Bornova Vet.Kont.Arst.Enst.

The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
Version. 1
R-Biopharm Group &
the development of immunoaffinity
columns for multi-mycotoxin analysis
Speaker:
Patrick Taylor
Date:
October 2008
Venue:
Izmir Turkey
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
Our markets
October 2008
Our business is exported to over 60 countries, managed by exclusive distributors like
Sincer in Turkey
R-Biopharm AG Headquarters is located in Darmstadt, Germany.
R-Biopharm Rhone is based in Glasgow, Scotland.
R-Biopharm also has global subsidiary sales companies in:
USA
R-Biopharm Inc.
France
R-Biopharm SA.
Italy
R-Biopharm Srl.
Argentina
R-Biopharm Latin America.
Brazil
R-Biopharm Brasil.
China
R-Biopharm China.
Australia
R-Biopharm Australia Pvt Ltd.
Version. 1
1
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
RB Group’un Dahil Olduğu Bazı Uluslararası Mikotoksin ARGE
Projeleri’ne Örnekler
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
October 2008
Examples of some International R&D Development Projects for
mycotoxins that RB Group is involved with
•
•
SafeNut - Brazil Nut Export Hazard Control
•
FQS 64 FSA LINK Project
‘Fate of Fusarium Toxins in the Cereal Food Chain’
•
FQS 64 FSA LINK Projesi
SafeNut - Brezilya Cevizi İhracat Tehlike Kontrolü
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
‘Tahıl Gıda Zincirinde Fusarium Toksinlerinin Akıbeti’
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
•
Development of Multi-Residue methods and products for international
manufacturers
•
Uluslararası üreticiler için Multi-Kalıntı metotlar ve ürünlerin geliştirilmesi
analysis
Validation of DZT
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
SafeNut Project
Brazil Nut Export Hazard Control
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
Version. 1
2
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
RIDASCREEN®
ELISA-Testkit
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
®
RIDA QUICK Aflatoxin
Negative result < 4 µg/kg (ppb)
Positive result > 4 µg/kg (ppb)
Version. 1
3
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
UK Food Standards Agency Funded Project
İngiltere Gıda Standatları Ajansı’nın Finanse Ettiği Proje
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
FQS 64 LINK Project
‘Fate of Fusarium Toxins in the Cereal Food Chain’
‘Tahıl Gıda Zincirinde Fusarium Toksinlerinin Akıbeti’
Background on AFT & OTA analysis
2005 - 2008
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
… an investigation of UK cereals industry to assess if a problem
exists
… İngiltere’deki tahıllarda bir problemin olup olmadığının tespiti
için bir araştırma
Validation of DZT
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
UK Funded Link Project
October 2008
UK FUNDED LINK PROJECT ON FUSARIUM MYCOTOXINS
İNGİLTERE’NİN FİNANSE ETTİĞİ FUSARIUM MİKOTOKSİNLERİ LİNK PROJESİ
Oat Process
In order to gather information on the Fusarium mycotoxins, the processes that the
Wheat Process
raw cereal undergoes were examined in order to determine the mycotoxin
Maize Process
levels throughout the process.
Brewing Process
Conclusions of Link Project
Fusarium mikotoksinleri hakkında bilgi toplamak için, ham tahıllara uygulanan
işlemler, proses süresince mikotoksin seviyelerini tespit etmek amacı ile
RIDA QUICK DON Trial
incelenmiştir.
Version. 1
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The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
UK Funded Link Project
October 2008
UK FUNDED LINK PROJECT ON FUSARIUM MYCOTOXINS
İNGİLTERE’NİN FİNANSE ETTİĞİ FUSARIUM MİKOTOKSİNLERİ LİNK PROJESİ
MAIZE
OATS
WHEAT
Milling (dry)
Milling
Breakfast Cereals
Milling
Baking (bread, cakes,
biscuits, crackers)
Extruded Snacks
Breakfast Cereals
Tortilla Snacks
Extruded Snacks
Oat Process
Wheat Process
Maize Process
Brewing Process
Conclusions of Link Project
RIDA QUICK DON Trial
Oat Flakes
Fusarium mycotoxins that are most commonly found in UK cereals include:
İngiltere’de en fazla bulunan Fusarium mikotoksinleri:
• Deoxynivalenol
• Fumonisin (maize only) (sadece mısır)
• T2 & HT2
• Zearalenone
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
Fusarium Mycotoxins
UK FUNDED LINK PROJECT ON FUSARIUM MYCOTOXINS
UK Funded Link Project
İNGİLTERE’NİN FİNANSE ETTİĞİ FUSARIUM MİKOTOKSİNLERİ LİNK PROJESİ
Oat Process
Unique combination of government, food manufacturers and academic experts.
Wheat Process
Devlet, gıda üreticileri ve akademik uzmanların ortak çalışması
Maize Process
Brewing Process
Conclusions of Link Project
INDUSTRIAL
ACADEM IC
ADVISORS
R-Biopharm
KAS Mycotoxins
Cam pden & Chorleywood
Food Research Association
Harper Adam s University
College
Food Standards Agency
PepsiCo (Quaker Oats)
RHM
Sm iths Flour Millers
University of Bristol
United Biscuits
Central Science Laboratory
DACSA
Prof Ron Walker
RIDA QUICK DON Trial
DEFRA
SNACMA
Home Grown Cereals
Authority
National Association of British
& Irish Millers
Maizecor
Kelloggs
Cereal Partners
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The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
UK Funded Link Project
Oat Process
October 2008
OATS PROCESS (CONCLUSIONS)
YULAF PROSESİ (SONUÇLARIN DEĞERLENDİRİLMESİ)
It was found that the processing of oats to oat flakes was highly effective in reducing
the tricothecene levels.
