DETERMINATION OF ENDECTOCIDES IN ANIMAL TISSUES, MILK AND EGGS BY LC WITH FLUORESCENCE DETECTION

Transcription

1 DETERMINATION OF ENDECTOCIDES IN ANIMAL TISSUES, MILK AND EGGS BY LC WITH FLUORESCENCE DETECTION CFIA Saskatoon Laboratory Centre for Veterinary Drug Residues References: Salisbury, C.D.C., J. AOAC Int., (1993) 76: Danaher et al, Analyst (2001) 126 (5) Scope: This method permits determination of residues of the major component of abamectin, doramectin, ivermectin, and moxidectin in muscle and liver tissue from cattle, equine, swine and sheep as well as milk and eggs. The method also allows for the determination of the residue eprinomectin in liver tissues from cattle, equine and swine as well as milk and eggs. 2. Principle: If applicable, the matrix is homogenized with acetonitrile. The sample is then centrifuged and the supernatant is passed through a neutral alumina column. The eluent is diluted with water, triethylamine is added and the mixture passed through a conditioned C18 SPE column. The analytes are eluted from the column with acetonitrile, which is evaporated to dryness. The dried residue is derivatized with 1- methylimidazole, trifluoroacetic anhydride and acetic acid at 65ºC. The analytes are determined by liquid chromatography using a XDB-C18 column and fluorescence detection. Suspect, nonderivatized samples are sent for LC-MS/MS confirmation. Approved By: B. Shurmer, CVDR Head (2017/04) Page 1 of 18

2 3. Apparatus: 3.1 Notes: Suppliers are listed for reference only. Other brands of equal performance may be substituted All volumetric glassware used throughout this method is Class A. 3.2 Balance, electronic, 0.01 g and g sensitivity (Mettler, VWR). 3.3 Centrifuge, capable of 3000 x g and refrigeration, with 15 ml and 50 ml tube carriers (Sorvall, Fisher Scientific). 3.4 Centrifuge tubes, polypropylene, 15 ml disposable (Falcon brand, VWR). 3.5 Centrifuge tubes, polypropylene, 50 ml disposable (Falcon brand, VWR). 3.6 Filter vials, 13 mm, 0.2 µm PTFE syringeless (# , Whatman, Fisher Scientific). 3.7 Homogenizer - Polytron PT 3100 with PTA 10TS generator (Brinkman Instruments, Fisher Scientific). 3.8 Liquid Chromatograph column, Zorbax Eclipse XDB-C18, 4.6 x 30 mm, 1.8 μm ( , Agilent, Mississauga, ON). 3.9 Liquid Chromatograph system, Waters 2695 HPLC with a Waters 2475 fluorescence detector (Waters Ltd., Mississauga, ON) Liquid dispensers, adjustable 1-10 ml and 5-25 ml (Brinkman Instruments) Nitrogen evaporator with heating bath, N-Evap (Organomation Assoc. Inc., Berlin, MA) Pipettors, adjustable, µl and µl plus tips (Eppendorf or equivalent) Shaker, two speed, flatbed (Eberbach, 6010, VWR) Solid Phase Extraction column, alumina, 4.5 g, 8 ml. Using a 35 mm long neck polypropylene funnel, pour ~ 4.5 g of neutral alumina (Brockman Activity III, see section 4.4) into an 8 ml SPE reservoir. Attach a 75 ml SPE reservoir and install on the vacuum manifold. Note: the exact weight of the alumina is not critical. The height of 4.5 g added to one reservoir can be marked and then used to estimate the amount needed for the remaining reservoirs Solid Phase Extraction column, C18, 500 mg, 6 ml (# , Bond Elut, Agilent) Solid Phase Extraction reservoir, 75 ml with adapters (#9434, 9430, VWR) Solid Phase Extraction reservoir, 8 ml with frit (#C612102, Chromatographic Specialties) Solid Phase Extraction vacuum manifold, (#57030-U (12 port), U (24 port) Supelco, Oakville, ON) Transfer pipets, 3mL disposable polyethylene (Falcon Brand, VWR) Vortex Mixer, multi tube, variable rate, pulsed (DVW-2500 Multi-tube, VWR). Approved By: B. Shurmer, CVDR Head (2017/04) Page 2 of 18

