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Clenbuterol and salbutamol in animal feed: Clean-up and end-methods testing study. Report

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ISSN 1018-5593 %r European Commission ber information CHEMICAL ANALYSIS CLENBUTEROL AND SALBUTAMOL IN ANIMAL FEED: CLEAN-UP AND END-METHODS TESTING STUDY Report EUR 16242 EN European Commission ber information CHEMICAL ANALYSIS CLENBUTEROL AND SALBUTAMOL IN ANIMAL FEED: CLEAN-UP AND END-METHODS TESTING STUDY L. LEYSSENS1, R. MOERMANS2, D. COURTHEYN3 1 Dr. L. Willems-Instituut, vzw Universitaire Campus B-3590 Diepenbeek 2 Rijksstation voor Nematologie. Centrum voor Biometrie Burgemeester van Gransberghelaan 96 B-9820 Merelbeke 3 Rijksontledingslaboratorium Braemkasteelstraat 59 B-9050 Gentbrugge Contractor Dr. L. Willems-Instituut, vzw Universitaire Campus B-3590 Diepenbeek Contract No MAT1-CT92-0022 PROGRESS REPORT Directorate-General Science, Research and Development 1995 EUR 16242 EN LEGAL NOTICE Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information Cataloguing data can be found at the end of this publication EUROPEAN COMMISSION Luxembourg: Office for Official Publications of the European Communities, 1995 ISBN 92-827-4120-6 © ECSC-EC-EAEC. Brussels · Luxembourg.

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ISSN 1018-5593
%r
European Commission
ber information
CHEMICAL ANALYSIS
CLENBUTEROL AND SALBUTAMOL IN ANIMAL FEED:
CLEAN-UP AND END-METHODS TESTING STUDY
Report
EUR 16242 EN European Commission
ber information
CHEMICAL ANALYSIS
CLENBUTEROL AND SALBUTAMOL IN ANIMAL FEED:
CLEAN-UP AND END-METHODS TESTING STUDY
L. LEYSSENS1, R. MOERMANS2, D. COURTHEYN3
1 Dr. L. Willems-Instituut, vzw
Universitaire Campus
B-3590 Diepenbeek
2 Rijksstation voor Nematologie. Centrum voor Biometrie
Burgemeester van Gransberghelaan 96
B-9820 Merelbeke
3 Rijksontledingslaboratorium
Braemkasteelstraat 59
B-9050 Gentbrugge
Contractor
Dr. L. Willems-Instituut, vzw
Universitaire Campus
B-3590 Diepenbeek
Contract No MAT1-CT92-0022
PROGRESS REPORT
Directorate-General
Science, Research and Development
1995 EUR 16242 EN LEGAL NOTICE
Neither the European Commission nor any person acting on behalf of the Commission is responsible
for the use which might be made of the following information
Cataloguing data can be found at the end of this publication
EUROPEAN COMMISSION
Luxembourg: Office for Official Publications of the European Communities, 1995
ISBN 92-827-4120-6
© ECSC-EC-EAEC. Brussels · Luxembourg. 1995
Printed in Belgium ß-AGONISTS IN ANIMAL FEED 2
CLENBUTEROL AND SALBUTAMOL IN ANIMAL FEED:
CLEAN-UP AND END-METHODS TESTING STUDY
1993 - 1994
Table of Contents page
ABSTRACT 5
LIST OF ABBREVIATIONS
PARTICIPANTS 6
CHAPTER 1: TECHNICAL OBJECTIVES 7
CHAPTER 2: DESIGN OF THE CLEAN-UP AND END-METHODS STUDY 7
2.1. Basic principles
2.2. Set 1 experiments (matrix: animal feed I)
2.2.1. Preparation of PRIMARY extracts of set 1 by one
laboratory
2.2.2. Clean-up and measurement of set 1 by four
laboratories
2.3. Set 2 experiments (matrices: dairy concentrate and 11
milk replacer - 18% crude fat)
2.3.1. Preparation of PRIMARY MATRIX extracts of
set 2 by one laboratory
2.3.2. Clean-up and measurement of set 2 by four
laboratories
CHAPTER 3: INFORMATION ON THE MATERIALS, PREPARATION OF BLANK 14
AND CONTAMINATED PRIMARY EXTRACTS, HOMOGENEITY STUDY AND STABILITY
3.1. Materials 14
3.1.1. Animal feed samples
3.1.2. Standards and internal standards
3.2. Preparation of primary extracts by means of the Official 17
Extraction method
3.2.1. Operating procedure
3.2.2. Batch processing and pooling