Wheat Process
Maize Process
Tricothecene seviyelerinin azaltılmasında yulafın “flake”e işlenmesinin son derece
etkili olduğu görülmüştür.
Brewing Process
Conclusions of Link Project
Reduction of tricothecene levels was of approximately 95%.
RIDA QUICK DON Trial
Tricothecene seviyeleri yaklaşık %95 azaltılmıştır.
However, the tricothecenes were concentrated in the pellet by-products.
Fakat, tricotheceneler pelet yan ürünlerinde konsantre halde bulunmaktadır.
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
UK Funded Link Project
October 2008
WHEAT PROCESS (RESULTS)
BUĞDAY PROSESİ (SONUÇLAR)
Oat Process
It was noted that Deoxynivalenol was detected in all samples.
Wheat Process
Tüm numunelerde Deoxynivalenol tespit edilmiştir.
Maize Process
Brewing Process
Zearalenone was detected infrequently.
Conclusions of Link Project
Zearalenone nadiren bulunmuştur.
RIDA QUICK DON Trial
Both Deoxynivalenol and Zearalenone were detected below the EC legislative
limits.
Deoxynivalenol ve Zearalenone’un her ikisi de AB limitlerinin altında bulunmuştur.
Version. 1
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The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
UK Funded Link Project
October 2008
WHEAT PROCESS (CONCLUSIONS)
BUĞDAY PROSESİ (SONUÇLARIN DEĞERLENDİRİLMESİ)
It was found that deoxynivalenol was reduced in white flour by approximately 30% .
Oat Process
Deoxynivalenol’un beyaz unda yaklaşık %30 azaldığı görülmüştür.
Wheat Process
Maize Process
It was found that deoxynivalenol levels were increased in bran flour .
Deoxynivalenol seviyelerinin kepek ununda arttığı görülmüştür.
Brewing Process
The same pattern was observed for all of the other tricothecenes.
Conclusions of Link Project
Diğer Tricothecenlerde aynı örnek görülmüştür.
RIDA QUICK DON Trial
In bread making, there was a small decrease of approximately 12%.
Ekmek yapımında yaklaşık %12 gibi küçük bir düşüş görülmüştür.
There was no loss of deoxynivalenol observed during biscuit making.
Bisküvi yapımında Deoxynivalenol kaybı gözlemlenmemiştir.
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
UK Funded Link Project
October 2008
MAIZE PROCESS (CONCLUSIONS)
MISIR PROSESİ (SONUÇLAR)
When the raw maize was processed into flaking grits during the breakfast cereal
Oat Process
Wheat Process
Maize Process
process, a 70% reduction of the mycotoxins was observed.
Ham mısırın kahvaltılık gevreğe işlenmesi sırasında mikotoksinlerin %70 azaldığı
gözlemlenmiştir.
Brewing Process
Conclusions of Link Project
RIDA QUICK DON Trial
Fumonisins were found to be destroyed during the cooking process, however this
raises other questions, i.e. what were they broken down to?
Fumonisinlerin pişirme esnasında parçalandığı görülmüştür, fakat bu durumda
başka sorular ortaya çıkar, neye parçalandılar?
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The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
October 2008
In the production of cornflakes there appears to be a small increase in
deoxynivalenol.
UK Funded Link Project
Mısır gevreği üretiminde Deoxynivalenol seviyesinde küçük bir artış görülmektedir.
Oat Process
Wheat Process
Maize Process
Brewing Process
However, in the tortilla making process there appears to be no loss of
deoxynivalenol.
Fakat, tortilla üretiminde Deoxynivalenol kaybı görülmemektedir.
Conclusions of Link Project
RIDA QUICK DON Trial
In the tortilla making process there is a 60% decrease in fumonisin observed.
Tortilla üretimi esnasında Fumonisin’de %60 düşüş görülmüştür.
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
Fusarium Mycotoxins
October 2008
CONCLUSIONS ON UK FUNDED LINK PROJECT ON FUSARIUM MYCOTOXINS
İNGİLTERE’NİN FİNANSE ETTİĞİ FUSARIUM MİKOTOKSİNLERİ LİNK PROJESİNİN SONUÇLARI
UK Funded Link Project
Oat Process
The effect on the mycotoxin depends on the process.
Mikotoksinlere etkiler prosese bağlıdır.
Wheat Process
However, it was found that Fumonisins are more susceptible during the process and
Maize Process
are partially destroyed, with the cooking process being the most effective in
Brewing Process
Conclusions of Link Project
RIDA QUICK DON Trial
reducing fumonisins.
Fakat, Fumonisinlerin prosese karşı en dayanıksız olduğu, kısmen parçalandığı ve
pişirme işleminin fumonisin seviyesinin düşürülmesinde en etkili yol olduğu
görülmüştür.
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The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
CONCLUSIONS ON UK FUNDED LINK PROJECT ON FUSARIUM MYCOTOXINS
Fusarium Mycotoxins
İNGİLTERE’NİN FİNANSE ETTİĞİ FUSARIUM MİKOTOKSİNLERİ LİNK PROJESİNİN SONUÇLARI
UK Funded Link Project
Oat Process
Deoxynivalenol, Zearalenone and T2 & HT2 all proved to be very stable.