3 3.21 Water Bath, covered with amber lid, Isotemp (Fisher Scientific). 4. Reagents: 4.1 Notes: Manufacturers listed for reference only. Other brands of equal purity may be substituted All water used throughout the method was purified by reverse osmosis followed by deionization, adsorption, and filtration Preparation instructions are provided for guidance purposes only and, unless noted otherwise, the volume required can be adjusted to allow for more or less solution as required. Preparation details are to be recorded in the reagent preparation log. 4.2 Acetic acid, glacial, reagent grade (#B , EMD). 4.3 Acetonitrile, non UV, HPLC grade (# , Caledon). 4.4 Alumina, neutral, Brockman Activity III: Combine neutral alumina (#A , Fisher Scientific) and water in the ratio of 94/6 (w/w) into an amber storage bottle. Cap and shake at high speed for 45 minutes or until free-flowing. Expires: 1 day (use immediately). 4.5 Formic acid, 98% (#FX0440-5, EMD) % formic acid (aq) : Place approximately 80 ml of polished water into a 100 ml volumetric flask. Add 100 μl of formic acid. Make to volume with water :70 formic acid (aq) (0.1%) : acetonitrile: Combine 30 ml of 0.1% formic acid (aq) with 70 ml of acetonitrile. 4.6 Methanol, HPLC grade (# , Caledon) Methylimidazole, 99% purity, purified by redistillation (#336092, Sigma-Aldrich) methylimidazole/acetonitrile (2+7, v/v): Combine 2 ml of 1-methylimidazole with 7 ml acetonitrile and mix. Prepare daily as needed. 4.8 Nitrogen, compressed, minimum purity %. 4.9 Triethylamine, 99% purity (#T-0886, Sigma-Aldrich) Triethylamine (0.1%) in 70:30 Water : Acetonitrile: Combine 105 ml water with 45 ml of acetonitrile. Add 150 μl of triethylamine and swirl to mix. Prepare just before use Trifluoroacetic anhydride, 99% purity (#T-8258, Sigma-Aldrich). 5. Standard Solutions: Trifluoroacetic anhydride/acetonitrile (2+7, v/v): Combine 2 ml trifluoroacetic anhydride with 7 ml acetonitrile and mix. Prepare daily as needed. Approved By: B. Shurmer, CVDR Head (2017/04) Page 3 of 18

4 5.1 Notes: Caution: Endectocides are physiologically active - they are weak teratogens. Avoid direct contact or inhalation of these drug standards. Wear disposable nitrile gloves, lab coat and protective eyewear In addition to following the safety procedures outlined in the Agency s Laboratory Safety Manual and the Saskatoon Laboratory s Safety Procedures (Safe Work Practices and Handling, Storage and Disposal of Chemicals and Hazardous Waste), the analyst must review the CVDR Job Hazard Analysis (JHA) for preparation of standards and relevant Material Safety Data sheets (MSDS s) Supplier information is provided for guidance purposes only. For current supplier details, contact the program supervisor Abamectin, doramectin, ivermectin and moxidectin were obtained from Sigma- Aldrich Canada Ltd Eprinomectin was obtained as a 100 ng/µl (100 µg/ml) solution from Sigma- Aldrich Canada Ltd Standard preparation instructions are provided for guidance purposes only and, unless noted otherwise, the volume required can be adjusted to allow for more or less solution as required. Preparation details are to be recorded in the standards preparation log To determine the weight of the standard required, the analyst must know the chemical form (hydrochloride, sodium salt, etc.) and assayed purity of the analytical standard material, taking both into account when determining the actual amount to weigh for a given concentration. Example: Corrected = Target Mass x 100 x Molecular Weight (chemical form, salt) Mass Purity, % age Molecular Weight (free base) The actual stock standard concentration may vary slightly from the target concentration. In that event, the amount required to prepare a given concentration of a working standard solution will need to be adjusted accordingly to ensure that the working solution concentration is maintained at the target value. Approved By: B. Shurmer, CVDR Head (2017/04) Page 4 of 18

