2019 Vet Drug Residues ERP - Review Book

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Expert Review Panel for Veterinary Drug Residues Wednesday, September 11, 2019

10:00AM – 1:30PM MDT Sheraton Downtown Denver 1550 Court Place, Denver, Colorado, 80202 Room: Governor’s Square 12

AGENDA

1. Welcome and Introductions Joe Boison, University of Saskatchewan, Chair of the Vet Drug Residues Expert Review Panel

2. Review of AOAC Volunteer Policies & ERP Process Overview and Guidelines Deborah McKenzie, AOAC INTERNATIONAL

3. Review of Methods For each method, the assigned ERP members will present a review of the methods and manuscripts, after which the ERP will discuss the method and reach consensus on the status for each method.

A. Candidate Method VDR-001,* VD4S154 a. ERP Member Reviews b. Discussion and Consensus 4. Final Action Requirements for Adopted Methods (if applicable) 5. Adjourn

Draft meeting agenda subject to change without notice *Items requiring a vote by ERP

VET DRUG RESIDUES EXPERT REVIEW PANEL

METHODS AND SMPR ACCESS

• AOAC SMPR 2018.010 • METHODACCESS [ERP ONLY - PASSWORD REQUIRED]

VDR-001 ERP EVALUATION OF VETERINARY DRUGS IN FOOD BY LC-MS/MS JOE BOISON INDEPENDENT CONSULTANT AND ERP CHAIR SHERATON DENVER DOWNTOWN HOTEL, GOVERNOR’S SQUARE 10:00 – 13:30 MDT SEPTEMBER 11, 2019

Table 1. SMPR

Residue concentration in Matrix

Acceptance Criteria

0 (Blank) 0.5 MRL 1.0 MRL

≤ 10% POD with 95% CL

30 per drug a 30 per drug a

≥ 10% with 95% CL ≥ 90% with 95% CL b

a Tested as drug cocktail(s).

b All incorrect results must be investigated for the determination of the concentration at which POD 90

with 95%

confidence is achieved.

Method Properties and Expected Attributes

1. Suitable methods will include blank check samples and check standards at 0.5 x MRL and 1.0 x MRL. 2. Method developers will provide information on how cutoffs were determined. 3. Method developers may prepare cocktails of multiple drug residues. 4. Method developers are cautioned that some drug residues may have additive or masking effects when combined and should be prepared to demonstrate that these concerns have been addressed with their submitted materials/data.

Method Properties and Expected Attributes (Cont’d)

4. Method developers should consider the stability of drug residues in cocktails and be prepared to demonstrate that these concerns have been addressed in their data submission package. 5. For identification, method developers must provide the precursor ion and at least two SRM transitions with ion ratios and retention parameters for each veterinary drug.

Method Properties and Expected Attributes (Cont’d )

6. The performance criteria in SMPR Table are for single laboratory validation. 7. Method developers contemplating a multi‐ lab validation study should contact AOAC for developing a collaborative study design.

Marker Residues To Be Monitored for Regulated Compliance in Milk

Target Testing Level (ppb) Screening Target Concentration (STC)

Analytical Range Top (ppb)

Regulated marker

Compounds

Albendazole sulfone Albendazole sulfoxide Albendazole 2- aminosulfone Albendazole oxide Albendazole sulfone Albendazole 2- aminosulfone sum of metabolites containing 2,4- DMA moiety

Chlortetracycline 4-epi- chlrotetracycline Cypermethrin Alpha- Cypermethrin Ciprofloxacin Enrofloxacin Eprinomectin (B1a) Oxfendazole sulphone Fenbendazole Oxfendazole sulphone Fenbendazole Sulfoxide

50 50 10 10 50 50

500 500 100 100 500 500

5 Chlortetracycline

500

Cypermethrin

500

Albendazole

500

Enrofloxacin

500

8 Eprinomectin

500

9 Febantel

500

Amitraz

Fenbendazole

300

3000

Cefapirin

100

Cefapirin

Deacetylcefapirin

300

Marker Residues To Be Monitored for Regulated Compliance in Milk (cont’d)

