Determination of Emamectin Benzoate and Other Avermectin Residues In

DeterminationDetermination ofof EmamectinEmamectin BenzoateBenzoate andand otherother AvermectinAvermectin ResiduesResidues inin FishFish TissuesTissues UsingUsing LC/MSLC/MS

Dayue Shang, Angelo Di Cicco, Nicole Gibbons, Monica Dyck and Helen Nicolidakis

Health Canada, HPFB, Western Region Laboratory, Burnaby, B.C. V5G 4P2 AvermectinsAvermectins z What are they?

z A group of compounds used as acaricides or parasiticides for animals or plants.

z In aquaculture – emamectin and ivermectin are used to control sea lice in farmed salmon and trout. TheThe SeaSea LiceLice IssueIssue z Sea lice produce larvae which attach to salmon and feed on blood and mucous, causing serious damage and death to fish. z Farmed fish in unnatural and crowded conditions may lead to more sea lice, which could have an impact on wild salmon. z Ivermectin has been approved by VDD to treat sea lice infestation. Emamectin is still being evaluated by VDD, but can be used under the Emergency Drug Release program on a case by case basis. z Human health and environmental impacts are being investigated. z CFIA conducts routine tests of about 100 samples per year. HealthHealth Canada’sCanada’s ConcernConcern z Health Canada’s concerns around avermectins:

z Accumulation of residues over time

z Long term effects, especially in people whose diet consists of considerable fish consumption

z Effects on most vulnerable individuals, e.g. children and people with compromised immune systems ChemicalChemical StructuresStructures

OCH3 OCH3 N HO H3C OCH3 OCH3 O O H3C O H3C O CH3 CH3 H H CH3 CH3 H H O O O O H3C O CH3 H3C O CH H O H 3 H H O H3C R H H3C R O O Emamectin O O Abamectin OH H B1a, R=C2H5 OH H B1b, R=CH3 B1a, R=C2H5 O CH3 B1b, R=CH3 H O OH CH3 H OH

OCH3

HO OCH3 O O CH3 CH3 H3C H H O O O CH3 H C 3 H O H H R H3C O O Ivermectin OH H

B1a, R=C2H5 O B1b, R=CH3 CH3 H OH ChemicalChemical StructuresStructures

H3CO N

CH3 CH3 H O CH3 OCH O 3 H HO H3C CH3 CH3 OCH 3 O O O H C O 3 CH3 CH3 OH H H H O O H3C O H O Moxidectin H O H3C CH3 H O O OH OH H Doramectin OCH3 H O H3C N CH3 OCH3 H OH O O O CH3 H3C H H O O O CH3 H3C O H H H R H3C Eprinomectin O O OH H B1a, R=C2H5 B1b, R=CH3 O CH3 H OH AvermectinsAvermectins z Some characteristics:

z Very hydrophobic

z Low toxicity

z Unknown cumulative effects SampleSample PreparationPreparation z Urgent need to develop a so-called “universal” sample preparation method for emergency situations. z More than two-thirds of the analysis time is spent on sample preparation. z Approximately 30% of the analytical error stems from the sample preparation step. z Optimizing the sample preparation procedure is crucial to the development of robust, reproducible, and accurate analytical methods. MythMyth No.No. 1:1: SPESPE cleanclean--upup andand concentrationconcentration isis alwaysalways neededneeded forfor aa goodgood LC/MS/MSLC/MS/MS methodmethod z Fact: an SPE cartridge normally has 3 to 5 plates while a HPLC column has a plate number of about 10,000. z Fact: it is often simply too much for a small SPE column to deal with the demand of a multi-residue method. z Solution: avoid SPE as much as possible when developing a multi-residue method. Instead, use a solvent removal unit to concentrate samples, followed by a good chromatographic separation. MythMyth No.No. 22:: OnlyOnly ASE,ASE, MSE,MSE, SFE,SFE, etc.etc. cancan efficientlyefficiently extractextract analytesanalytes fromfrom solidsolid matricesmatrices z Fact: many of the new apparatus require elaborate preparation steps, e.g. packing of 24 columns for ASE process. z Fact: tubing and re-used cartridges are potential sources of cross contamination. z Solution: design a method to test multiple solvents to find a good solvent system for the job. For trace analysis, disposable containers are recommended for sample preparation. AA reviewreview ofof samplesample preparationpreparation techniquestechniques z From several review papers, the techniques used for the detection of over 100 pharmaceutical compounds in biological and environment matrices (100% = all cases): z 98 % liquid-liquid (L-L) z 77 % protein precipitation z 45 % SPE z 26 % on-line and automated SPE z 8 % MSPE z 3 % ASE z 2 % Microwave z 1 % Other (MSPD, SFE, etc) z Your best bet: ------MythMyth No.No. 33:: LiquidLiquid--liquidliquid extractionextraction hashas limitedlimited useuse whenwhen dealingdealing withwith solidsolid matricesmatrices z Fact: solid matrices usually can be liquefied with the help of homogenizers, and liquid samples may be laden with solid particulates. z Fact: L-L has been used in the determination of trace organics in solid samples for many years, eg. PCBs and Dioxins. z Solution: design a method to test multiple solvents and buffers to find a good solvent system; miniaturize the L-L process using disposable centrifuge tubes. FactorsFactors toto considerconsider inin developingdeveloping aa LL--LL extractionextraction methodmethod z All these factors have to be considered together: z Distribution coefficient z Solvent miscibility z pH adjustment z Temperature z Fatty substance z Solvent toxicity z Agitation z Solvent removal AA SystematicSystematic ApproachApproach