Deoxynivalenol, Zearalenone ve T2 & HT2 toksinlerinin hepsinin çok stabil olduğu
bulunmuştur.
Wheat Process
In some cases, the concentration of the mycotoxin can be lowered due to the dilution
Maize Process
effect of other ingredients (i.e in beer production).
Brewing Process
Conclusions of Link Project
RIDA QUICK DON Trial
Bazı durumlarda mikotoksin konsantrasyonunun diğer bileşenlerin seyreltme etkisi ile
azalabilmektedir (ör. bira üretimi).
In some cases, these mycotoxins were actually concentrated in grits or bran.
Bazı durumlarda bu mikotoksinler iri çekilmiş hububat ve kepekte yoğunlaşmaktadır.
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
RESULTS FROM RIDA QUICK DON COLLABORATIVE TRIAL
RIDA QUICK DON DENEMESİNDEN SONUÇLAR
RIDA QUICK DON performed will in the UK Funded Fusarium link project collaborative study. Only 2
spurious results out of 120 analysis.
İngiltere’nin finanse ettiği Fusarium link projesi çalışmasında RIDA QUICK DON isteneni gerçekleştirmiştir.
120 analizde sadece 2 hatalı sonuç vermiştir.
SAM PLE
DON (ppb) HPLC
RESULTS
LAB 1
LAB 2
LAB 3
LAB 4
LAB 5
LAB 6
LAB 7
LAB 8
LAB 9
LAB 10
LAB 11
LAB 12
M AIZE 680
750
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
M AIZE 730
< 10
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
M AIZE 731
500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
OATS 833
500
> 500
< 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
BARLEY 1
< 10
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
WHEAT 786
< 10
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
WHEAT 1002
> 100
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
M AIZE 327
100
< 500
< 500
< 500
< 500
< 500
> 500
< 500
< 500
< 500
< 500
< 500
< 500
OATS 456
< 10
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
< 500
M AIZE 73
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
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The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
Fusarium Mycotoxins
UK Funded Link Project
Oat Process
RIDA QUICK DON COLLABORATIVE TRIAL - UK FUNDED LINK PROJECT ON
FUSARIUM MYCOTOXINS
RIDA QUICK DON DENEMESİNDEN SONUÇLAR
Wheat Process
Lateral flow tests can often be fast and easy to use.
Maize Process
Brewing Process
Lateral flow testler hızlı ve kullanımı kolaydır.
Often more attractive commercially for cereal industry.
Conclusions of Link Project
Tahıl endüstrisi için ticari olarak daha caziptir.
RIDA QUICK DON Trial
Suitable for use as field tests or on the factory or mill floor.
Saha testleri, fabrika ya da değirmende kullanıma uygundur.
Require little equipment or technical expertise.
Az ekipman ve teknik uzmanlık gerektirir.
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
MULTİ-MİKOTOKSİN ANALİZLERİNİN ZEMİNİ
•
There is a market need for multiple analysis of relevant mycotoxins and
mycotoxin combinations.
•
İlgili mikotoksinler ve mikotoksin kombinasyonları için çoklu analizlere
gereksinim duyulmaktadır.
•
•
EU Legislation for food often requires analysis of more than one mycotoxin.
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
October 2008
BACKGROUND ON MULTI-MYCOTOXIN ANALYSIS
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
AB Gıda Mevzuatı genellikle birden falza mikotoksin analizine gereksinim
duymaktadır.
… cereals tahıllar
… dried fruits kuru meyveler
… spices baharatlar
Benefits of IAC
Version. 1
10
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
BACKGROUND ON MULTI-MYCOTOXIN ANALYSIS
MULTİ-MİKOTOKSİN ANALİZLERİNİN ZEMİNİ
•
As more analysis is being carried out routinely there is a need for faster and
more cost effective analysis.
•
Rutinde daha fazla analiz yapıldıkça daha hızlı ve uygun maliyetli analizlere
ihtiyaç duyulmaktadır.
•
For analysis of multi-mycotoxin it is recommended to use immunoaffinity
columns for clean-up of complex matrices in conjunction with either HPLC or
LCMSMS, depending on the customer requirements.
•
Multi-mikotoksin analizleri için kompleks matrislerin HPLC ya da LC MSMS
öncesi hazırlanmasında immunoaffinity kolonların kullanılması tavisye
edilmektedir.
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
October 2008
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
October 2008
HPLC ANALYSIS – HPLC ANALİZLERİ
HPLC analysis
LCMSMS analysis
•
HPLC is the most commonly used method for quantitative analysis of
mycotoxins.
•
Mikotoksin analizlerinde HPLC en sık kullanılan metottur.
•
•
It is the most favoured technique in FAPAS rounds.
•
Many labs have validated extraction methods in place using immunoaffinity
columns.
•
Birçok laboratuvar ekstraksiyon metotlarını immunoaffinity kolon kullanarak
valide etmiştir.
•
CEN/AOAC methods by HPLC have been already approved and validated for
different mycotoxins.
•
Farklı mikotoksinler için HPLC metotları CEN/AOAC tarafından onaylanmıştır.
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
FAPAS roundlarında en çok kabul gören tekniktir.
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
Version. 1
11
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
October 2008
ADVANTAGEDS OF MULTI-MYCOTOXIN COLUMNS
USED IN CONJUNCTION WITH HPLC
MULTİ-MİKOTOKSİN KOLONLARININ HPLC İLE BİRLİKTE KULLANIMININ
AVANTAJLARI
•
•
•
•
•
•
•
•
•
Customers can use existing equipment and techniques.