Target Testing Level (ppb) Screening Target Concentrati on (STC)

Analytic al Range Top (ppb)

Regulated marker

Compounds

Flunixin

5-hydroxyflunixin Sum of C1, C1a, C2, C2a Ivermectin B1a 4- Methylaminoantip yrin N-methyl-1,3- propanediamine Albendazole oxide Albendazole sulfone Albendazole 2- aminosulfone Neomycin B

200

Gentamicin( s) Ivermectin

500

Metamizole

250

Morantel

250

Neomycin

75 50 50

750 500 500

Netobimin

500

Marker Residues To Be Monitored for Regulated Compliance in Tissue

Target Testing

Level (ppb) Screening Target Concentra tion (STC)

Analytical Range Top

Compounds

Regulated marker

Albendazole sulfone Albendazole sulfoxide Albendazole 2- aminosulfone Albendazole oxide Albendazole sulfone sum of metabolites containing 2,4-DMA moiety Dichloroisoeverninic acid Albendazole 2- aminosulfone

50 50 25 50 50 50

500 500 250 500 500 500

Albendazole

Amitraz

25 30 30 75

250 300 300 750

3 Avilamycin 4 Azaperone

Azaperone Azaperol Bacitracin A

Bacitracin (A, B, C)

Marker Residues To Be Monitored for Regulated Compliance in Tissue

Target Testing

Level (ppb)

Analytical Range Top

Regulated marker

Screenin g Target Concentr ation (STC)

Compounds

Carprofen Carprofen glucuronide Cefapirin

250 250

2500 2500

Carprofen

7 Cefapirin

25 25

250 250

Deacetylcefapirin

8 Ceftiofur

Ceftiofur

500 500

5000 5000 500

Desfuroylceftiofur Chlortetracycline 4-epi- chlrotetracycline Cypermethrin α-Cypermethrin

50 50 10 10 50 10 50

Chlortetracycline

500 100 100 500 100 500

10 Cypermethrin

11 Doxycycline 12 Enrofloxacin

Doxycycline Ciprofloxacin Enrofloxacin

Marker Residues To Be Monitored for Regulated Compliance in Tissue

Target Testing Level (ppb) Screening Target Concentrat ion (STC)

Analytical Range Top

Compounds

Regulated marker

13 Eprinomectin Eprinomectin (B1a)

25 25 50 25 50 50 50

250 250 500 250 500 500 500

Oxfendazole sulphone Fenbendazole Oxfendazole sulphone Fenthion and metabolites Florfenicol Florfenicol-amine Flubendazole 2-amino 1H- benzimidazol-5-yl- (4fluorophenyl) methanone

14 Febantel

Fenbendazole

16 Fenthion 17 Florfenicol

Flubendazole

19 Flunixin

Flunixin

10 20 25

100 200 250

5-hydroxyflunixin

20 Gentamicin(s) Sum of C1, C1a, C2, C2a

Marker Residues To Be Monitored for Regulated Compliance in Tissue

Target Testing Level (ppb) Screening Target Concentrat ion (STC)

Analytical Range Top

Compounds

Regulated marker

21 Ivermectin

Ivermectin B1a 4-Methylamino antipyrin Monepantel sulfone N-methyl-1,3- propanediamine Neomycin B Albendazole oxide Albendazole sulfone Albendazole 2- aminosulfone Dinitrocarbanilide (DNC)

500

Metamizole

150

1500

22 Monepantel 23 Morantel 24 Neomycin

500

250

2500 500

50 50

500

Netobimim

500

100

1000

26 Nicarbazin

Marker Residues To Be Monitored for Regulated Compliance in Tissue

Target Testing Level (ppb) Screening Target Concentrat ion (STC)

Analytical Range Top

Regulated marker

Compounds

Methylphenylquin olin-carboxylic acid Oxfendazole Sulphone Fenbendazol Oxytetracycline 4-epi- oxytetracycline