5 5.2 Abamectin, Doramectin, Moxidectin and Ivermectin Stock Solutions (100 g/ml): Weigh g of abamectin, doramectin, moxidectin and ivermectin into separate 100 ml volumetric flasks, dissolve and bring to volume with acetonitrile. Mix by inversion before transferring each into separate 50 ml polypropylene tubes, 1 tube per stock solution. Discard the excess solutions. Prepare every twelve months and store in a temperature monitored freezer set at -20ºC. 5.3 Working Solution Preparation Example: **Note: The actual volume transferred must be corrected according to the actual stock solution concentration. Example: 500 ng/ml Moxidectin from a 103 µg/ml stock solution. Actual vol. = 500 L x 100 µg/ml / 103 µg/ml = 485 L 5.4 Mixed Working Solution (500 ng/ml): Transfer 500 L of the eprinomectin, abamectin, doramectin, moxidectin and ivermectin stock solutions into a 100 ml volumetric flask and bring to volume with acetonitrile. Mix by inversion before transferring into two 50 ml polypropylene tubes. Store one unopened for validation purposes. Prepare every twelve months and store in a temperature monitored freezer set at -20ºC. 5.5 LC Standards (0, 2, 10, 20 and 30 ng/g tissue equivalents (TE)): Pipette 0, 20, 100, 200 and 300 µl of the 500 ng/ml mixed working standard solution into 15 ml polypropylene centrifuge tubes. Cap and set aside in the dark until step Fortified Recovery Sample (5 ng/g TE): Fortify a blank sample with 50 µl of the 500 ng/ml mixed working standard solution, Step Negative Control Sample: Do not fortify one of the blank tissue samples. This sample will serve as the negative control for the run. 5.8 Positive Control Sample (8 ng/g TE): Fortify a blank tissue sample from a different source than that used for the recovery and the negative control samples with 80 µl of the 500 ng/ml mixed working standard solution, Step LOD Sample (2 ng/g TE): Fortify a blank tissue sample from the same source that was used for the positive control sample with 20 µl of the 500 ng/ml mixed working standard solution, Step Extraction and Clean-up Procedure: 6.1 Note: In addition to following the safety procedures outlined in the Agency s Laboratory Safety Manual and the Saskatoon Laboratory s Safe Work Practices and Handling, Storage and Disposal of Chemicals and Hazardous Waste, the analyst must review the relevant Job Hazard Analyses (JHA s) and Material Safety Data Sheets (MSDS s). Endectocides are weak teratogens. Wear protective gloves at all times. 6.2 Weigh 5.0 g ± 0.05 g test samples into separate 50 ml Falcon tubes. Also weigh two blank Approved By: B. Shurmer, CVDR Head (2017/04) Page 5 of 18

6 samples from one source and two blank samples from a second source into separate 50 ml Falcon tubes. Fortify the first two blank samples at 8 and 2 ng/g TE (steps 5.8 and 5.9, respectively). Fortify one of the other blank samples at 5 ng/g TE (step 5.6). Process the remaining blank sample as a negative control. 6.3 Prepare LC standards as described in Step For liver samples, add 15 ml acetonitrile to each tube and homogenize with a Polytron until just blended. Rinse the probe into the Falcon tube with approximately 5 ml acetonitrile. Shake tubes at high speed for 10 minutes on a flatbed shaker. Centrifuge at 2500 x g for 5 minutes. For egg and milk samples, add 15 ml acetonitrile to each tube and shake on low speed for 10 minutes on a flatbed shaker. Centrifuge at 2500 x g for 5 minutes. 6.5 Add 5 ml of acetonitrile to the reservoir of alumina (prepared as in 3.14). Allow the solvent to draw through the bed by gravity feed to waste. Vacuum may be applied at the end if necessary. Critical Control Point: Acetonitrile rinse necessary to properly prepare bed and remove interferences. 6.6 Place 50 ml Falcon tubes in the vacuum chamber to collect the extract. Decant the supernatant from 6.4 into the reservoir of the rinsed alumina column, allowing the extract to pass through the alumina into the 50 ml Falcon tube. 6.7 Add 15 ml acetonitrile to the sample plug, re-cap the tube and re-suspend the plug with a vortex mixer. For liver and egg samples, the plug may need to be broken up with a spatula first. For milk samples, only the vortex is needed. For liver samples, shake on high speed for 10 minutes on a flatbed shaker and centrifuge at 2500 x g for 5 minutes. For milk and egg samples, shake on low speed for 10 minutes on a flatbed shaker and centrifuge at 2500 x g for 5 minutes. 6.8 Add the supernatant to the reservoir and allow to pass through the alumina by gravity (vacuum can be applied if needed). Use vacuum to collect the remaining solvent extract. Collect the eluent in the same 50 ml Falcon tube used in step Prepare a set of 150 ml Erlenmeyer flasks by first adding 70 ml of water to each flask. Add 100 μl triethylamine to the water and gently swirl to mix. For each sample, transfer the acetonitrile eluent from step 6.8 to the prepared flask and swirl to mix Remove the reservoirs from the alumina cartridges and rinse them with RO water. Attach these reservoirs to the C18 SPE columns to be used in the next step Condition a C18 SPE column with 5 ml acetonitrile followed by 5 ml of the triethylamine (0.1%) in 70:30 water:acetonitrile solution. Immediately follow with the sample extract, using vacuum assist as necessary. Allow the sample to pass through the SPE column at a flow rate of approximately 4-5 ml/min. Approved By: B. Shurmer, CVDR Head (2017/04) Page 6 of 18