Müşteriler halihazırdaki ekipman ve teknikleri kullanabilirler
One extraction method for the analysis of different mycotoxins.
Farklı mikotoksinlerin analizleri için tek ekstraksiyon metodu.
One set of HPLC conditions for the analysis of different mycotoxins.
Farklı mikotoksinlerin analizleri için tek HPLC koşulları.
Simultaneous detection of more than one mycotoxin.
Birden fazla mikotoksinin aynı anda tespiti.
More efficient use of time – Zamanın daha etkin kullanımı
… reduced time for sample preparation. – numune hazırlama için daha az
analysis
zaman
Validation of DZT
… reduced HPLC run time. – daha kısa HPLC süresi
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
LCMSMS ANALYSIS - LCMSMS ANALİZLERİ
HPLC analysis
•
•
•
•
•
•
•
•
•
•
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
October 2008
LCMSMS is one of the most advanced techniques for mycotoxin analysis.
LCMSMS mikotoksin analizleri için en gelişmiş tekniklerden biridir.
Many analysts and official labs are moving to this technique.
Birçok analit ve resmi laboratuvar bu tekniğe geçmektedir.
Particularly suitable for analysis of multi parameters.
Özellikle çoklu parametrelerin analizleri için uygundur.
One extraction method for the analysis different mycotoxins.
Farklı mikotoksinlerin analizleri için tek ekstraksiyon metodu.
Simultaneous detection of more than one mycotoxin.
Birden fazla mikotoksinin aynı anda tespiti.
analysis
Validation of DZT
Benefits of IAC
Version. 1
12
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
October 2008
ADVANTAGEDS OF MULTI-MYCOTOXIN COLUMNS USED IN CONJUNCTION
WITH LCMSMS
LCMSMS İLE BİRLİKTE KULLANILAN MULTİ-MİKOTOKSİN KOLONLARININ
AVANTAJLARI
•
•
•
No requirement for derivatisation of samples
•
LCMSMS öncesi immunoaffinity kolon uygulaması ile kompleks numunelerin
daha iyi ekstraksiyonu.
•
•
•
Improved sensitivity using immunoaffinity columns
Örneklerin türevlendirilmesine gerek yoktur
Better clean up of complex samples using LCMSMS in conjunction with
immunoaffinity columns
İmmunoaffinity kolonların kullanılması ile artan hassasiyet.
More efficient use of time – Zamanın daha verimli kullanılması
… reduced time for sample preparation. - Örnek hazırlama zamanı kısadır
… reduced HPLC run time. HPLC analiz zamanı kısadır
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
RBR IACs columns
October 2008
Contain antibodies specific for the analysed compound
Analiz edilen bileşiğe spesifik antikorlar içerir
Concentrate from samples for analysis
Analiz için örneği konsantre eder
Removal of contaminants, matrix components & pigments
Bulaşanları, analize engel olabilecek pigmentleri ve komponentleri uzaklaştırır
Provide excellent clean-up for more complex matrices
Kompleks yapıdan mükemmel arındırma sağlar
Can be used in combination with any analytical method
Herhangi bir analitik metod ile birlikte de kullanılabilir
Version. 1
13
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
MULTI-MYCOTOXIN ANALYSIS WITH IMMUNOAFFINITY COLUMNS
İMMUNOAFFİNİTY KOLONLAR İLE MULTİ-MİKOTOKSİN ANALİZİ
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
•
•
R-Biopharm Rhone are currently developing a number of new multi mycotoxin
immunoaffinity columns for use with HPLC or LCMSMS.
R-Biopharm Rhone HPLC ya da LCMSMS ile kullanılmak üzere yeni multimikotoksin immunoaffinity kolonlar geliştirmektedir.
… EASI-EXTRACT ® T-2 & HT-2 for use in conjunction with HPLC
… HPLC ile birlikte kullanılan EASI-EXTRACT ® T-2 & HT-2
… AFLAOCHRA PREP ® for use in conjunction with HPLC
… HPLC ile birlikte kullanılan AFLAOCHRA PREP ®
… DZT for use in conjunction with LCMSMS
… LCMSMS ile birlikte kullanılan DZT
… Other requested combinations soon ...
… İstenen diğer kombinasyonlar yakında...
Validation of DZT
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
October 2008
PRINCIPLE OF MULTI-MYCOTOXIN IMMUNOAFFINITY COLUMNS
MULTİ-MİKOTOKSİN İMMUNOAFFİNİTY KOLONLARIN PRENSİBİ
SAMPLE
WASHING
ELUTION
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
The sample extract
(containing the toxin)
is passed through the
column.
The antibody isolates and
concentrates the toxin
and retains it in the
column
Numune ekstraktı
Kolonda bulunan
antikorlar toksini izole
ederek kolonda
tutulmasını sağlar
(toksin ihtiva eden)
kolondan geçirilir.
Passage of pure
methanol through the
column denatures the
antibody and
releases the toxin
Kolondan geçirilen
saf metanol ile
antikorlar denatüre
edilerek toksinin
serbest hale geçmesi
sağlanır
Version. 1
14
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
MULTI-MYCOTOXIN ANALYSIS OF T-2 & HT-2
T-2 ve HT-2 MULTİ-MİKOTOKSİN ANALİZLERİ
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
•
•
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
•
•
•
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
•
T-2 & HT-2 are amongst the most dangerous of the fusarium mycotoxins.
T-2 ve HT-2 Fusarium mikotoksinleri arasında en tehlikeli mikotoksinler
arsındadır.