Olaquindox

250

28 Oxfendazol

420 50 50 25 0.5

4200 500

Oxytetracycline

500 250

30 Penethamate Penicillin G

Pyrantel

N-methyl-1,3- propanediamine

750

32 Ractopamine

Roxarsone (Arsanilic acid) Arsenic

250

2500

Salicylate Aluminium Salicylate Sodium

Salicylic Acid

100

1000

Salicylic Acid Spiramycin Neo-Spiramycin Sum of all substances belonging to the sulfonamide

200 100 100

2000 1000 1000

36 Spiramycin

SULFONAMID E

500

Marker Residues To Be Monitored for Regulated Compliance in Tissue

Target Testing Level (ppb) Screening Target Concentrat ion (STC)

Analytical Range Top

Regulated marker

Compounds

Tetracycline

50 50 50 50 50 50 50 50 25 25 1

500 500 500 500 500 500 500 500 250 250 10

Tetracycline

4-epi-tetracycline Thiabendazole 5-hydroxy- thiabendazole 8-alpha- hydroxymutilin Toltrazuril sulfone Beta-trenbolone Ketotriclabendazo le Tiamulin

Thiabendazole

Tiamulin

41 Toltrazuril 42 Trenbolone

Triclabendazol e

44 Tylvalosin

Tylvalosin

3-O-acetyltylosin

Zoalene

1500 1500

15000 15000

45 Zoalene

3-amino-5-nitro-o- toluamide

(Dinitolmide)

ERP REVIEWS

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-09-09 02:30:09

Name

JOE BOISON

E-mail

jboison@shaw.ca

Organization

Independent Consultant

Title of Method

VETERINARY DRUGS IN FOODS BY LC-MS/MS

AOAC Candidate Method Number (e.g. ALN-01)

VDR-001

Applicable SMPR

VDR-001

I. Summary of the Method

The method uses 4 methods to screen for and confirm a complement of 154 veterinary drug residue in milk-based products (skimmed milk powder, fat-filled milk powder, whey protein, lactose, casein, infant formula, infant cereals and baby foods), meat-based and fish-based products (fresh, powdered, cooked, infant cereals and baby foods by LC-MS/MS. The method involves the extraction of the food matrix with a mixture of water, acetonitrile and formic acid followed by a liquid-liquid partitioning with a mixture of sodium sulfate, sodium chloride and citrate salts. After centrifugation, the resulting acetonitrile supernatant is diverted to 4 portions. The first portion can be split into 2 to permit the analysis of 99 multi-analyte drug residue (1 portion) and 6 avermectins (2nd portion). YE S. 105 VETERINARY DRUGS – [SHOULD BE REVISED TO ACCOUNT FOR MONITORING THE APPROPRIATE MARKER RESIDUES] - milk based products including milk fractions, intact formula, growing up formula, adult formula, infant cereals, and baby foods - meat and fish-based products including powdered fresh, cooked, infant cereals and baby foods 23 BETA LACTAMS – - for powdered samples (infant formula, milk fractions, infant cereals, meat and fish powders…….) - for fresh meat/seafood/fish products; - baby food jars (eg., puree) - 12 (14) AMINOGLYCOSIDES – Sample preparation described for - milk-based products – milk fractions, infant formulae, infant cereals, baby foods - meat-based, fish-based, seafood-based products including powdered, fresh, , infant cereals and baby foods cooked meat/fish/seafood - egg-based products including egg white and whole egg powders [GENTAMYCIN SHOULD BE COUNTED AS 1 EVEN THOUGH THE 3 ISOMERS ARE BEING MEASURED BECAUSE THEIR PRESENCE NEED TO BE SUMMED UP] 5 TETRACYCLINE RESIDUES & THEIR EPIMERS – - Applicable matrices not spelled out as the previous methods. [SHOULD BE INCLUDED IN THE SOP]

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing.

2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR.

Residue concentration in Matrix N Acceptance Criteria 0 (Blank) 30 ≤10% POD with 95% CL 0.5 MRL 30 per druga ≥ 10% with 95% CL 1.0 MRL 30 per druga ≥ 90% with 95% CL

aTested as drug cocktail(s). bAll incorrect results must be investigated for the determination of the concentration at which POD90 with 95% confidence is achieved.

3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method.

