Agenda Item 7 CX/RVDF 07/17/10 June 2007 JOINT FAO/WHO ... fileE Agenda Item 7 CX/RVDF 07/17/10 June...

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Agenda Item 7 CX/RVDF 07/17/10 June 2007

JOINT FAO/WHO FOOD STANDARDS PROGRAMME

CODEX COMMITTEE ON RESIDUES OF VETERINARY DRUGS IN FOODS

Seventeenth Session

Breckenridge, Colorado, United States of America, 3 -7 September 2007

METHODS OF ANALYSIS AND FOR RESIDUES OF VETERINARY DRUGS IN FOODS

Comments in response to CL 2006/04-RVDF submitted by Australia, Norway, Sweden and the United States of America

AUSTRALIA

Australia welcomes the opportunity to comment on these matters. Australia provides the following information in response to the request for information/comments on the compendium of methods prepared by the 16th session of CCRVDF.

Australia requests removal of the following methods from the compendium as the information is mostly either out of date or not supported:

1. Dihydrostreptomycin/streptomycin – Australian Government Analytical Laboratories, GPO Box 1844, Canberra ACT 2601, Australia.

2. Doramectin – Australian Government Analytical Laboratories, GPO Box 1844, Canberra ACT 2601, Australia.

3. Eprinomectin – Australian Government Analytical Laboratories, GPO Box 1844, Canberra ACT 2601, Australia.

4. Levamisole – Australian Government Analytical Laboratories, GPO Box 1844, Canberra ACT 2601, Australia.

5. Moxidectin – Australian Government Analytical Laboratories, GPO Box 1844, Canberra ACT 2601, Australia.

6. Neomycin – Animal Research Institute, Chemical Residue Laboratory, 665 Fairfield Road, Yeerongpilly QLD 4105, Australia.

7. Thiabendazole (liver cattle, liver pig and liver sheep) – Amdel. 36-40 Halloran St., Lilyfield NSW 2040, Australia.

8. Triclabendazole –Amdel. 36-40 Halloran St., Lilyfield NSW 2040, Australia.

NORWAY

Thank you for inviting Norway to submit comments on CL 2007/04-RVDF. Norway has noticed that the document contains only one method of analysis for fish. For your information please find the attached table showing several accredited methods currently applied by the National Institute of Nutrition and Seafood Research (NIFES) in the analysis of residues of therapeutic agents in fish in Norway. The list is updated May 2007.

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NIFES is accredited in accordance with ANSI/ISO/IEC 17025 - General Requirements for the Competence of Testing and Calibration Laboratories.

The methods are currently in Norwegian, but could be provided in English if necessary.

Table showing the methods currently applied by National Institute of Nutrition and Seafood Research (NIFES) in the analysis of residues of therapeutic agents in fish. The list is updated May 2007.

NIFES is accredited in accordance with ANSI/ISO/IEC 17025 - General Requirements for the Competence of Testing and Calibration Laboratories.

Abbreviations used in the table: APCI: Atmospheric Pressure Ionization, HPLC: High Performance Liquid Chromatography, GC: Gas Chromatography, MS: Mass Spectroscopy, DAD: Diode Array Detection, ES: Electro Spray, FD: Fluorescent Detection, UVD: Ultra Violet Detection.

Agent Method LOD (Limit of detection, ppb)

LOQ (Limit of quantification, ppb)

Azamethiphos LC-MS (API-ES) 1.5 4.0 Chloramfenicol muscle LC-MS (API-ES) 0.3 1.0 Cypermetrin (high–cis) GC/MS 5.0 10.0 Deltamethrin GC/MS 10.0 20.0 Dichlorvos GC/MS 5.0 10.0 Diflubenzuron LC-MS (API-ES) 10.0 20.0 Emamectin benzoate HPLC/MS (APCI) 2.5 5.0 Fenbendazole HPLC/MS (APCI) 2.5 5.0 Florfenicol muscle HPLC/MS (ES) 0.2 0.5 Florfenicol feed HPLC/MS (ES) 0.4 1.0 Flumequine LC-MS (API-ES) 10.0 20.0 Ivermectine HPLC/MS (APCI) 25.0 50.0 Leuco malachite green HPLC/MS (APCI) 1.0 2.0 Malachite green HPLC/DAD 1.0 2.0 Furazolidone HPLC-MS/MS 0.5 1.5 Furaltadone HPLC-MS/MS 0.5 1.5 Nitrofurantoine HPLC-MS/MS 0.5 1.5 Nitrofurazone HPLC-MS/MS 0.5 1.5 Oxolinic acid LC-MS (API-ES) 10.0 30.0 Oxytetracycline LC-MS (API-ES) 2.0 5.0 Praziquantel HPLC/DAD 50.0 100.0 Metronidazol LC-MS (API-ES) 5.0 10.0 Teflubenzuron LC-MS (API-ES) 5.0 15.0

SWEDEN

Analytical Method Information Summary

A. Descriptive Information 1. Name of drug or chemical: Aminoglykosides 2. Drug or chemical class: Antimicrobial (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use:__Infection diseases 4. Analyte(s) measured: Streptomycin, Dihydrostreptomycin (specify if metabolite) 5. Intended use of the method:

a. Screening _______Yes__________________________________ b. Routine_________Yes___________________________________ c. Reference________No___________________________________ d. Confirmatory_____Yes__________________________________