7 6.12 When the extract has passed through the C18 column (to waste), remove the reservoir and adaptor and continue drawing air through the column for at least 10 minutes. This ensures complete removal of all water which is necessary for complete derivitization. Place a labelled 15 ml polypropylene centrifuge tube in the collection area of the vacuum block. Elute the analytes from the C18 column with 5 ml of acetonitrile under gravity. Once eluted, apply vacuum to collect the remaining solvent Evaporate the standards and samples to dryness on an N-evap at 65ºC. Note: Some oily residue may remain. This can trap moisture but will not affect derivatization. Trapped moisture can be removed by directing the nitrogen stream to swirl the residue at the bottom of the tube. Critical Control Point: water interferes with the derivitization reaction, ensure complete water removal Add in order: 225 μl of a freshly prepared solution of 1-methylimidazole/acetonitrile (2 + 7, v/v, step 4.7.1), 225 μl of a freshly prepared solution of trifluoroacetic anhydride/acetonitrile (2 + 7, v/v, step ), to each tube. Critical Control Point: The derivatized analytes have been reported to be light sensitive, avoid prolonged exposure to direct light during derivatization Cap and pulse vortex for 2 minutes at 1200 rpm (using multi-tube vortexer), incubate at room temperature (in the dark) for 10 minutes Add 50 µl glacial acetic acid, cap and pulse vortex for 2 minutes at 1200 rpm Place tubes in rack and put in water bath (with amber lid) set at 65ºC for 30 minutes. Remove from heat and let samples sit at 5ºC or less for 3-4 minutes before proceeding. Caution: Filtration of hot (65ºC) derivatizing reagents is dangerous. Ensure samples are cooled to at least room temperature prior to filtration For each sample, filter approximately 350 µl using PTFE syringeless filter vials. 7. LC Determination: 7.1 HPLC parameters: Injection Volume: Column: Column temperature: Mobile Phase Flow rate: Run time: 10 μl Zorbax Eclipse XDB-C18, 4.6 x 30 mm, 1.8 μm 50 º C A - Acetonitrile B - Water Variable, 1.0 ml/min to 2.2 ml/min 22 minutes Gradient: Time (min) Flow %A %B Curve Initial Fluorescence Detection parameters Approved By: B. Shurmer, CVDR Head (2017/04) Page 7 of 18