YES. The terms specified in the SMPR are used and applied appropriately. 32 milk based samples are analyzed for 23 beta lactams, 30-31 samples were analyzed for 23 antimicrobials, 20 samples for HA for 23 antimicrobials. A minimum of 30 replicate samples was to be analyzed for each vet drug. Negative control samples tested negative for the 23 antibiotics Appropriate number of replicate samples used in the estimation of the analytical parameters of the 4 methods

YES

YES. Minor editorial revisions will be provided in the text to the method authors

The SMPR does not require the use of reference standards but the method authors chose to subject the methods to the following Proficiency testing round robins: lyophilized bovine milk, Progetto Trieste 2015, 3rd Round MI1532-2; chicken muscle, Progetto Trieste, 2015, 4th Round, MI 1542-1; lyophilized bovine milk, Progetto Trieste, 2015 4th Round MI 1543-1; lyophilized cod muscle Progetto Trieste 2016, 1st Round FI 1610-2; rabbit muscle, Progetto Trieste 2016, 1st Round M 1625-1; Material B, Salmon muscle RIKILT 2016-02, porcine muscle RIKILT 2017-01, porcine muscle RIKILT 2017-01

3. Is there information demonstrating that the

YES

method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 5. Based on the supporting information, what are the pros/strengths of the method?

YES

Except for a few typographical and editorial errors, the method was written clearly and concisely. Recommendations for minimum editorial revision

1. Methods have the capacity and ability to screen for the presence and or absence of the specifiedd beta-lactams in the specified matrices. 2. For the aminoglycosides, Gentamicin monitored as sum of C1, C1a, C2, C2a; 3. Protonated ion and at least 1 transition ion but no identification ion and retention time provided for each aminoglycoside vet drug 4. For the beta-lactams monitoring cephapirin as desacetyl cephapirin 5. Ionization and retention parameters for the beta lactams provide for guidance 6. A minimum of 60 samples analyzed in replication for repeatability assessment

6. Based on the supporting information, what are the cons/weaknesses of the method?

1. The method appears to monitor the parent drugs for all the antibiotics involved with no indication that the marker residues that had been identified for regulatory monitoring by International organizations were being monitored. [Seek clarification from method authors] 2. For the multi-residue method containing 105 compounds, it appears that there were some vet drugs for which the appropriate marker residues were not monitored. 3. These included – enrofloxacin as ciprofloxacin, febantel as oxfendazole sulphone, flubendazole as 2-amino 1H-benzimidazol-5-yl-(4- fluorophenyl) methanone; florfenicol as florfenicol amine, fenbendazole as oxfendazole sulphone and fenbendazole sulfoxide; nicarbazin as dinitrocarbanilide (DNC), oxfendazole as oxfendazole sulphone; phenylbutazone as oxyphenbutazone, spiramycin as a mix of spiromycin and neo-spiramycin, carprofen as the glucoronidase; Triclabendazole as ketotriclabendazole. 4. Monitoring ceftiofur as ceftiofur (should be monitored as desfuroylceftifur) 5. Chromatographic retention times provided but no Ionization parameters for the 105 compound method;

7. Any general comments about the method?

Check with method authors to ensure that the appropriate marker residues are being monitored and not merely the parent drugs for convenience.

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

Recommend first action.

If possible, the authors should include in a Table the recommended MARKER RESIDUE that should be monitored for these APPROVED veterinary drugs in these food matrices.

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-08-30 10:27:22

Name

Stefan Ehling

E-mail

stefan.ehling@abbott.com

Organization

Abbott

Title of Method

Screening and Identification Method for Regulated Veterinary Drug Residues in Food

AOAC Candidate Method Number (e.g. ALN-01)

VDR-001

Applicable SMPR

2018.010

I. Summary of the Method

The method achieves screening and confirmation of 154 veterinary drug residues in a broad range of milk and meat-based foods. It uses liquid chromatography-mass spectrometry (LC/MS) to ensure high selectivity and sensitivity of detection. Four different work streams (one of them with two substreams) are used to accommodate a great variety of compounds with different physicochemical properties. The results are reported as either "negative" or "suspect".

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used.