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6. Test matrix__Honey (e.g. muscle, kidney, urine, etc) 7. Summary of principal steps in sample preparation: __Homogenization of the sample 8. Summary of principal steps in extraction procedure: Extraction with water_ 9. Summary of principal steps in analyte clean-up procedure: Clean-up_with cation exchange solid phase extraction columns, Waters Sep-Pak Acell Plus CM 100 mg/1cc. 10. Measurement procedure:

a. Chemical 1. Instrumentation _____HPLC__ 2. Detector system ______MS/MS_ 3. Chromatographic column ____HILIC, Phenomenex, Synergy Polar-RP, 4 µm, 50 x 2 mm (if applicable)

b. Immunochemical/Immunoassay 1. Technique: _____________________________________________________________ (e.g. Elisa, RIA, Immunochromatog, etc) 2. Critical reagents: ________________________________________________________ (e.g. antibody specificity and availability) 3. Special equipment required: ________________________________________________

c. Microbiological 1. Technique: _____________________________________________________________ 2. Organism: ______________________________________________________________ 3. Media: _________________________________________________________________ 4. Special equipment required:________________________________________________

11. Sample/Analyte Stability Warning (if applicable): _____________________________________________________________ 12. Literature References available: a) Kaufmann Anton. Letter to the Editor. Rapid Mass Commun. Mass Spectrom. 2003; 17: 2575-2577._ b) Becker M et.al. Tandem mass spectrometric determination of streptomycin, dihydrostreptomycin and spectinomycin in bovine kidney using hydrophilic interaction chromatoggraphy (HILIC). First_______ International Symposium on recent Advances in Food analysis, Prague Czech republic, November 5-7,_ 2003. 13. Contact for Information: a. Name ____Carina_Branzell b. Country __Sweden_ c. Affiliation _National food Administration d. Address ___Box 622, SE-751 26 Uppsala_ e. Telephone _+46 (0)18 17 55 00___ f. FAX ______+46 (0)18 10 58 48__ g. Email [email protected] __

B. Method Performance

1. a. Limit of Detection (LOD) (µg/kg) ____1.1-2.5__ How was LOD determined?___3 x noise____ b. Limit of Quantification (LOQ) (µg/kg)____10___ How was LOQ determined?__The lowest validated level__________________ c. Method sensitivity____________________________________________ (The smallest difference in concentration that can be measured) 2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes_X__ No_____

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4. How is recovery estimated? _Matrix matched calibration curve_ (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested ___10, 20 and 30 µg/kg______ b. Concentration(s) measured __98-105 % (Bias)__ __ c. Recovery (%) ______61-82 %______

6. Precision using fortified control tissue a. Concentration(s) tested __10, 20 and 30 µg/kg_________________________ b. Repeatability (within lab CV) ___4-8 %_______________________________ c. Reproducibility (between lab CV) __4-17 %_____(between day)____________

7. Precision using tissue containing incurred drug residues a. Concentration(s) tested __106 µg/kg____z-score = 0.1 (FAPAS)___________ b. Repeatability (within lab CV) __Not applicable ____________ ___________ c. Reproducibility (between lab CV) _ Not applicable ___________ ___________

8. Selectivity of the method This information is often referenced as “Specificity”. Selectivity refers to the ability of the method to provide accurate measurement of the analyte of interest when other chemicals or drugs are also resident in the laboratory sample. Data of interest in this regard are the effects of:

a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along with the analyte of interest:___Sulfonamides and tetracyclines __ b. Contaminants that are likely to be present in the sample ________________________________

9. Type of Validation studies a. Single laboratory __X___ b. Multi-laboratory _____ c. AOAC or other official procedure _____

C. Information relevant to laboratory implementation

1. Training and experience recommended for analyts 2. Critical steps in the method 3. Information on availability of unusual reagents or equipment 4. Special reagent or sample stability concerns 5. Reagent handling and safety concerns (if any) 6. Literature references or other useful information

OUTLINE OF SCIENTIFIC ISSUES COMMONLY CONSIDERED IN THE DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS

1. Determinative (Quantitative) Method

A. Purpose of the Method

*Scope of application (intended use) *Target tissue *Marker residue (analyte) *Limit of quantification (LOQ), Limit of Detection (LOD) or other Lowest Validated Level B. Experimental data

*Reagents (purity, strength, grade) *Apparatus and Equipment *Analytical Standards (quality, concentration and solvents) *Tissue Samples (procedure for preparation for analysis) *Analyte Extraction Procedures *Analyte Clean-up *Instumental Procedures and Calibrations

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*Calculations C. Quality Assurance

*Storage Stability of the Analyte in Tissue *Quality Control Samples *System Suitability Criteria *Readiness to perform assessment *Data Acceptability Criteria

2. Confirmation Procedure

*Sample preparation *Instrumental procedures and calibrations *Standards employed *Criteria for positive identification

3. Validation considerations

*Accuracy *Recovery *Precision ( repeatability and reproducibility) *Sensitivity and LOQ *Specificity