3.2.3. Addition of clenbuterol and salbutamol to the pool of
primary extracts
3.2.4. Packaging, numbering, labeling, storage and shipment of
primary extracts
3.3. Homogeneity testing of PRIMARY extracts 19
3.3.1. Operating procedure
3.3.2. Results
3.3.3. Conclusions
3.4. stability of primary extracts 22 ß­AGONISTS IN ANIMAL FEED
CHAPTER 4: CLEAN­UP OF PRIMARY EXTRACTS 2 3
4.1. Schematic overview of clean­upmethods2 3
4.1.1. clean­up A (clenbuterol/salbutamol):
Mixed mode solid­phase clean­up (Certify)
4.1.2. Clean­up Β (clenbuterol): Chem­Elut
4.1.3. clean­up Β (salbutamol): t
4.1.4. clean­up c : Immuno­affinity
4.1.5. Clean­up D : Extrelut + RIDA­column
4.1.6. p D (salbutamol): t + n
4.2. Pooling of final extracts 2 8
4.3. Addition ofinternalstandards28
4.4. Packaging, numbering,labeling, storage and shipment29
of final extracts
CHAPTER 5: MEASUREMENT OF FINAL EXTRACTS 30
5.1. Measurement methods 30
5.1.1.LaboratoryA:GC­MSMS­measurementsof
clenbuterol/salbutamol
5.1.2.yB:HPLC­measurementsofclenbuterol
5.1.3.LaboratoryB:GCMS­measurements of salbutamol
5.1.4.yC:s of clenbuterol/salbutamol
5.1.5.yD:s of clenbuterol
5.1.6. LaboratoryD:3 of salbutamol
5.2. Calibration andsequence36
5.3. Check of calibration curves obtained at each laboratory 36
5.4. Handling of practical problems 36
CHAPTER 6: RESULTS 38
6.1. Rawresults38
6.2. Graphicalevaluationof results43
6.3. Analysisofvariance52
6.3.1.Clenbuterol:set 1A
6.3.2.:set 2A
6.3.3.Salbutamol:setIB
6.3.4.:set23
6.4. Discussion of clenbuterol results (Sets 1A and 2A) 69
6.5. n of salbutamol s (Sets IB and 2B)70
6.6. Overall conclusion 71
CHAPTER 7.ADDITIONALOBSERVATIONS72
7.1. Co­extractabilityofboth analytes: 72
Salbutamolrecoveryin clenbuterol extracts (sets 1Aand2A)
Clenbuterolyin salbutamols (sets IBand2B)
7.2. ElectronImpactversus chemicalIonisation78ß-AGONISTS IN ANIMAL FEED
CHAPTER 8: CONCLUSIONS 79
8.1. conclusions to the clean-up and end-methods testing study
8.2. Overall conclusions
REFERENCES
ANNEXE 1 : Draft procedure ß­AGONISTS IN ANIMAL FEED
ABSTRACT
It was the objective of the clean­up and end­methods testing study to complete
the development of an Official "modular", confirmatory Community method for the
detection of ß­agonists in animal feed. Clenbuterol and salbutamol were chosen as
representative analytes for the group. Homogeneous pools of primary extracts
(blank and contaminated) from three different animal feed matrices were prepared
in one laboratory by means of the community extraction module. The Official
extraction method was based on the conclusions of the first part of this work
carried out in the "BCR extraction testing study ­ clenbuterol and salbutamol in
animal feed (1992 ­ 1993; report EUR 15273 EN)". The primary extracts were
further processed by four laboratories by means of 4 different clean­up schemes.
The final extracts thus obtained were cross­distributed between the same
laboratories and measured either by GCMS or HPLC.
Two laboratories (B and D) applied different clean­up schemes for clenbuterol and
salbutamol. All clean­up schemes for clenbuterol were found compatible with all
end­methods. In contrast, for salbutamol clean­up method D was found not
compatible with the end­methods applied by laboratory·Β and C. The results of
this study have clearly demonstrated that both the clean­up methods for
clenbuterol and for salbutamol, applied by laboratory Β yielded superior
recoveries with an acceptable standard deviation. Therefore, in conclusion to
this study, the participating laboratories recommend the clean­up schemes applied
by laboratory Β to serve as the Official Community clean­up methods.