Yes, with the exception of 3 compounds which were removed from the scope of the method.

Yes

4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 3. Is there information demonstrating that the

Yes

Yes, with the exception of 3 compounds which were removed from the scope of the method.

2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 5. Based on the supporting information, what are the pros/strengths of the method?

Yes

-LC/MS ensures high degree of selectivity and sensitivity. -Different work streams offer optimal performance for a variety of compounds with different physicochemical properties. -Each sample is analyzed twice to demonstrate absence of analyte in the unspiked sample and presence of analyte after spiking. -Design of validation scheme using cutoff value is original, sound, and effective. -Extensive use of blanks, positive controls, negative controls, and proficiency test samples greatly enhance confidence in generated results.

6. Based on the supporting information, what are the cons/weaknesses of the method?

-3 compounds had to be removed from the scope of the method. If these are to be included, additional work streams will have to be developed.

7. Any general comments about the method?

The method is of high quality, scientifically sound, and properly validated.

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

Yes, I recommend the method as a First Action. It meets or exceeds the SMPR for the vast majority of compounds.

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-09-06 19:39:36

Name

Ujwal Patil

E-mail

ujwalpatil@eurofinsus.com

Organization

Eurofins CAL

Title of Method

Screening and Identification Method for Regulated Veterinary Drug Residues in Food

AOAC Candidate Method Number (e.g. ALN-01)

VD4S154

Applicable SMPR

2018.010

I. Summary of the Method

Current method include protocol for screening and identification of a variety of groups of veterinary drugs in assorted matrices. Veterinary drugs were divided into four different streams based on chemical structures, extraction and analytical separation parameters. Different sample preparation protocols were used for each stream. LC-MS/MS was used for analysis of samples. Method was validated according to Community Reference Laboratories Residues (CRLs) guidelines.

Yes.

Every method contains safety precautions section which recommended handling chemicals according to MSDS sheet.

Yes.

Validation for multifamily, aminoglycosides and tetracyclines involved use of incurred samples. Incurred sample was not included for validation of beta lactams, possibly due to availability. During routine analysis, an independent source of verification can be added , if incurred samples are not available.

There are some analytes which have STC set at higher than the lowest MRL. Additionally, comment should be made about the equivalence of validation protocol used by the authors (CRL) to validation guidance in SMPR.

Blank checks and check standards were part of method validation.Cutoff determination calculations were provided.

Required data is provided to prove that the system suitability tests and controls worked as specified in SMPR.

Yes.

Based on the data provided, method is rugged and easy. Dividing veterinary drugs into different groups add more reliability to analysis especially for challenging groups such as aminoglycosides and beta lactams. Method is organized and very detailed. It includes specific handling directions for certain group of analytes that could significantly affect the outcome. Since naturally incurred samples/CRM are difficult o find for all/majority of veterinary drugs, an independent source of verification should be added during routine analysis.

6. Based on the supporting information, what are the cons/weaknesses of the method?

7. Any general comments about the method?

None.

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

I am convinced by the method's ability to screen and identify drug residues in assorted matrices discussed in the document. Proposed method meets the SMPR requirements. Method is technically sound and rugged.

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-09-06 16:35:28

Name

Sherri Turnipseed

E-mail

sherri.turnipseed@fda.hhs.gov

Organization

US FDA

Title of Method

Veterinary Drug Residues

AOAC Candidate Method Number (e.g. ALN-01)

VD4S154

Applicable SMPR

2018.010

I. Summary of the Method

The submission describes and documents a suite of methods to screen for 154 veterinary drug residues in milk and meat products There are four different analytical methods (defined as "streams") that would be performed for each sample to cover many the veterinary drug residues specified in the SMPR The four methods are divided by drug class with the first method "Stream A" incorporating the majority (107) of the veterinary drugs covered by the submission including the major drug classes of sulfonamides, quinolones, macrolides, avermectins, benzimidazoles and several others. The sample cleanup is divided into two portions, one for the avermectins and one for the remaining analytes. Stream B method includes 23 β -lactam (penicillin and cephalosporin) analytes. Stream C method is for the very polar aminoglycoside analytes (N=14). Stream D method is designed for the 5 major tetracycline residues and their related epimers. Other Comments: • The methods use a relatively large volumes of buffer or acetonitrile for extraction (20-45 mL). • The documentation regarding the different choices for QuEChERS salts and standard mixes (preparing manually and/or purchasing pre-made kits from manufacturers) is very helpful. This not only demonstrates method ruggedness but also allows for flexibility in implementation.