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Analytical Method Information Summary A. Descriptive Information 1. Name of drug or chemical:_Quinolones, macrolides, tetracyclines, sulfonamides, penicillin G 2. Drug or chemical class:__Antimicrobial______ (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use:__Infectious diseases________ 4. Analyte(s) measured: Enrofloxacin, ciprofloxacin (metabolite), difloxacin, danofloxacin, tylosin, acetylisovaleryltylosin, 3-orto-valeryltylosin (metabolite), spiramycin 1, tetracycline, oxyteracycline, chlortetracycline, sulfadiazine, sulfathiazole, sulfamethazine, sulfadoxine, sulfaclozine, Penicillin G (specify if metabolite) 5. Intended use of the method: a. Screening ___Yes______ b. Routine_____Yes_________ c. Reference___No_______ d. Confirmatory__Yes___________ 6. Test matrix___Kidney, muscle, egg_______ (e.g. muscle, kidney, urine, etc) 7. Summary of principal steps in sample preparation: Homogenization of the sample__________ 8. Summary of principal steps in extraction procedure: Extraction with acetonitrile. Centrifugation and five-fold dilution with water______ 9. Summary of principal steps in analyte clean-up procedure: _None_____________ 10. Measurement procedure:

a. Chemical 1. Instrumentation _HPLC___________ 2. Detector system __MS/MS________

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3. Chromatographic column _Genesis C18, 4µm, 2.1 x 50 mm_______ (if applicable)

b. Immunochemical/Immunoassay 1. Technique: __________________ (e.g. Elisa, RIA, Immunochromatog, etc) 2. Critical reagents: ______________________________________________ (e.g. antibody specificity and availability) 3. Special equipment required: ________________________________________________

c. Microbiological 1. Technique: _______________ 2. Organism: ___________ 3. Media: __________________ 4. Special equipment required:________________________________________________

11. Sample/Analyte Stability Warning (if applicable): __Some of the analyte are light sensitive. Penicillin G samples and solutions should be stored at -70ºC.___ ____ 12. Literature References available: a) Granélli K, Rosén J, Simultaneous Determination of Penicillin G, Tetracyclines and Sulfonamides in Kidney by LC-MS/MS, 2002, Poster, 4th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, Belgium.____ __ b) Granélli K, A rapid method for the determination of macrolides, including acetylvaleryltylosin, in pig muscle by liquid chromatography-tandem mass spectrometry, 2006, Poster, 5th International symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, Belgium. 13. Contact for Information: a. Name ____Carina Branzell____ b. Country __Sweden_______ c. Affiliation _National Food Administration___ d. Address ___Box 622, SE-751 26 Uppsala___ e. Telephone ___+46 (0)18 17 55 00_______ f. FAX ________+46 (0)18 10 58 48___ g. Email [email protected] ______


1. a. Limit of Detection (LOD) (µg/kg) _LOD < ¼ MRL_________ How was LOD determined?__ LOD was defined as the fortification level where S/N > 3 b. Limit of Quantification (LOQ) (µg/kg)____1/2 MRL______________________

How was LOQ determined?__The lowest validated level____________________ c. Method sensitivity____________________________________________

(The smallest difference in concentration that can be measured) 2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes_X___ No_____ 4. How is recovery estimated _Matrix matched calibration curve___ (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested ___1/2 MRL, MRL and 1.5 MRL________ b. Concentration(s) measured ___Bias ± 10 %________ ____________ ____________ c. Recovery (%) _Tetracyclines 47-61 %. The others > 71 %__________ ____________

6. Precision using fortified control tissue a. Concentration(s) tested __1/2 MRL, MRL and 1.5 MRL ____________ ____________ b. Repeatability (within lab CV) ____3-34 %_______ ____________ ____________ c. Reproducibility (between lab CV) _ Not applicable_ ____________ ____________

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7. Precision using tissue containing incurred drug residues a. Concentration(s) tested __Participated in proficiency test with satisfying result____ b. Repeatability (within lab CV) ___________ ____________ ___________ c. Reproducibility (between lab CV) ___________ ____________ ___________


a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along with the analyte of interest ______________________________________. b. Contaminants that are likely to be present in the sample ______________________

9. Type of Validation studies a. Single laboratory __X___ b. Multi-laboratory _____ c. AOAC or other official procedure _____







*Reagents (purity, strength, grade) *Apparatus and Equipment *Analytical Standards (quality, concentration and solvents) *Tissue Samples (procedure for preparation for analysis) *Analyte Extraction Procedures *Analyte Clean-up *Instumental Procedures and Calibrations *Calculations C. Quality Assurance



*Sample preparation *Instrumental procedures and calibrations *Standards employed

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*Criteria for positive identification



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A. Descriptive Information 1. Name of drug or chemical: Tetracyclines, sulfonamides, quinolones, macrolides, ß-lactams 2. Drug or chemical class: Antimicrobial__ (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use: Infectious diseases______ 4. Analyte(s) measured: Chlortetracycline, oxytetracycline, tetracycline, doxycycline, sulfadoxine, sulfametazine, sulfathiazole, sulfadiazine, enrofloxacin, ciprofloxacin (metabolite), difloxacin, danofloxacin, acetylisovaleryltylosin, 3-O-acetyltylosin (metabolite), tylosin, spiramycin I, penicillin G, amoxicillin, ampicillin (specify if metabolite) 5. Intended use of the method:

a. Screening ______Yes_____ b. Routine________Yes___ c. Reference_______No__ d. Confirmatory____No___