These mycotoxins have a strong impact on the health of animals and humans
due to their immunosuppresive effects.
Bu mikotoksinler insan ve hayvan sağlığı üzerinde kuvvetli etkilere sahiptir
(bağışıklık sistemi).
Legislation is currently pending for T-2 & HT-2. However it is thought that the
two mycotoxins will be evaluated together since T-2 is readily metabolised to
HT-2.
T-2 ve HT-2 için yasal düzenleme beklenmektedir. Fakat T-2 HT-2’ye
metabolize olduğundan iki mikotoksin birlikte değerlendirilecektir.
analysis
Validation of DZT
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
PENDING LEGISLATION FOR T-2 & HT-2
T-2 ve HT-2 İÇİN BEKLENEN YASAL DÜZENLEME
LCMSMS analysis
Multi-mycotoxin analysis with IAC
T-2 &HT-2 IN FOOD LEGISLATION (PENDING)
Principle of IAC
Background on T2 & HT2 analysis
COMMODITY
MAXIMUM LEVEL
Validation of EASI-EXTRACT T2 &
Unprocessed cereal
100ppb
Malting barley
200ppb
Unprocessed oats
500ppb
Cereal for infant food
50ppb
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Infant food
100ppb
Dietetic nutrition for infants
500ppb
Dietetic nutrition without gluten
100ppb
Benefits of IAC
Version. 1
15
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
VALIDATION OF EASI-EXTRACT
& HT-2
EASI-EXTRACT ® T-2 & HT-2 NİN VALİDASYONU
In response to customer requests RBR have developed an application note for the
analysis of oats in conjunction with Nestle.
Müşteri isteği üzerine RBR Nestle ile birlikte yulaf analizi için uygulama notu
geliştirmiştir.
1)
25g of sample + 125ml 90% MeOH + 2.5g NaCl
2)
Blend and filter
3)
Dilute 20ml of filtrate with 80ml 2% NaCl solution
4)
Gently shake for 5 minutes
Derivatisation with AFLAOCHRA PREP
5)
Filter through GMF
Background on T2, HT2, ZEA & DON
6)
Pass 25ml of diluted filtrate through column
Validation of DZT
7)
Wash with 20ml H2O
Benefits of IAC
8)
Elute with MeOH
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
analysis
October 2008
® T-2
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
October 2008
VALIDATION OF EASI-EXTRACT ® T-2 & HT-2
EASI-EXTRACT ® T-2 & HT-2 NİN VALİDASYONU
For type A trichothecenes such as T-2 and HT-2 derivatisation is required to allow
detection by fluorescence.
T-2 ve HT-2 gibi A tipi Trichothecenelerin floresan detektör ile tespiti için
türevlendirme gerekmektedir.
1) Evaporate the sample eluate or standards under an air stream at 50°C.
1) Örnek eluatı ya da standartlar 50°C’de hava akımı altında evapore edilir.
2) Add 50ul of D-MAP and 50ul of 1-AN to the residue and vortex for 1
minute
2) 50ul of D-MAP ve 50ul of 1-AN eklenerek 1 dk. vortexlenir
3) Leave the mixture to react in a heater block at 50°C for 15 minutes
3) Karışım 50°C’de 15 dk reaksiyona bırakılır
4) Cool mixture on ice for 10 minutes
4) Karışım 10 dk. Buz içerisinde soğutulur
5) Dry mixture under steam at 50°C
5) Karışım 50°C’de buharla kurutulur
6) Reconstitute in 1ml of mobile phase 1ml mobil faz içerisinde çözülür
7) Inject 100ul onto an HPLC 100ul HPLC’ye enjekte edilir
Version. 1
16
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
October 2008
® T-2
VALIDATION OF EASI-EXTRACT
& HT-2
Summary of the results obtained during the validation of the oat protocol were as
follows -
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
T-2 TOXIN
Principle of IAC
Background on T2 & HT2 analysis
Spiking Level
50ppb
100ppb
200ppb
200ppb
Sample ID.
4022
4022
4022
4011
Mean Recovery (%)
(n = 6)
102.5
97.8
94.9
101.9
%CV
5.0
3.7
6.6
3.7
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
HT-2 TOXIN
PREP
Derivatisation with AFLAOCHRA PREP
Spiking Level
50ppb
100ppb
200ppb
200ppb
Sample ID.
4022
4022
4022
4011
102.4
118.2*
128.4*
104.2
5.5
9.6
6.8
2.8
Background on T2, HT2, ZEA & DON
analysis
Mean Recovery (%)
(n = 6)
Validation of DZT
Benefits of IAC
%CV
* High recoveries due to natural contamination of samples
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
VALIDATION OF EASI-EXTRACT ® T-2 & HT-2
1,200
071106 #5 [modified by Administrator]
mV
Fluorescence
1,200
1,000
1,000
800
800
600
071106 #9 [modified by Administrator]
mV
Fluorescence
600
2 - HT-2 - 19.098
2 - HT-2 - 19.042
400
400
1 - T-2 - 10.034
200
200
0
0
-200
0.0
min
5.0
10.0
15.0
20.0
25.0
SPIKED OATS - Original IFU method
30.0
35.0
-200
0.0
1 - T-2 - 10.073
min
5.0
10.0
15.0
20.0
25.0
30.0
35.0
SPIKED OATS – Application note prepared in conjunction with
Nestle
Version. 1
17
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
MULTI-MYCOTOXIN ANALYSIS OF AFLATOXIN & OCHRATOXIN
AFLATOKSİN VE OKRATOKSİNİN MULTİ-MİKOTOKSİN ANALİZLERİ
LCMSMS analysis
Multi-mycotoxin analysis with IAC
•
Principle of IAC
Background on T2 & HT2 analysis
•
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
•
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
•
Legislation is constantly increasing for aflatoxin and ochratoxin with more
commodities being included in EU legislation.