8 Excitation wavelength: 352 nm Emission wavelength: 457 nm 7.3 System Suitability: Prior to the injection of the sample set, inject 3 spiked samples or one spiked sample in triplicate, prepared either fresh or from a previous run. If acceptable (see 7.4 for suitability criteria), proceed with the sample set. 7.4 Suitability criteria for acceptability of chromatograms: Analytes are to be detected with retention parameters comparable to the presented typical chromatograms (see Appendices). Minor tailing of the peaks is acceptable, but excessive tailing and/or ghost peaks is an indication of a contaminated column. If for any reason the system suitability criteria are not met and cannot be readily resolved, notify the responsible program supervisor of the issue. Document actions taken in the instrument log. 8. Calculations: 8.1 Calibration curve generation and quantitation calculations are done using Empower software. A linear curve function is used to produce the calibration curve for each analyte. 8.2 The calculation for the concentration of endectocide in the samples includes a correction for sample weight and for recovery as follows: ENDT (ng/g) = (Sample peak area) - (y-intercept) x 5.00 g x 100 Slope of calibration curve Sample Wt (g) % Recovery 9. Confirmatory Analysis for Endectocides by LC-MS/MS Note: The confirmatory analysis procedure described here can be conducted on any triple quadrupole mass spectrometer that is capable of matching or exceeding the sensitivity requirements of the Waters TQD LC-MS/MS system. Instrument conditions are provided for reference purposes only. Contact the program supervisor for current run condition details. 9.1 Suspect samples at or above the detection (as noted in 10.2) are to be confirmed by LC-MS/MS. Suspect samples requiring confirmation must be re-extracted following the deviations listed below. See below also for samples to submit for confirmation: Note: These samples are not derivatized and are reconstituted in the mobile phase used for the MS analysis At step 6.13, evaporate the samples just to dryness. A small amount of water can remain in the oily residue it will not affect the MS analysis. Over drying can result in analyte loss Add 1000 µl of 30:70 0.1% formic acid (aq): acetonitrile to each sample Vortex the tubes for 1 minute at 1200 rpm using a multi-tube vortexer Sonicate the tubes for 5 minutes. Approved By: B. Shurmer, CVDR Head (2017/04) Page 8 of 18

9 9.1.5 Filter approximately 350 µl using PTFE syringeless filter vials. 9.2 Sample Submission A set of standard solutions Fortified tissue sample of the same tissue type as the suspect positive, fortified at the level closest to the level of the suspect positive Blank tissue sample of the same tissue type as the suspect positive LOD fortified sample of the same tissue type as the suspect positive Suspect positive sample(s). 9.3 To confirm any of the analytes detected and quantified in Section 8, set up the triple quadrupole mass spectrometer in the Multiple Reaction Monitoring mode using the following instrument parameters: 9.4 Waters Acquity UPLC operation conditions UPLC column: Acquity UPLC HSS PFP, 1.8 μm, 2.1 x 30 mm Column temperature: 45 C Flow rate: ml/min Injection volume: 2.00 µl Run time: 5.20 minutes Mobile phase: A - Methanol B % Formic Acid Gradient conditions: Time (min) A% B% Curve Waters TQD MS/MS Operation Conditions Ionization mode: ESI positive Desolvation temperature: 500ºC Collision gas: 0.18 ml/min Capillary (kv): 1.00 RF Lens (V): 0.30 Desolvation Temperature: 500 ºC Cone Gas Flow: 50 L/hr Approved By: B. Shurmer, CVDR Head (2017/04) Page 9 of 18

10 Desolvation Gas Flow: 1100 L/hr LM1 Resolution: HM1 Resolution: Ion Energy 1: 0.17 LM2 Resolution: HM2 Resolution: Ion Energy 2: Multiple reaction monitoring (1) (2) : Compound Time Window Monitored Reactions Dwell Time Cone Voltage Collision Energy (min) Precursor > Product Ion (sec) (V) (ev) Eprinomectin > > Abamectin (3) > > > Doramectin (3) > > Moxidectin > > Ivermectin (3) > > (1) Nominal values only. Actual values will be determined for each MS/MS system used. (2) Bolded transitions used for quantitation. (3) [Mona] + adduct monitored. 10. Test Reporting: 10.1 Results are reviewed by program supervisor before reporting or re-analysis of samples Method Characteristics (1) Analytical Range 2-30 ng/g Detection limit: All Analytes, All Species, All matrices 2 ng/g Reporting Limit: All Analytes, All Species, All matrices 2 ng/g Tolerances: See Health Canada s website for the most current information regarding Maximum Residue limits (MRL s), and/or banned substances. For banned substances, any level detected and confirmed will be reported. (1) The Measurement Uncertainty (MU) should be recalculated whenever a change that affects method accuracy, precision or sensitivity occurs. This information is to be prepared by the responsible program chemist. 11. Quality Assurance Plan: 11.1 Performance Standards (see Quality Manual for definitions) Acceptable Repeatability CV 15% Approved By: B. Shurmer, CVDR Head (2017/04) Page 10 of 18