6. Test matrix______Muscle and kidney____ (e.g. muscle, kidney, urine, etc) 7. Summary of principal steps in sample preparation: ___Homogenization_____ 8. Summary of principal steps in extraction procedure: ___Extraction with 70 % methanol__ 9. Summary of principal steps in analyte clean-up procedure: _A five-fold dilution with water____ 10. Measurement procedure:

a. Chemical 1. Instrumentation ____HPLC_____________________________________________ 2. Detector system ____MS/MS, Quattro LC, Micromass___________________________ 3. Chromatographic column __Genisis C18, 50 x 2.1 mm, 4 µm______________________ (if applicable)

b. Immunochemical/Immunoassay 1. Technique: __________________________________ (e.g. Elisa, RIA, Immunochromatog, etc) 2. Critical reagents: _________________________________________________________ (e.g. antibody specificity and availability) 3. Special equipment required: ________________________________________________

c. Microbiological 1. Technique: ___________________ 2. Organism: __________________________ 3. Media: _____________________________________ 4. Special equipment required:________________________________________________

11. Sample/Analyte Stability Warning (if applicable): ___ß-Lactams are light sensitive and should be stored at – 70° C. 12. Literature References available: Granelli K. and Branzell C., Analytica Chimica Acta 586 (2007) 289-295_______________________ 13. Contact for Information:

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a. Name ___Kristina Granelli_____ b. Country ___Sweden_____ c. Affiliation ___National Food Administration___ d. Address _____Box 622, SE-751 26 Uppsala___ e. Telephone ___+46 18 175500____ f. FAX ________+46 18 105848__ g. Email [email protected]___


1. a. Limit of Detection (LOD) (mg/kg) __LOD < ¼ MRL How was LOD determined?_LOD was defined as the fortification level where S/N > 3 b. Limit of Quantification (LOQ) (mg/kg)__Not applicable________ How was LOQ determined?___________________________________ c. Method sensitivity____________________________________________ (The smallest difference in concentration that can be measured)

2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes_X__ No_____ 4. How is recovery estimated __By the use of a matrix matched standard_______________ (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested ___MRL__________ b. Concentration(s) measured ___No false positives, no false negatives c. Recovery (%) ____Muscle: tetracyclines 50 – 60 %, the others > 70 %, Kidney: tetracyclines, macrolides and quinolones 30 – 70 %, the others > 70 %_______

6. Precision using fortified control tissue a. Concentration(s) tested __MRL_____ ____________ ____________ b. Repeatability (within lab CV) ___< 20 %____ ____________ ____________ c. Reproducibility (between lab CV) __Not applicable____________

7. Precision using tissue containing incurred drug residues a. Concentration(s) tested _Participated in proficiency test with satisfying results b. Repeatability (within lab CV) ___________ ____________ ___________ c. Reproducibility (between lab CV) ___________ ____________ ___________


a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along with the analyte of interest____Not applicable, only screening b. Contaminants that are likely to be present in the sample

9. Type of Validation studies a. Single laboratory _X__ b. Multi-laboratory _____ c. AOAC or other official procedure _____



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A. Descriptive Information

1. Name of drug or chemical: ß-estradiol 2. Drug or chemical class: steroid hormone (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use: 4. Analyte(s) measured: ß-estradiol (specify if metabolite) 5. Intended use of the method:

a. Screening: Bovine serum b. Routine: Bovine serum c. Reference__________________________________________ d. Confirmatory:

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6. Test matrix: Bovine serum (e.g. muscle, kidney, urine, etc) 7. Summary of principal steps in sample preparation:Serum is ready to use 8. Summary of principal steps in extraction procedure: 9. Summary of principal steps in analyte clean-up procedure: 10. Measurement procedure:

a. Chemical 1. Instrumentation : 2. Detector system: 3. Chromatographic column:

b. Immunochemical/Immunoassay 1. Technique: DELFIA (e.g. Elisa, RIA, Immunochromatog, etc) 2. Critical reagents: _DELFIA kit for Estradiol from Wallac OY__________________________________________________________________________ (e.g. antibody specificity and availability) 3. Special equipment required: ________________________________________________

c. Microbiological 1. Technique: _____________________________________________________________ 2. Organism: ______________________________________________________________ 3. Media: _________________________________________________________________ 4. Special equipment required:________________________________________________ 11. Sample/Analyte Stability Warning (if applicable): _____________________________________________________________ 12. Literature References available: ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ 13. Contact for Information:

a. Name : Gunnel Alfredsson b. Country: Sweden c. Affiliation: National Food Administration d. Address : Box 622 751 26 UPPSALA, Sweden e. Telephone :+46 18 175612 f. FAX : +46 18 105848 g. Email : [email protected]


1. a. Limit of Detection (LOD) (ng/kg) 14 How was LOD determined? From the kit calibration curve b. Limit of Quantification (LOQ) (ng/kg) 14 How was LOQ determined: From the kit calibration curve c. Method sensitivity____________________________________________ (The smallest difference in concentration that can be measured)

2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes__X__ No 4. How is recovery estimated ? From spiked serum (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested: 20, 40, 75 ng/kg c. Recovery (%) 92 %

6. Precision using fortified control tissue

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a. Concentration(s) tested: 20, 40, 75 ng/kg b. Repeatability (within lab CV) : 9 c. Reproducibility (between lab CV) ___________ ____________ ____________

7. Precision using tissue containing incurred drug residues a. Concentration(s) tested ___________ ____________ ___________ b. Repeatability (within lab CV) ___________ ____________ ___________ c. Reproducibility (between lab CV) ___________ ____________ ___________

8. Selectivity of the method: High This information is often referenced as “Specificity”. Selectivity refers to the ability of the method to provide accurate measurement of the analyte of interest when other chemicals or drugs are also resident in the laboratory sample. Data of interest in this regard are the effects of:

a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along with the analyte of interest: _steroid hormones________ b. Contaminants that are likely to be present in the sample _____________