Aflatoksin ve Okratoksin için düzenlemeler, daha fazla gıda maddesinin AB
Mevzuatında yer alması ile birlikte sürekli artmaktadır.
Analysts are requiring to analyse samples for both aflatoxins and ochratoxin
therefore it it was decided that RBR would focus on the main commodities that
were covered by legislation for both toxins –
Analistlerin numunelerde hem Aflatoksin hem de Okratoksin analizlerine ihtiyaç
duymasından dolayı RBR her iki toksinin de mevzuat kapsamında olduğu
ürünlere yönelmiştir
… cereals tahıllar
… dried fruit kuru meyve
… spices baharatlar
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
October 2008
CURRENT LEGISLATION FOR AFLATOXIN & OCHRATOXIN
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
AFLATOXIN & OCHRATOXIN IN FOOD LEGISLATION
Principle of IAC
Background on T2 & HT2 analysis
COMMODITY
TOTAL AFT
OTA
Validation of EASI-EXTRACT T2 &
Cereals including processed products for human
consumption or as an ingredient
4ppb
3ppb
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
Raw cereal grains
5ppb
Maize subject to sorting
10ppb
Spices
10ppb
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
10ppb
Dried figs
8 – 10ppb
Other dried fruits
1 – 2ppb
Validation of DZT
Benefits of IAC
Version. 1
18
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
PREP ®
VALIDATION OF AFLAOCHRA
AFLAOCHRA PREP ® VALİDASYONU
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
In response to market requirements RBR developed methods for the analysis of
cereals, dried fruit and spices for the detection of aflatoxin and ochratoxin.
RBR, piyasa gereksinimini karşılamak amacı ile, tahıllar, kuru meyveler ve
baharatlarda Aflatoksin ve Okratoksin tespiti için metotlar geliştirmiştir.
HT2
Background on AFT & OTA analysis
1)
25g of sample + 100ml 80% MeOH + 5g NaCl
Validation of AFLAOCHRA PREP
2)
Blend and filter
PREP
3)
Dilute 2ml of filtrate with 18ml PBS (or 10% Tween 20 in PBS)
Derivatisation with AFLAOCHRA PREP
4)
Pass 10ml of diluted filtrate through column
5)
Wash with 20ml PBS
6)
Elute with MeOH
HPLC conditions for AFLAOCHRA
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
VALIDATION OF AFLAOCHRA PREP ®
Summary of the results obtained during the validation for cereals were as follows -
LCMSMS analysis
Multi-mycotoxin analysis with IAC
WHEAT (4ppb TOTAL AFT, 3ppb OTA)
Principle of IAC
Background on T2 & HT2 analysis
TOXIN
AFT B1
AFT B2
AFT G1
AFT G2
OTA
Validation of EASI-EXTRACT T2 &
RECOVERY
(%)
100.8
104.4
104.3
105.9
104.4
% CV
2.1
1.2
2.4
1.7
3.4
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
MAIZE (FAPAS T04105, 10.7ppb TOTAL AFT)
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
TOXIN
AFT B1
AFT B2
AFT G1
AFT G2
OTA
RECOVERY
(ppb)
4.58
2.29
2.17
0.90
ND
% CV
0.7
0.4
0.9
2.3
ND
Benefits of IAC
Version. 1
19
4ppb AFT 3ppb OTA Spiked wheat matrix 1 – AFLA/OCHRA PREP
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
VALIDATION OF AFLAOCHRA PREP®
30.0
291107 Spiked wheat matrix #23 [modified by Administrator]
mV
Thin spike 1
Emission
EM:442 nm
3 - B2 - 11.252
1 - G2 - 7.688
15.0
17.5
2 - G1 - 9.374
Ex = 365, Em = 442, Az
4 - B1 - 13.972
Ex = 333, Em = 463, Az
5 - OTA - 27.069
20.0
10.0
-5.0
0.0
min
2.5
5.0
7.5
10.0
12.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
40.0
4ppb AFT 3ppb OTA Spiked wheat matrix 1 – AFLAOCHRA PREP ®
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
October 2008
VALIDATION OF AFLAOCHRA PREP ®
Summary of the results obtained during the validation for dried fruits were as follows
-
Multi-mycotoxin analysis with IAC
SULTANAS (4ppb TOTAL AFT, 10ppb OTA)
Principle of IAC
Background on T2 & HT2 analysis
TOXIN
AFT B1
AFT B2
AFT G1
AFT G2
OTA
Validation of EASI-EXTRACT T2 &
RECOVERY
(%)
84.3
84.8
77.1
78.0
87.9
% CV
3.8
2.6
2.9
3.7
3.5
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
SULTANAS (FAPAS T1760, 4ppb TOTAL AFT, 50ppb OTA)
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
TOXIN
AFT B1
AFT B2
AFT G1
AFT G2
OTA
RECOVERY
(%)
90.5
87.5
86.2
82.9
83.6
% CV
3.4
2.0
1.7
2.2
3.8
Benefits of IAC
Version. 1
20
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
VALIDATION OF AFLAOCHRA PREP®
060308 Spiked sultanas matrix #13 [modified by Administrator]
mV
5.0
1 - G2 - 6.357
2 - G1 - 7.726
Emission
EM:442 nm
5 - OTA - 24.895
3 - B2 - 9.293
2.5
-1.0
0.0
Thin spike 1
Ex = 333, Em = 463, Az
9.0
4 - B1 - 11.563
min
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
40.0
4ppb AFT 10ppb OTA Spiked sultana matrix 1 – AFLAOCHRA PREP ®
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
VALIDATION OF AFLAOCHRA PREP ®