11 Acceptable Reproducibility CV 20% Acceptable Recovery Acceptable correlation coefficient 90 ± 30% (ivermectin, moxidectin, abamectin, doramectin; all matrices) 100 ± 35% (eprinomectin; all matrices) r 0.995, all analytes No false positives. No false negatives if present 2 ng/g No systematic bias and no individual bias 20% 11.2 Critical Control Points Procedure Item Acceptable control 6.5 acetonitrile rinse solvent rinse is necessary to remove contaminants present in the Alumina that may co-elute with Eprinomectin and also to prepare the SPE bed for proper removal of matrix interferences 6.13 evaporation of acetonitrile dryness must be attained as moisture will interfere with the derivatization reaction 6.14 light sensitive the derivatized analytes are light sensitive and prolonged exposure to direct light during derivatization should be avoided Readiness to Perform (Training Plan): Notes: (1) As part of the analyst qualification process, an observation run is to be completed, whereby the analyst has the opportunity to work with an experienced analyst who is knowledged in the application of the procedure. (2) Since calibration standards are chemical standard samples, Phase I and Phase II can be combined Phase I/II: Phase I provides the analyst with the opportunity to demonstrate competency on instrument setup and evaluation of instrument system suitability data. Phase II provides the analyst with the opportunity to demonstrate competency on the analytical procedure, evaluation of results and reporting. Phase I/II is to include: Two liver runs each set up on different days. Each run is to include the system suitability injections, the standard calibration curve, a negative control, recovery spike, and four analyst spikes, prepared in duplicate. *One set of duplicates is to be at the limit of detection in order to assess analyst proficiency at the reporting limit for non-approved analytes At the completion of Phase I/II, submit the following information for review and Approved By: B. Shurmer, CVDR Head (2017/04) Page 11 of 18

12 approval to proceed to Phase III: For each run, the worksheet/report (reviewed and approved by the program supervisor) which is to include the system suitability evaluation (analyst comments) as they relate to acceptance of instrument output, the run sequence, regression analysis(es), all chromatograms as well as a summary of the analyst spike recoveries (and/or accuracy results) and precision data All runs must be accounted for (including those which did not meet the test method acceptability criteria) Phase III provides for an evaluation of the analyst s ability to obtain and produce an analytical result which is unknown to them and is to include: Two liver runs set up on separate days, each run to include the system suitability injections, the calibration curve, the recovery sample, the LOD spike, the positive and negative quality control samples, and a minimum of 6 check samples (whose levels are to be blind to the analyst), set up by the program supervisor or designate. To extend qualification to include muscle, milk and egg matrices, a minimum of one additional phase III run in each matrix is required At the completion of Phase III, submit the following information for review: For each run, the worksheet/report (reviewed and approved by the supervisor), which is to include the system suitability evaluation (analyst comments) as they relate to acceptance of instrument output, the run sequence, regression analysis(es), all chromatograms, as well as a quantitative presentation of the check sample results All runs must be accounted for (including those which did not meet the test method acceptability criteria) Submissions for review of the analyst qualification include: The completed analyst qualification flow chart (CVDR-A-0046). For the phase I and II data, summaries of the analyst spike results (including an assessment of the recovery and precision data). For the phase III data, a quantitative presentation of the check sample results. All runs must be accounted for. The summaries are to be traceable to the run identifiers and traceable to the analytical instrument used at analysis Acceptability criteria. See 11.1, Performance Standards Upon successful completion of the requirements for qualification, the Section Head will authorize the analyst to perform the specific test method via a Readiness to Perform letter. Records of completion are to be maintained by the respective program supervisor. See QM-S Intralaboratory Check Samples Approved By: B. Shurmer, CVDR Head (2017/04) Page 12 of 18

13 Minimum System Requirements: Repeat samples - one sample analysed in duplicate (within the same run), for every 20 samples analysed. Positive Control Samples - at least one positive control is included in every run. The level is as defined within the text of the method. Limit of Detection (LOD) Samples - at least one LOD sample is included in every run. The level is defined within the text of the method. Negative Control Samples - at least one negative control in included in every run Records are to be updated and reviewed for trends by the analyst with every run. Those records include: All repeat sample results. Positive Quality control results are recorded in a table and when sufficient data is available to generate control limits, the positive QC results are plotted into Individual, X and moving Range, R Statistical Process Control charts. Qualification and validation data may be used to supplement charts Acceptability criteria. See Section 11.1, Performance Standards. Positive Control and Recovery Sample results, when plotted into an SPC chart, are to be reviewed. See CVDR-S-0004, Test Method Quality Control Samples, for details. When criteria are not met and/or trends observed, consult with the responsible supervisor. Investigate and identify probable cause, documenting this information with the run, noting actions taken on the necessary control charts/table Interlaboratory Check Sample: When available, check samples are obtained from the Proficiency Testing Unit, CVDR Uniform Analytical Standards: When each new stock standard solution is prepared (Section 5) it is validated for use as per CVDR-S-0014 and is to include a review of the individual preparation for possible cross contamination as well as a review of the concentration of the prepared stocks (prepared at the working standard concentration) relative to the current/old mixed working standard solution For old and new stock preparations, pipette 10 µl of each individual stock solution into separate tubes and dilute with approximately 2 ml of acetonitrile. 300 µl of each of these dilutions are transferred to separate polypropylene tubes and steps are completed See CVDR-S-0014 for repeatability criteria of responses Working standards are validated against the previous working standard by comparing Approved By: B. Shurmer, CVDR Head (2017/04) Page 13 of 18