9. Type of Validation studies a. Single laboratory X b. Multi-laboratory _____ c. AOAC or other official procedure _____









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1. Name of drug or chemical: Beta-agonists 2. Drug or chemical class:______________________________________________________________ (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use: Illegal 4. Analyte(s) measured: Brombuterol, Clenbuterol, Cimaterol, Cimbuterol, Mabuterol, Mapenterol, Ractopamine, Sabutamol, Tulobuterol and Zilpaterol (specify if metabolite) 5. Intended use of the method:

a. Screening Yes b. Routine Yes c. Reference______________ d. Confirmatory Yes

6. Test matrix Liver, muscle and urine (e.g. muscle, kidney, urine, etc) 7. Summary of principal steps in sample preparation: Liver and muscle are homogenised, proteas treated and hydrolysed with glucuronidase. Urine only hydrolysed with glucuronidase. 8. Summary of principal steps in extraction procedure: No 9. Summary of principal steps in analyte clean-up procedure: SPE extraction with MIP4SPE for beta-agonists 10. Measurement procedure:

a. Chemical 1. Instrumentation HPLC Agilent 1100 2. Detector system LC-MS/MS API 4000 Q-trap 3. Chromatographic column Cromolith Performance RP-18e 100x4,6mm (if applicable)

b. Immunochemical/Immunoassay 1. Technique: _____________________________________________________________ (e.g. Elisa, RIA, Immunochromatog, etc) 2. Critical reagents: _________________________________________________________ (e.g. antibody specificity and availability) 3. Special equipment required: ________________________________________________

c. Microbiological 1. Technique: _____________________________________________________________

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2. Organism: ______________________________________________________________ 3. Media: _________________________________________________________________ 4. Special equipment required:________________________________________________

11. Sample/Analyte Stability Warning (if applicable): _________________________________ 12. Literature References available: 13. Contact for Information:

a. Name Stina Söderlund b. Country Sweden c. Affiliation National Food Administration d. Address Box 622, 751 26 UPPSALA, Sweden e. Telephone +46 (0)18 175333 f. FAX +46 18 105848 g. Email [email protected]


1. a. Limit of Detection (LOD) (µg/kg) CCα=0,04 – 0,68 How was LOD determined?From calibration curve in matrix b. Limit of Quantification (LOQ) (µg/kg)0,5 How was LOQ determined?From CV c. Method sensitivity____________________________________________ (The smallest difference in concentration that can be measured)

2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes____ No X 4. How is recovery estimated Internal deuteated standard (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested .5-5.0 µg/kg 1.0-10.0 µg/kg 2.0-20.0 µg/kg b. Concentration(s) measured ___________ ____________ ____________ c. Recovery (%) 70-130 %

6. Precision using fortified control tissue a. Concentration(s) tested 0.5-5.0 µg/kg 1.0-10.0 µg/kg 2.0-20.0 µg/kg b. Repeatability (within lab CV) 3-56 c. Reproducibility (between lab CV) ___________ ____________ ____________



a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along with the analyte of interest__________________. b. Contaminants that are likely to be present in the sample _____________________



1. Training and experience recommended for analyts 2. Critical steps in the method

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3. Information on availability of unusual reagents or equipment 4. Special reagent or sample stability concerns 5. Reagent handling and safety concerns (if any) 6. Literature references or other useful information

OUTLINE OF SCIENTIFIC ISSUES COMMONLY CONSIDERED IN THE DEVELOPMENT ANDVALIDATION OF ANALYTICAL METHODS










------------------------------------------------------------------------


1. Name of drug or chemical: Chloramphenicol 2. Drug or chemical class: Antibiotic (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use: Illegal 4. Analyte(s) measured: Chloramphenicol (specify if metabolite)

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5. Intended use of the method: a. Screening: Honey b. Routine: Honey c. Reference___________________ d. Confirmatory: All matrixes

6. Test matrix: Muscle, urine, egg, milk, honey (e.g. muscle, kidney, urine, etc) 7. Summary of principal steps in sample preparation: hydrolysis with Helix Pomatia(urine, milk), other matrixes are homogenised, honey is diluted with water 8. Summary of principal steps in extraction procedure:Extraction with ethylacetate 9. Summary of principal steps in analyte clean-up procedure: SPE extraction withBondElute C18 10. Measurement procedure:

a. Chemical 1. Instrumentation : HPLC Waters Alliance 2690 2. Detector system: LC-MS/MS QUATTRO ULTIMA 3. Chromatographic column: Genesis C18 4µm, 2,1x50 mm


c. Microbiological 1. Technique: _____________________________________________________________ 2. Organism: ______________________________________________________________ 3. Media: _________________________________________________________________

4. Special equipment required:________________________________________________ 11. Sample/Analyte Stability Warning (if applicable): _____________________________________________________________ 12. Literature References available: 13. Contact for Information:



1. a. Limit of Detection (LOD) (µg/kg) CCα = 0,06 How was LOD determined? From calibration curve in matrix b. Limit of Quantification (LOQ) (µg/kg) 0,3 How was LOQ determined? From CV c. Method sensitivity____________________________________________ (The smallest difference in concentration that can be measured)

2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes____ No X 4. How is recovery estimated ? Internal deuterated standard (e.g. external standard; internal standard. etc) 5. Accuracy

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a. Concentration(s) tested: 0,15;0,3; 0,45 µg/kg c. Recovery (%) 91-124 %