Summary of the results obtained during the validation for spices were as follows -
LCMSMS analysis
Multi-mycotoxin analysis with IAC
PEPPER (10ppb TOTAL AFT, 15ppb OTA)
Principle of IAC
Background on T2 & HT2 analysis
TOXIN
AFT B1
AFT B2
AFT G1
AFT G2
OTA
Validation of EASI-EXTRACT T2 &
RECOVERY
(%)
88.1
90.3
81.2
79.0
90.6
% CV
4.4
3.6
3.2
2.6
3.2
TOXIN
AFT B1
AFT B2
AFT G1
AFT G2
OTA
RECOVERY
(%)
92.5
94.9
98.1
91.0
81.0
% CV
1.7
0.7
1.8
2.3
1.3
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PAPRIKA (40ppb TOTAL AFT, 50ppb OTA)
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
Version. 1
21
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
VALIDATION OF AFLAOCHRA PREP®
70.0
181207 PEPPER SPIKED MATRIX #22 [modified by Administrator]
mV
Thin spike 1
Emission
EM:442 nm
5 - OTA - 27.376
4 - B1 - 14.193
1 - G2 - 7.783
2 - G1 - 9.506
Ex = 365, Em = 442, Az
40.0
Ex = 333, Em = 463, Az
3 - B2 - 11.408
20.0
-10.0
0.0
min
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
40.0
10ppb AFT 15ppb OTA Spiked pepper matrix 1 – AFLAOCHRA PREP ®
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
October 2008
HPLC CONDITIONS FOR AFLAOCHRA PREP ®
HPLC analysis
LCMSMS analysis
HPLC CONDITIONS
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Mobile Phase A
Water: methanol (55:45 v/v)
Mobile Phase B
Water: methanol (20:80 v/v)
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Flow Rate
Column temperature
To 1 litre add119mg of potassium bromide and 350l 4N Nitric Acid
To 1 litre add 119mg of potassium bromide and 350l 4N Nitric Acid
0.8ml/min
40oC
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
End
Time
0
14.0
16.0
29.5
31.0
40.0
Time
0
17
Excitation (nm)
362
333
Start
Gradient Program
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
Fluorescence
conditions
%A
100
100
35
35
100
100
%B
0
0
65
65
0
0
Emission (nm)
442
463
Version. 1
22
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
DERIVATISATION OF AFLATOXINS WITH AFLAOCHRA
October 2008
PREP ®
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
• The KOBRA® CELL (KOk BRomine
Apparatus Cell) is a unique system which
offers a popular alternative derivatisation.
• KOBRA® CELL
• Electrochemical cell consisting of a platinum
working electrode and a stainless steel
auxiliary electrode separated from another by
a membrane. These layers are sandwiched
between rigid plastic housings.
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA
PREP
Background on T2, HT2, ZEA & DON
• Electrochemical cell, fitted between the HPLC
column and the detector that generates the
derivatisation agent, bromine, on-line from
potassium bromide and nitric acid present in
the mobile phase
analysis
Validation of DZT
• The derivatisation results in a significant
increase in the fluorescent signals of the
modified forms of aflatoxin B1 and G1.
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
KOBRA CELL switched on
KOBRA CELL
installation
Detector
Column
Heater
HPLC
ColumnOut
KC # 123
KC # 123
‘Fingertight’
Fittings
‘Union’
Fittings
waste
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23
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
MULTI-MYCOTOXIN ANALYSIS OF DON, ZEARALENONE ,T-2 & HT-2
DON, ZEARALENONE ,T-2 ve HT-2 MULTİ-MİKOTOKSİN ANALİZLERİ
•
DON, Zearalenone, T-2 and HT-2 were chosen because of their co-occurrence
and legislative significance.
•
DON, Zearalenone, T-2 ve HT-2 ortak bulunma ve yasal önemlerinden dolayı
seçilmiştir.
•
The prototype has been developed to minimise matrix effects on the result, aid
sensitivity and provide clean, easy to interpret chromatography.
•
Sonuçlarda matriks etkisini minimize etmek, hassaslığı desteklemek ve temiz,
değerlendirilmesi kolay bir kromotografi sağlamak için prototip geliştirilmiştir.
•
Prototype product has been evaluated for approximately 1 year by over 15
companies.
•
Prototip ürün 15’ten fazla firma tarafından yaklaşık 1 yıl değerlendirilmiştir.
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
October 2008
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
October 2008
VALIDATION OF DZT – DZT VALİDASYONU
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
In response to market requirements RBR have started developing and
evaluating their first multi-mycotoxin columns for use in conjunction with
LCMSMS. All methods have been validated by UK Central Science
Laboratories (CSL).
RBR, piyasa gereksinimini karşılamak amacı ile, LCMSMS ile birlikte
kullanılmak üzere ilk multi-mikotoksin kolonlarını geliştirmeye ve
değerlendirmeye başlamıştır. Tüm metotlar İngiltere Merkez Bilim
Laboratuvarları (CSL) tarafından valide edilmiştir.
