500000
400000
150000
300000
100000
200000
T2 toxin
100000
HT2 toxin
50000
0
0
2.5
5
7.5
10
ng inj
12.5
0
0
2.5
5
ng inj
7.5
10
12.5
Matrix assisted calibration is necessary for reliable quantitative
analyses
Matrix assisted calibration can be avoided using Internal Standards
13C
24
T2 toxin and
(Biopure, Austria)
13C
24
HT2 toxin are available starting from 2008
HPLC-MS/MS Method - Improved
Limit of quantification (LoQ) of the method, recovery and repeatability
(RSDr) for raw cereal spiked with T2 and HT2 toxins
LoQ (µg/kg)
CEREAL
T2 toxin
HT2 toxin
T2+HT2
1.4 - 2.5
1.9 - 5.1
3.6 - 6.6
T2 toxin
CEREAL
HT2 toxin
Recovery*
(%)
RSDr*
(%)
Recovery*
(%)
RSDr*
(%)
>70
<7
>72
<9
* Spiking range 10-500 µg/kg, n= 3 replicates
HPLC-MS/MS Method – Improving sensitivity
Extraction
Extraction
Ground
Groundsample
sample(50
(50g)g)with
withmethanol:water
methanol:water
(9:1
(9:1v/v,
v/v,100
100mL,
mL,11ggNa
NaCl)
Cl)
Filtration
(dilution with water)
Immunoaffinity
Immunoaffinitycolumn
column
clean-up
clean-up
HPLC-MS/MS
HPLC-MS/MSdetermination
determination
LoQ (µg/kg)
T2
HT2
Combined
LoQ
SPE
(20 mg inj)
2.5
3.6
6.1
IAC
(100 mg inj)
0.3
0.5
0.8
In-House Validation of HPLC-MS/MS improved method
Limit of quantification (LoQ), recovery and repeatability (RSDr)
for wholewheat pasta, cornflakes, oat based breakfast cereal spiked
with T-2 and HT-2 toxins at 40 µg/kg and 200 µg/kg.
LoQ (µg/kg)
wholewheat pasta
cornflakes
T2 toxin
HT2 toxin
T2+HT2
1.5 - 3.1
4.5 - 5.9
6.0 - 7.9
T2 toxin
wholewheat pasta
cornflakes
HT2 toxin
Recovery
(%)
RSDr (%)
Recovery
(%)
RSDr
(%)
>89
<7.5
>98
<7.5
HPLC-MS/MS Method - Improved
LoD (µg/kg)
wholewheat
pasta
cornflakes
oat based
breakfast cereal
Spike Levels
(µg/kg)
T2
HT2
1.0
1.6
0.5
0.7
1.5
2.0
Uf *
(µg/kg)
T2
HT2
40
8
8
200
36
36
40
8
8
200
36
36
40
8
8
200
36
36
Uf = [ (LoD\2)2 + (α × C)2 ]1\2
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.
Acknowledgements
UK Food Standards Agency for financial support
All project participants for their support and
enthusiasm
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
Lattanzio et al., J. Food Prot. (submitted)
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.
16
H3C
H
10
H
O
9
11
8
(CH3)2CHCH2OCO
7
6
H
1
2
13
5
15CH
12
2 14
CH3
OCOCH3
T-2 toxin
O
3
4
OH
PBS
(shaking, 1 h, r.t.)
16
H3C
11
H
8
(CH3)2CHCH2OCO
enzymes
(carboxylesterases)
7
6
15
H
1
O
9
H
O COCH3
H
10
2
3
13
5
O
12
CH2 14
CH3
OCOCH3
4
OH
H
OH
HT-2 toxin
Carboxyl esterase activities toward pesticide and xenobiotic esters have recently found in maize and sorghum
(Gershater et al, 2006, Phytochemistry, 67:2561).
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.
conversion yield: 100%
HT-2
5000
100
4000
80
3000
60 %
2000
40
1000
20
0
30
60
90
time (min)
100
4000
80
3000
60 %
2000
40
1000
20
0
0
0
120
30
120
BARLEY
6000
HT-2
T2
HT-2
5000
100
5000
100
4000
80
4000
80
3000
60 %
3000
60 %
2000
40
2000
40
1000
20
1000
20
0
0
0
30
60
90
time (min)
120
ng
ng
T2
OAT
60
90
time (min)
6000
HT-2
5000
0
0
T2
WHEAT
6000
ng
ng
T2
MAIZE
6000
0
0
0
30
60
90
time (min)
120
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.
WHEAT
control
+ 20 mg maize proteins
200
µg/kg
160
120
80
40
0
0 min
30 min
60min
90 min
120 min
180 min
OATS
BARLEY
control
control
200.0
160
160.0
120
120.0
µg/kg
µg/kg
200
80
80.0
40
40.0
0.0
0
0 min
30 min
60min
90 min
120 min
180 min
0 min
30 min
60min
90 min
120 min 180 min
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 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 cereals
T-2
HT-2
Perspectives of application:
Extraction
Phosphate buffer (pH 7.4)
and maize protein extract
9 Antibody – based methods
ELISA
Fluorescence polarization
Strip test
Filtration
Immunoaffinity column
clean-up
LC-MS/MS
HPLC-FD (pre-column derivatisation with 1-AN)
Recoveries and repeatability (LC-MS/MS)
Spiking level,
T2 + HT2 (µg/kg)
Recovery, % (RSDr, %)*
20
40
100
200
400
95 (4.0)
96 (6.9)
97 (0.2)
94 (1.6)
72 (2.1)
Wheat
20
40
100
200
400
68 (5.7)
69 (2.4)
79 (5.6)
84 (7.3)
67 (3.3)
Oats
20
40
100
200
400
87 (3.4)
73 (9.4)
68 (5.6)
66 (8.1)
61 (1.0)
Maize
*
HT2
Limit of
quantification
(signal-to-noise ratio of
1:10)
maize 1.6 µg/kg
wheat 1.3 µg/kg
oats 1.3 µg/kg
RSDr, relative standard deviation (n = 3).
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.
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.
JOINT FAO/WHO EXPERT COMMITTEE
ON FOOD ADDITIVES
“… 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.
Conclusions – ISPA-CNR activities

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.

T2 and HT2 Toxins: Occurrence, Food Processing and Improved

Transkript

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