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing.

The suite of methods submitted is applicable and meets the general requirements of the SMPR 2018.010. The SMPR states that “A single method is not required to cover all drug/matrix combinations, but method developers should strive to include as many relevant drug residues as possible for each matrix claimed. Method developers may choose to claim one or more matrices”. Many of the residues listed in Table 1 of the SMPR are included in the scope of the methods submitted. However, the list of analytes covered by the submitted methods and those listed in the SMPR do not match completely. Most of the analytes listed in the SMPR, but not covered by the suite of methods submitted, can be classified as pesticides (organophosphate and pyrethroid compounds), hormones, or metabolites of primary analytes. In addition, many drugs listed on the SMPR list but not included in the suite of methods submitted have MRLs only for chicken. It does appear that chicken was included in the “meat-based products” category of matrices validated (see description on page 208 of submission), so not including some of these drugs may be a gap in addressing the SMPR that should be discussed. There are also several (~30) analytes included in the 154 compound list of the methods submitted that were not part of the SMPR Table 1 including several quinolones, β -lactams and macrolide compounds. Some prohibited substances, such as chloramphenicol and phenylbutazone, are also included in the submitted methods. The analytical methods meet the requirements of the SMPR. The SMPR stated that the methods should be liquid chromatography mass spectrometry and the methods submitted are based on this technique. Although there is a suite of methods submitted (using 4-5 different sample preparation/ instrument conditions) all are LC-MS/MS triple quadrupole methods utilizing electrospray ionization and selected reaction monitoring (SRMs) with two transitions per analyte. The definitions specified in SMPR are used and applied appropriately in the method. The two definitions included in the SMPR are Maximum Residue Limit (MRL) and Probability of Detection (POD). The MRLs from Table 1 of the SMPR were used by the method developers to optimize and validate the appropriate residues levels for analytes in their methods. Probability of Detection values were determined for each analyte and are listed in Annex-1 Validation. In the introduction to Annex -1, it might be helpful to include the citation to the AOAC document describing POD (Appendix H cited in the SMPR) as well as an example calculation, even if it very simple (POD = x/N where x is number found positive and N is total number tested). The methods as written contain all appropriate precautions and warning related to safety and potentially hazardous reagents or procedures. Each of the four method streams contains a section (“Section 4 Safety Precautions” on pages 12, 72, 111, 152) that addresses any possible safety concerns that might be encountered when preforming the methods.

2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used.

III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method.

The definitions specified in the SMPR are used applied appropriately in the supporting documentation. The validation data supplied in Annex 1 specifically addresses the MRL and POD for each compound. Other definitions used in this package, particularly “Screening Target Concentration (STC)” and “Cut-off level” are defined several times throughout the supporting information. The relationship (fold change) between the STC and the MRL is shown in Annex-1 with the STC levels typically between 0.1X and 0.5X of the MRL which is appropriate for a screening method. False positive/false negative values are also calculated in addition to POD. The manuscripts cited at the beginning of the submission (in the Notes to ERP) primarily cite false positive/false negative values to characterize the screening method results. To validate the method the developers primarily used fortified samples. There were several representative types of each matrix class. For example, milk products included milk fractions, formulas, cereals and baby foods and meat products included fresh, powdered, processed from several species of animals. Adequate information was provided with each method stream for sample preparation. The validation plan in terms of levels (blanks, spikes at 1 STC (~ 0.1-0.5 MRL) and spikes at 2 STC) and numbers of samples (60-80) was specified in Section 7.1 of each method stream and meets the requirements of the SMPR. The results are shown in Annex 1 (it was noted that the exact numbers differ slightly between the Validation Plans and the results in Annex 1 – maybe results from different laboratories?). Also, I did not understand how the Table on page 54 of the submission package relates to the number of samples specified in the Validation Plans and/or documented in Annex 1. Certified reference materials are not available for these drug/matrix combinations. There were examples of analysis of proficiency testing samples included with the validation. For example, a proficiency sample for containing spectinomycin was tested with the screening method and the correct results of “Suspect” was obtained. Other examples of proficiency sample testing are included in attached manuscripts (e.g. tetracyclines).