6. Precision using fortified control tissue a. Concentration(s) tested : 0,15;0,3; 0,45 µg/kg b. Repeatability (within lab CV) : 11-16 c. Reproducibility (between lab CV) ___________ ____________ ____________



a. Drugs of similar structureor drug class or other veterinary drugs that may also be used along with the analyte of interest_______________________________. b. Contaminants that are likely to be present in the sample ___________________________________



1. Training and experience recommended for analyts 2. Critical steps in the method 3. Information on availability of unusual reagents or equipment 4. Special reagent or sample stability concerns 5. Reagent handling and safety concerns (if any) 6. Literature references or other useful information OUTLINE OF SCIENTIFIC ISSUES COMMONLY CONSIDERED IN THE DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS





*Storage Stability of the Analyte in Tissue *Quality Control Samples

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*System Suitability Criteria *Readiness to perform assessment *Data Acceptability Criteria





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1. Name of drug or chemical: tetracyclines and betalactams 2. Drug or chemical class: antimicrobial 3. Veterinary Use: infectious diseases 4. Analyte(s) measured: Tetracycline, Oxytetracycline, Chlortetracycline,Doxycycline, Penicillin-G,Amoxicillin, Ampicillin,Cefazolin, Cloxacillin, Ceftiofur, Cefoperazone, Nafcillin 5. Intended use of the method:

a. Screening _____________yes____________________________ b. Routine_______________yes_____________________________ c. Reference______________no____________________________ d. Confirmatory___________no____________________________

6. Test matrix milk ______________________________________ 7. Summary of principal steps in sample preparation: Homogenize 8. Summary of principal steps in extraction procedure: none 9. Summary of principal steps in analyte clean-up procedure: No clean-up________________________________________ 10. Measurement procedure:

b. Immunochemical/Immunoassay 1. Technique: Twinsensor receptorbased immunoassay 2. Critical reagents: Twinsensor kit_________________________ 3. Special equipment required: ______Heatsensor heating block

11. Sample/Analyte Stability Warning (if applicable): Betalactams requires -70° storage and are light sensitive. 12. Literature References available: 13. Contact for Information:

a. Name __Åsa Lundström______________________________________________ b. Country _Sweden_____________________________________________ c. Affiliation _F/K1 d. Address Naional Food Administration, Box 622, 751 26 Uppsala____ e. Telephone _4618-175702___________________________________________

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f. FAX _________________________________________________ g. Email [email protected]______________________________________________


1. a. Limit of Detection (LOD) (mg/kg) : MRL________________________ How was LOD determined? spiked samples__________________________________

b. Limit of Quantification (LOQ) (mg/kg) MRL__________________________ How was LOQ determined?___spiked samples ________________________________ c. Method sensitivity not applicable The method is not quantitative

2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes____ No_X____ 4. How is recovery estimated _________________________________________ (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested _____MRL______ ____________ ____________ b. Concentration(s) measured __no false positives, no false negatives____________ c. Recovery (%) The method is not quantitative___________ ____________ ____________

6. Precision using fortified control tissue a. Concentration(s) tested _______MRL____ ____________ ____________ b. Repeatability (within lab CV) ___not applicable________ ____________ ____________ c. Reproducibility (between lab CV) not applicable___________ ____________ ____________

7. Precision using tissue containing incurred drug residues

a. Concentration(s) tested ___________ ____________ ___________ b. Repeatability (within lab CV) ___________ ____________ ___________ c. Reproducibility (between lab CV) ___________ ____________ ___________


a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along _with the analyte of interest : b. Contaminants that are likely to be present in the sample ________________________________

9. Type of Validation studies a. Single laboratory _yes____ b. Multi-laboratory _____ c. AOAC or other official procedure _____


1. Training and experience recommended for analyts 2. Critical steps in the method 3. Information on availability of unusual reagents or equipment: Twinsensor kit sold by Unisensor SA,Belgium 4. Special reagent or sample stability concerns 5. Reagent handling and safety concerns (if any) 6. Literature references or other useful information

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1. Name of drug or chemical: Corticosteroids 2. Drug or chemical class: Antiinflammatory (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use: Infection diseases, metabolic diseases, inflammatory diseases, bovine mastitis 4. Analyte(s) measured: (specify if metabolite) Dexamethasone(DEX), Betamethasone(BET), Flumethasone(FLU), Methylprednisolone(MPRE), Prednisolone(PRE) 5. Intended use of the method:

a. Screening : YES b. Routine: YES c. Reference: NO

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d. Confirmatory: YES 6. Test matrix: milk and liver from bovine 7. Summary of principal steps in sample preparation: Milk-none Liver-homogenized in mixer 8. Summary of principal steps in extraction procedure: Milk- protein precipitation, centrifugation, filtration Liver-enzymatic hydrolysis, methanol extraction, evaporation, dilution with water. 9. Summary of principal steps in analyte clean-up procedure: SPE Oasis HLB 3 ml, 60 mg 10. Measurement procedure:

a. Chemical 1. Instrumentation: HPLC 2. Detector system: Micromass Quattro Ultima , LC-MS/MS MRM mode 3. Chromatographic column: Hypercarb, 2.1x100mm, 5 um (if applicable)


c. Microbiological 1. Technique: _____________________________________________________________ 2. Organism: ______________________________________________________________ 3. Media: _________________________________________________________________ 4. Special equipment required:________________________________________________