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
1)
Extract sample in 75% MeOH
PREP
2)
Blend and filter
3)
Dilute 1 or 2ml of filtrate with PBS to final volume of 50ml
4)
Pass diluted filtrate through column
5)
Wash with 20ml H2O
6)
Elute with MeOH
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
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The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
VALIDATION OF DZT
Summary of the results obtained by CSL during their validation were as follows –
LCMSMS analysis
Multi-mycotoxin analysis with IAC
CORNFLAKES
Principle of IAC
Background on T2 & HT2 analysis
TOXIN
DON
HT-2 Toxin
T-2 Toxin
Zearalenone
Validation of EASI-EXTRACT T2 &
SPIKING LEVEL
500 ppb
50 ppb
50 ppb
50 ppb
RECOVERY(%)
(n=6)
86.7
94.0
82.4
95.8
% CV
6.5
3.9
2.4
3.7
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PASTA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
TOXIN
DON
HT-2 Toxin
T-2 Toxin
Zearalenone
SPIKING LEVEL
750 ppb
75 ppb
75 ppb
75 ppb
RECOVERY(%)
(n=6)
83.0
97.3
83.8
95.2
% CV
8.2
3.3
4.4
3.4
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
October 2008
VALIDATION OF DZT
Summary of the results obtained by CSL during their validation were as follows –
LCMSMS analysis
Multi-mycotoxin analysis with IAC
ANIMAL FEED
Principle of IAC
TOXIN
DON
HT-2 Toxin
T-2 Toxin
Zearalenone
Validation of EASI-EXTRACT T2 &
SPIKING LEVEL
250 ppb
25 ppb
25 ppb
25 ppb
HT2
RECOVERY (%)
(n=6)
79.1
96.9
83.8
92.0
% CV
5.0
4.1
5.2
1.3
Background on T2 & HT2 analysis
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
OATS
PREP
TOXIN
DON
HT-2 Toxin
T-2 Toxin
Zearalenone
Background on T2, HT2, ZEA & DON
SPIKING LEVEL
1750 ppb
100 ppb
100 ppb
100 ppb
analysis
RECOVERY (%)
(n=6)
93.0
99.5
88.6
89.3
% CV
6.4
6.3
10.2
3.3
Derivatisation with AFLAOCHRA PREP
Validation of DZT
Benefits of IAC
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25
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
VALIDATION OF DZT
TIC: from Sample 19 (Job12356_019) of Data36450.wiff (Turbo Spray), Smoothed, Smoothed
Max. 1257.8 cps.
15.94
1250
TIC: from Sample 19 (Job12355_019) of Data36449.wiff (Turbo Spray), Smoothed, Smoothed
Max. 2728.9 cps.
15.94
2729
2600
1200
1150
10.32
2400
1100
1050
16.86
2200
1000
950
2000
16.85
900
16.01
850
1800
800
Intensity, cps
750
1600
700
10.33
650
1400
600
16.0516.91
550
1200
500
450
1000
15.39
400
16.98
15.60
800
350
15.39
300
250
17.02
200
14.21
600
400
150
17.11
13.80
100
14.21
11.42
200
17.13
50
0
1
2
3
4
5
6
7
8
9
10
Time, min
11
12
13
14
15
16
17
18
SPIKED BABY FOOD
(200ppb DON, 20ppb each HT2,T2 and ZEA)
0
1
2
3
4
5
6
7
8
9
10
Time, min
11
12
13
14
15
16
17
18
SPIKED OATS
(1750ppb DON, 100ppb each HT2,T2 and ZEA)
Version. 1
Bornova Vet.Kont.Arst.Enst.
The development of immunoaffinity columns for multi-mycotoxin analysis
General background
HPLC analysis
LCMSMS analysis
Multi-mycotoxin analysis with IAC
Principle of IAC
Background on T2 & HT2 analysis
Validation of EASI-EXTRACT T2 &
HT2
Background on AFT & OTA analysis
Validation of AFLAOCHRA PREP
HPLC conditions for AFLAOCHRA
PREP
Derivatisation with AFLAOCHRA PREP
Background on T2, HT2, ZEA & DON
analysis
Validation of DZT
Benefits of IAC
October 2008
BENEFITS OF USING R-BIOPHARM MULTI-MYCOTOXIN IMMUNOAFFINITY COLUMNS
R-BIOPHARM MULTİ-MİKOTOKSİN İMMUNOAFFİNİTY KOLONLARI KULLANIMININ AVANTAJLARI
•
•
•
•
•
•
•
•
•
•
•
•
RBR immunoaffinity columns used by many governmental labs and public
analysts.
RBR immunoaffinity kolonları birçok devlet laboratuvarı tarafından kullanılmaktadır.
Performance far exceeds requirements of EC and other international legislation.
Performans AB ve diğer uluslar arası mevzuatın gereksinimlerini fazlasıyla
aşmaktadır.
Suitable for a huge range of commodities and all those considered for
legislation.
Mevzuatta olan tüm gıda maddeleri için uygundur.
Methods validated externally and internally.
Metotlar eksternal ve internal olarak vailde edilmiştir.
Technical support always available from a very experienced team.
Çok deneyimli takımdan teknik desteğe her zaman ulaşılabilir.
RBR can also provide reference material, standards and application notes.
RBR referans materyal, standartlar ve uygulama notları da sağlamaktadır.
Version. 1
26
The development of immunoaffinity columns for multi-mycotoxin analysis
October 2008
Please visit RBAG at:
www.r-biopharm.com
Version. 1
Bornova Vet.Kont.Arst.Enst.
27