3. Is there information demonstrating that the

As mentioned above in II.1 of this review report, many of the residues listed in Table 1 of the SMPR are included in the scope of the methods submitted. However, the list of analytes covered by the submitted methods and those listed in the SMPR do not match completely. There are many pesticides listed in the SMPR Table that are not included in methods described in this submission. Although, some pesticides might have been included in the “multi-family” method, others especially the pyrethroids, would most likely require an additional method stream. Because published methods are available which cover a larger scope of pesticides in these matrices, this submission is justifiably more focused on veterinary drug residues. Some explanation might be needed as to why some of the drugs with MRLs for poultry were not considered since chicken is included in the scope of validated matrices. Also, as mentioned previously, some drug metabolites listed on the SMPR are not included. In general, this is not a critical gap if the analytes selected adequately capture the potential incidence of contamination with drug residues. For example, it may not be necessary to include all the listed metabolites of albendazole. However, in other instances, the lack of a drug metabolite that may act as the marker residue (e.g. florfenicol amine) could result in missed non- compliant samples. For the analytes that were chosen to be included in this suite of methods, the vast majority met the performance characteristics specified in the submission. Only a few (3) compounds in the multi-family did not meet these criteria, and these analytes were removed from the scope. No additional precautionary statements were needed regarding safety. Critical steps in the method were adequately described in the detailed analytical flow sheets for each method stream. Some additional information regarding sample through-put would be useful. For each method stream, Section 10.5 specifies the time of analysis required for each batch of 20 samples (30-48 hours for each stream). In addition, knowing how many different LC-MS/MS instruments and how many analysts are required to run a sample set by all 4 method streams would allow a laboratory to allocate the necessary resources to implement these methods. The hormones listed in the SMPR, especially those with low MRLs, might also be difficult to include in the multi-residue method.

IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method?

2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones.

The method specifies system suitability and QC controls that should be performed with the method. The positive and negative controls that should be run with each batch are described for each method stream. In general, these include a solvent based QC standard corresponding to 1X STC (~0.5X MRL) or 2X STC levels of analytes. Matrix based QC samples include a blank matrix, and a positive control that is fortified at 1X STC and also at 2X STC to simulate actual samples analyzed unfortified and fortified, respectively.

In addition, the strategy of analyzing each sample “as is” as well as fortified is described and helps mitigate matrix effects for any given sample.

Information was provided to demonstrate how cutoff values were determined.

The “identification criteria” initially described initially on page 29 may not be adequate. The criteria in the submission states “Each analyte surveyed is identified when the following criteria are fulfilled: A signal is visible at two diagnostic transition reactions and the retention time of the analyte in the extract should correspond to that of the QC solutions within a ± 0.2 min tolerance “, but does not require any calculation of ion ratios. See additional comments below in part IV.6. There is adequate information to demonstrate that the system suitability tests, controls samples worked appropriate as expected. The criteria needed for system suitability (RSD of solvent QC standards < 30%) as well as matrix negative and positive QC samples analyzed with each batch (analytes in negative QC need to be < STC and positive controls need to have all analytes characterized as “suspect”). Based on all of the validation data provided, these QC guidelines seem appropriate and criteria were met as expected.

3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 5. Based on the supporting information, what are the pros/strengths of the method?

The method was written clearly and concisely. The method submitters did an excellent job of presenting a large amount of data in a very organized, easy to follow format. In my experience it is difficult to document these large multi- residue methods in a thorough, yet assessable way, and I was very impressed by this submission.

The methods include most of the analytes listed in the SMPR.