14 instrumental response Pipette 200 μl of both old and new mixed working solutions (step 5.4) into separate polypropylene tubes. Steps are then completed See CVDR-S-0014 for repeatability criteria of responses Records of the preparation details, the supporting chromatograms from the validation run(s), the calculation results (% difference of the retention times, the repeatability data for the response comparisons) and conclusions with regards to acceptance of the new standard preparations are kept in the Standards log book Sample Acceptability and Stability Matrix of choice: Condition upon receipt: Sample Storage: liver, muscle, milk or egg cold, not spoiled 2 months frozen at -20ºC 12. Revision Status Previous Version Previous Version Revision Date Paragraph revised, deleted, added Reason for update (Original Author): Revision Author if different CVDR-M /11 Header/Footer Updated version and date K.Souster Title Updated to include milk and eggs 1 Updated to include milk and eggs 2 Removed disclaimer statement 3.12 Deleted Pasteur pipettes; renumbered 3.19 Added transfer pipets 4 Added catalogue numbers for some reagents; renumbered Added preparation instructions for 0.1% formic acid (aq) Added preparation instructions for 30:70 0.1% formic acid (aq) : Acetonitrile 4.7 Added Methanol to reagents list Added instructions for preparing 1- methylimidazole:acn solution Added instructions for preparing triethylamine solution Added instructions for preparing trifluoroacetic anhydride:acn solution Updated contact 5.2 Combined all analytes into one preparation with an expiry date of 1 year 5.4 Updated to correct typo (500 Approved By: B. Shurmer, CVDR Head (2017/04) Page 14 of 18

15 Previous Version Previous Version Revision Date Paragraph revised, deleted, added Reason for update ml changed to 500 µl) 5.4 Updated to reflect change in practice of expiration date of standards (changed from 3 months to 12 months) Renumbered (Original Author): Revision Author if different 6.2 Removed reference to liver Updated to reflect change in sample vials and filters 6.4 Updated to include instructions for milk and egg 6.5 Updated step reference 6.7 Updated to include instructions for milk and egg 6.10 Added step for washing reservoirs with RO water 9 Updated to reflect change from Micro to TQD MS-MS system 9.4 Updated operation conditions to reflect change from Waters Alliance system to Waters Acquity system 9.5 Updated to reflect change in operation conditions from Waters Micro to Waters TQD MS-MS system 9.6 Updated MRM 10.2 Changed tissues to matrices 11.2 Update step references; removed references to step Updated to better reflect familiarization procedure and to include instructions for milk and eggs 11.7 Added milk and egg to matrix of choice 12 Updated revision table Appendix I Chromatograms Created to reflect codes used in Sample Manager Updated to include chromatograms for both milk and eggs Appendix I: Codes for Use in Sample Manager Test Analysis Code ENDT-SA Analyte Component Code Eprinomectin EPR Moxidectin MOX Abamectin ABA Doramectin DORA Approved By: B. Shurmer, CVDR Head (2017/04) Page 15 of 18

16 Ivermectin IVR Typical Endectocides Liver Chromatograms Approved By: B. Shurmer, CVDR Head (2017/04) Page 16 of 18

17 Typical Endectocides Egg Chromatograms Typical Endectocides Milk Chromatograms Approved By: B. Shurmer, CVDR Head (2017/04) Page 17 of 18

18 Approved By: B. Shurmer, CVDR Head (2017/04) Page 18 of 18