11. Sample/Analyte Stability Warning (if applicable): Keep standard solutions away from light 12. Literature References available: Rapid multi-residue method for the quantitative determination and confirmation of glucocorticosteroids in bovine milk using liquid chromatography-electro spray ionization-tandem mass spectrometry. M McDonald et al., Analytica Chimica Acta 588 (2007) 20-25. 13. Contact for Information:

a. Name: P. Sjöberg b. Country: Sweden c. Affiliation: National Food Administration http://www.slv.se d. Address: Box 622, SE-751 52 Uppsala e. Telephone: Central: +46 18 17 55 00 / Direct +46 18 17 57 21 f. FAX: Central: +46 18 10 58 48 g. Email: [email protected]


1. a. Limit of Detection (LOD) (µg/kg) Not Determined for milk, lower than 1/16 of MRL for all substances for liver. How was LOD determined? S/N > 3 for spiked samples at 1/16 times the MRL b. Limit of Quantification (LOQ) (µg/kg) Milk – 0.15 µg/kg for DEX, BET, FLU, MPRE, 0,3 µg/kg for PRE Liver – DEX 1 µg/kg, BET 1 µg/kg, FLU 2 µg/kg (no MRL), MPRE 5 µg/kg, PRE 5 µg/kg. How was LOQ determined? LOQ is defined as the lowest validated level c. Method sensitivity

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(The smallest difference in concentration that can be measured) 2. JECFA MRL ?? 3. Are analytical data corrected for recovery? Yes__X__ No_____ 4. How is recovery estimated: Matrixbased calibrationcurve (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested: Milk/liver 0,5xMRL, 1xMRL, 1,5xMRL b. Concentration(s) measured: Bias -27%-+21% c. Recovery (%) Absolute recovery: Milk 15-21%, Liver 56-82%

6. Precision using fortified control tissue a. Concentration(s) tested: 1xMRL, different samples b. Repeatability (within lab CV) Within day: milk 6-21%, liver 6-16% c. Reproducibility (between lab CV) Between day: milk 9-34%, liver 12-39%

7. Precision using tissue containing incurred drug residues

a. Concentration(s) tested ___________ ____________ ___________ b. Repeatability (within lab CV) ___________ ____________ ___________ c. Reproducibility (between lab CV) ___________ ____________ ___________

8. Selectivity of the method This information is often referenced as “Specificity”. Selectivity refers to the ability of the method to provide accurate measurement of the analyte of interest when other chemicals or drugs are alsoresident in the laboratory sample. Data of interest in this regard are the effects of:

a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along with the analyte of interest: This method separates chromatographically DEX and BET which have the same Molecular Weight and same fragmentation pattern on LC-MS/MS. b. Contaminants that are likely to be present in the sample: Not Determined

9. Type of Validation studies a. Single laboratory YES b. Multi-laboratory _____ c. AOAC or other official procedure _____


1. Training and experience recommended for analyst: HPLC, LC-MS/MS, Solid Phase Extraction 2. Critical steps in the method 3. Information on availability of unusual reagents or equipment 4. Special reagent or sample stability concerns 5. Reagent handling and safety concerns (if any) 6. Literature references or other useful information

------------------------------------------------------------------------


1. Name of drug or chemical: tetracyclines 2. Drug or chemical class: antimicrobial 3. Veterinary Use: infectious diseases 4. Analyte(s) measured: Tetracycline, Oxytetracycline, Chlortetracycline 5. Intended use of the method:

a. Screening _____________yes____________________________ b. Routine_______________yes_____________________________

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c. Reference______________no____________________________ d. Confirmatory___________no____________________________

6. Test matrix____________egg, muscle of fish and shellfish, honey____________- 7. Summary of principal steps in sample preparation: Homogenize 8. Summary of principal steps in extraction procedure: Add buffer, shake, centrifugate 9. Summary of principal steps in analyte clean-up procedure: No clean-up___________ 10. Measurement procedure:

b. Immunochemical/Immunoassay 1. Technique: Tetrasensor receptorbased immunoassay 2. Critical reagents: Tetrasensor kit_________________________ 3. Special equipment required: ______no__________________________________________

11. Sample/Analyte Stability Warning (if applicable): ______no_______________________________________________________ 12. Literature References available: Alfredsson G, Branzell C, Granelli K, Lundström Å: Simple and rapid screening and confirmation of tetracyclines in honey and egg by a dipstick test and LC-MS/MS.Analytica Chimica Acta 529(2005)47-51 13. Contact for Information:

a. Name __Åsa Lundström______________________________________________ b. Country _Sweden_____________________________________________ c. Affiliation _F/K1 d. Address Naional Food Administration, Box 622, 751 26 Uppsala____ e. Telephone _4618-175702___________________________________________ f. FAX _________________________________________________ g. Email [email protected]______________________________________________


1. a. Limit of Detection (LOD) (mg/kg) : MRL________________________ How was LOD determined? spiked samples__________________________________

b. Limit of Quantification (LOQ) (mg/kg) Not applicable, only screening__ How was LOQ determined?___________________________________ c. Method sensitivity not applicable The method is not quantitative

2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes____ No_X____ 4. How is recovery estimated _________________________________________ (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested _____MRL______ ____________ ____________ b. Concentration(s) measured __no false positives, no false negatives____________ c. Recovery (%) The method is not quantitative___________ ____________ ____________

6. Precision using fortified control tissue a. Concentration(s) tested _______MRL____ ____________ ____________ b. Repeatability (within lab CV) ___not applicable________ ____________ ____________ c. Reproducibility (between lab CV) not applicable___________ ____________ ____________