The method developers’ approach and strategy both in terms of how the classes of analytes were divided between the different method streams and how the screening was performed (fortifying each samples) was logical and thorough. Implementing these methods will allow for a complete investigation of samples to determine compliance or non-compliance for the presence of these 154 veterinary drug residues at the levels of interest. The data package submission was well -organized and easy to follow. The validation information was included and supported the methods’ applicability to the requirements of the SMPR.

6. Based on the supporting information, what are the cons/weaknesses of the method?

Because each sample is analyzed with 4-5 different method streams and is analyzed at least twice (fortified and unfortified), the number of analyses required for each sample is fairly high (N=10/sample plus any QC samples for each batch) and may prevent optimum sample efficiency. Although the different method streams described ensure a thorough investigation of each sample for the 154 drug residues, a more universal, generic screening procedure would be more practical for many laboratories. With a screening method, it is not necessary to optimize extraction efficiencies and linearity. Rather, it is just important to establish reproducible detection and identification for the residues at the levels of concern. As such, a more generic extraction and instrumental method might have been sufficient. Although it may always be necessary to treat the aminoglycosides separately, it may have been possible to include β -lactams and tetracyclines in the “multi-family” method stream. An increase in work-flow efficiencies may be worth the loss in method performance for these analytes if there are still adequately detected for screening purposes. The MS data as presented does not support established identification criteria (EU, FDA). Although this may not be required for a screening procedure, ion ratios were requested as part of the SMPR validation guidance. In the peer- reviewed articles, the method authors did provide a rationale for not including ion ratios in their criteria (to minimize false negative rates). However, performing additional data analysis to determine if established identification criteria are met would make the method more applicable to a wider variety of laboratories including regulatory agencies. I would concur that the suite of methods submitted in response to this SMPR should move forward as a First Action Official Method. The methods are logical and well documented and would be of value to many working with the analysis of veterinary drugs. However, I also think that other multi-analyte veterinary drug methods will still be of value to the community because the methods submitted here do not cover all analytes or matrices of interest. Also, if sample through-put is a priority, using several separate analytical method streams is also not as efficient as more generic, universal multi-residue veterinary drug methods currently available. I believe that this method should be adopted as First Action and published in the Official Methods of Analysis of AOAC International. The methods submitted generally meet the requirements specified in the 2019.010 SMPR. Because of the number of method streams, any new matrix would require a great deal of additional validation.

7. Any general comments about the method?

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-08-31 11:55:10

Name

TOMASZ TUZIMSKI

E-mail

tomasz.tuzimski@umlub.pl

Organization

Medical University of Lublin, Poland

Title of Method

Veterinary Drug Residues submitted by Thierry Delatour

AOAC Candidate Method Number (e.g. ALN-01)

VD4S154

Applicable SMPR

AOAC SMPR® 2018.010

I. Summary of the Method

The proposed method described determination 154 drug residues (such as 107 typical veterinary drugs, 23 Beta-Lactam residues, 14 aminoglycosides, 5 compounds from tetracyclines group (plus related epimers) in a broad range of food matrices including milk-based, meat-based, and fish-based ingredients, and their products by liquid chromatography - triple quadrupole tandem mass spectrometry (LC-MS/MS). The developed method was evaluated following the definitions of Standard Method Performance Requirements (SMPRs®) for Screening and Identification Method for Regulated Veterinary Drug Residues in Food (AOAC SMPR® 2018.010) with respect to Limit of Detection (LOD) and the Limit of Quantitation (LOQ), Linearity, Repeatability, Reproducibility, Recovery.

YES: The applicability of the method is adequate to the applicability of the SMPR.

YES: The analytical techniques in the method are adequate and meet the SMPR.

YES: Definitions, which are specified in the SMPR, were listed in the description, also were applied appropriately in the method.

Yes: The method contains all appropriate precautions and warnings related to the method’s reagents, components, instrumentation, or method steps that may be hazardous.

YES: The definitions specified in the SMPR were used and applied appropriately in the main document and supporting documentation.

YES: There are information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Material stated in the SMPR.

YES: There are information demonstrating that the method performs within the SMPR Method Performance Requirements table specifications for all analytes in the SPMR applicability statement.

In my opinion there is no need.

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