LIST OF ABBREVIATIONS
SDS : sodium­n­dodecylsulphate
PBS: phosphate buffered saline
PBS­2: double concentrated phosphate buffered saline
GC­MS: Gas Chromatography ­ Mass Spectrometry
HPLC: High­Performance Liquid Chromatography
CV: Coefficient of variation
EI : Electron Impact
CI: Chemical Ionisation
PCB: Polychlorbiphenyl
ST. DEV: standard deviation ß-AGONISTS IN ANIMAL FEED
PARTICIPANTS
Dr. Y. Berthoz
Lab. Interrégional de la Concurrence, de la Consommation et de la Repression des
Fraudes (DGCCRF)
26, Avenue du Coëtlogon
F-35000 Rennes (France)
Dr. D. courtheyn
Rijksontledingslaboratorium (ROL)
Braemkasteelstraat, 59,
B-9050 Gentbrugge (Belgium)
Dr. L. Leyssens (Coordinator)
Dr. L. Willems-Instituut (DWI)
Universitaire Campus
B-3590 Diepenbeek (Belgium)
Dr. R. Schilt
State Institute for Quality Control of Agricultural Products
(RIKILT)
Bornsesteeg, 45,
Postbus 230
NL-6 70 0 AE Wageningen (The Netherlands) ß-AGONISTS IN ANIMAL FEED
CHAPTER 1. TECHNICAL OBJECTIVES
It was the objective of this project to complete the development of an Official
"modular" Community method for the detection of ß-agonists in animal feed. The
first part of this work has been carried out in the "BCR extraction testing study
- clenbuterol and salbutamol in animal feed (1992 - 1993)". The results of this
study have extensively been discussed in the report prepared by D. Courtheyn et
al. (document EUR 15273 EN).
The work programme includes a clean-up and end-methods testing study involving 4
laboratories (results reported in this progress report), and a final
intercomparison study involving approx. 20 laboratories (to be organized near the
end of 1994). The conclusions of both the clean-up and end-methods study and the
already completed extraction testing study allow to compose an Official Community
analytical method for the detection of ß-agonists in animal feed. The objective
of the forthcoming 2nd Intercomparison exercise (the first one was organised in
1990) will be the evaluation of the Official Community method by 15 different
laboratories.
CHAPTER 2. DESIGN OF THE CLEAN-UP AND END-METHODS TESTING STUDY
The objective of the clean-up and end-methods testing study was the
intercomparison of 4p schemes and two end-methods for primary feed
extracts of clenbuterol and other ß-agonists (cimaterol, mabuterol, salbutamol,
terbutalin etc..) that:
a) are compatible with the extraction module(s) selected as a
result of the BCR extraction testing study;
b) aree with both end-methods (GC-MS, HPLC);
c) lead to the most acceptable yield and the lowest number of co-
extractives (i.e. the cleanest extract).
2.1. Basic principles
In this study, 4 laboratories were involved. Each laboratory, including the
coordinator performed two sets (i.e. on different days) of clean-ups on PRIMARY
extracts which were prepared and distributed by the coordinator. Thus, FINAL s were obtained. These were cross-distributed for measurements by the
coordinator and the three other laboratories. A schematic overview of the
practical organization of this study is given in Figures 1-4 (next pages). A
detailed description is given below.
2.2. Set 1 experiments (animal feed I as matrix)
2.2.1. Preparation of PRIMARY extracts of set 1 by one laboratory
(Fig. 1)
A number of 20 g aliquots of animal feed I (blank) were extracted at the
coordinator's laboratory by means of the Official extraction method (Fig.l, 2).
Two homogeneous pools of extracts were prepared by mixing the extracts obtained
(Fig.1,3). To one pool, clenbuterol and salbutamol were added to obtain a pool of
"contaminated" extracts with a clenbuterol content equivalent to 100 /Jg/kg feed
clenbuterol and a salbutamol content equivalent to 500 pg/kg feed (Fig.1,4). The
other pool was used as a pool of "blank" extracts. The two pools of extracts were
then divided into fractions equivalent to 4 g feed (Fig.1,5).