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a. Drugs of similar structure or drug class or other veterinary drugs that may also be used along with the analyte of interest : _______________________________ b. Contaminants that are likely to be present in the sample _______________________________

9. Type of Validation studies a. Single laboratory _yes____ b. Multi-laboratory _____ c. AOAC or other official procedure _____


1. Training and experience recommended for analyts 2. Critical steps in the method 3. Information on availability of unusual reagents or equipment: Tetrasensor kit sold by Unisensor SA,Belgium 4. Special reagent or sample stability concerns 5. Reagent handling and safety concerns (if any) 6. Literature references or other useful information









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1. Name of drug or chemical: Trenbolone_ 2. Drug or chemical class:_____steroid_______ (e.g. antimicrobial, anthelmintic, etc) 3. Veterinary Use:________illegal___________________ 4. Analyte(s) measured:__alfa-trenbolne, beta-trenbolone_______________ (specify if metabolite) 5. Intended use of the method:

a. Screening _________________________________________ b. Routine____________________________________________ c. Reference__________________________________________ d. Confirmatory_confermatory______________________________________

6. Test matrix: urine from bovines and pigs (e.g. muscle, kidney, urine, etc) 7. Summary of principal steps in sample preparation: hydrolysis with Helix Pomatia 8. Summary of principal steps in extraction procedure: ____________________________________ 9. Summary of principal steps in analyte clean-up procedure: SPE extraction + IAC ___________________________ 10. Measurement procedure: a. Chemical 1. Instrumentation _HPLC________________________________________________________ 2. Detector system __LC-MS/MS QUATTRO ULTIMA_______________________________________________________ 3. Chromatographic column _Zorbax C18, 5µ, 2,1x50_________________________________________________ (if applicable) b. Immunochemical/Immunoassay 1. Technique: _____________________________________________________________ (e.g. Elisa, RIA, Immunochromatog, etc) 2. Critical reagents: _________________________________________________________ (e.g. antibody specificity and availability) 3. Special equipment required: ________________________________________________ c. Microbiological 1. Technique: _____________________________________________________________ 2. Organism: ______________________________________________________________ 3. Media: _________________________________________________________________ 4. Special equipment required:________________________________________________ 11. Sample/Analyte Stability Warning (if applicable): _____________________________________________________________ 12. Literature References available:___________________________________________________________ 13. Contact for Information:

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1. a. Limit of Detection (LOD) (µg/kg) CCα = 0,3 How was LOD determined? From calibration curve in matrix b. Limit of Quantification (LOQ) (µg/kg) 0,5 How was LOQ determined? From CV c. Method sensitivity____________________________________________ (The smallest difference in concentration that can be measured) 2. JECFA MRL___________________________________________________ 3. Are analytical data corrected for recovery? Yes____ No X 4. How is recovery estimated ? Internal deuterated standard (e.g. external standard; internal standard. etc) 5. Accuracy

a. Concentration(s) tested: 0,5; 1,0; 1,5 µg/kg c. Recovery (%) 72-128 %

6. Precision using fortified control tissue a. Concentration(s) tested : O,5; 1,0; 1,5 µg/kg b. Repeatability (within lab CV) : 8-22 (α-Trenbolon), 6-19 ( ß-trenbolon) c. Reproducibility (between lab CV) ___________ ____________ ____________



a. Drugs of similar structure: Nortestosteron, other steroids or drug class or other veterinary drugs that may also be used along with the analyte of interest _________________________________________ b. Contaminants that are likely to be present in the sample ___________________________________



1. Training and experience recommended for analyts

2. Critical steps in the method

3. Information on availability of unusual reagents or equipment

4. Special reagent or sample stability concerns

5. Reagent handling and safety concerns (if any)

6. Literature references or other useful information

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*Storage Stability of the Analyte in Tissue *Quality Control Samples *System Suitability Criteria *Readiness to perform assessment *Data Acceptability Criteria 2. Confirmation Procedure

*Sample preparation *Instrumental procedures and calibrations *Standards employed *Criteria for positive identification 3. Validation considerations


UNITED STATES OF AMERICA

In Circular Letter, CL 2007/04-RVDF, dated January 2007, the Secretary, Codex Alimentarius Commission, invited governments and international organizations with observer status with Codex to provide comments on the compendium of methods prepared by the 16th CCRVDF and in particular to:

• review the content for any missing information;

• review the methods on which information was provided by their delegation and to update any addresses given as sources for the information;

• advise on any methods on which they may no longer be able to provide information;

• provide information by using Annex 3 on any methods which will address the current gaps either “validated method required” (see Annex 2) or supporting information to advance a method submitted buy another delegation which has “provisional” status to “full recommendation”. Annex 4 contains a list of some of the key performance requirements for methods fused for screening, quantitative determination or confirmation;

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• provide information on new methods which may provide alternative to the analysis of compounds for which methods have already been recognized by the Committee.

The United States delegation has completed its review of the compendium of methods and has no substantive comment to make on the content. The United States delegation wishes to acknowledge and thank the delegation from Canada for its leadership in stewarding the development of this compendium. The United States believes that this compendium serves a very useful purpose, particularly for developing countries and wishes to express its gratitude for the vision of the Canadian delegation to make this compendium a reality. We will continue to review the compendium and add additional methods as they become available.

Agenda Item 7 CX/RVDF 07/17/10 June 2007 JOINT FAO/WHO ... fileE Agenda Item 7 CX/RVDF 07/17/10 June...

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