Rapid Optimization Procedure for Extraction Method (Flowchart No. 2) (To be used in conjunction with Flowcharts no. 1 and 3) If matrix Add 10 ml of hexane, is oily rotate, freeze, and Polytron the samples at 1600 Divide the samples into 2 groups of 4. discard organic layer Spike 0.5 ml of 1 Add 20 ml of rpm for 30 sec. Add 0.5 ml of selected buffer to each before adding MTBE. Prepare Weigh out 5 g of ppm standard 0.01M EDTA Wash the polytron generator sample in Group 1, according to the START standards blank matrix into mixture and 0.5 (up to the 25 probe in a 400 ml beaker results outlined in flowchart no. 1. and 8 - 50 ml ml of 1 ppm IS ml mark) to containing a solution of 50% Similarily prepare group 2 with another solvents. centrifuge tubes. into each of the 8 each of the IPA and 1% formic acid. selected buffer. Usually one buffer for Add approx. 15 ml of samples. 16 samples. (Replace solution after every 5 acidic coompounds and the other for Ethyl acetate to the 40 samples). neutral and basic. If matrix ml mark. is not oily

Cap the sample. Add 100 ul of Sonicate at Take the samples out Decant the organic layer to Make volume to 2 ml Vortex for 30sec. until all the Centrifuge Put the samples into diethylene glycol as a Filter with 13 35o C for and put into a tray with clean and labelled 15 ml with 10 mM NH OAc/ particulates are suspended. for 6 min. the - 80o C freezer for keeper. 4 mm Acrodisc 20 min. ice on the bottom. Wait centrifuge tubes. Make up with 100 uM disodium Use a disposable spatula to at 5350 25 min. (until aqueous Evaporate off the GHP filters Vortex for for 5 min. until the to 12 ml. Place them in EDTA. Vortex for 10 disturb the solid bed if the rpm. layer is frozen. organic layer (~ 25 (small purple). 30 sec. organic layer is clear. the vacuum centrifuge. sec. particulates are not min.). suspended.

Add 1 ml of D.I. water to 0.5 ml IS Save the aqueous solution at Phase 3: Go to and 0.5 ml of STD mix to make a -20o C for future use. Flowchart No. 3 solvent standard.

Spike 2 series of Make volume to 2 Set up the sample and Process the If results are Extract with the samples of 4, each with ml with 10mM run the sequence for data and END of accepable, START of selected buffersand 0.2, 0.4, 0.6, and 0.8 ml NH4OH/MeOH with LC/MS analysis. Note: prepare continue to follow the previous Phase 1 Phase 2 of 1 ppm STD and 0.5 ml 100 umM of sodium injection volume 10 ul. charts. Phase 2. procedure. of internal std. EDTA.

TABLE 1 TABLE 2

1a 2a 3a 4a PREPARATION OF BUFFER SOLUTIONS Extraction 15 ml Ethyl 15 ml Ethyl 15 ml Ethyl 15 ml Ethyl Process data. 1M of Phosphate Buffer at Vortex, filter, Select the best END of Solvent acetate acetate acetate acetate 1M Citrate Buffer at pH 4.5 pH 7.4 and inject. extraction Phase 2 Buffer 0.5 ml 0.5 ml of 0.5 ml of 1 0.5 ml of solvent. Add 13.6 g of potassium Formic 1M citrate M triethylamine Add 19.2 g. of citric acid to 80 phosphate to 80 ml d.i. water. ml of d.i. water. Dropwise, Acid buffer pH phosphate Dropwise adjust pH to 7.4 with adjust pH to 4.5 with 1M 1M NaOH soution. Make to 4.5 pH 7.4 aqueous NaOH solution volume with water to 100 ml Proceed to Polytron 20 ml 20 ml 20 ml 20 ml 0.01M flowchart no.3 Solution 0.01M 0.01M 0.01M EDTA EDTA EDTA EDTA

BuffersBuffers RuleRule thethe LL--LL ExtractionExtraction

1a 2a 3a 4a Extraction 15 ml Ethyl 15 ml Ethyl 15 ml Ethyl 15 ml Ethyl Solvent acetate acetate acetate acetate Buffer 0.5 ml 0.5 ml of 0.5 ml of 1 0.5 ml of Formic Acid 1M citrate M triethylamine buffer pH phosphate 4.5 pH 7.4 Polytron 20 ml 0.01M 20 ml 0.01M 20 ml 20 ml 0.01M Solution EDTA EDTA 0.01M EDTA EDTA

SampleSample PreparationPreparation z Weigh 5 g of sample into 50 ml centrifuge tube z Add 30 ml of 50% acetonitrile with 10 mM EDTA, then add 0.5 mL of 1 M citrate buffer (pH 4.5) z Polytron 30 seconds z Sonicate sample for 20 minutes at 35 °C, vortex 30 seconds z Centrifuge for 4 minutes at 5000 rpm z Transfer supernatant to another 50 ml centrifuge tube. SampleSample PreparationPreparation (cont’d)(cont’d) z Reduce the extract to 10 mL with SpeedVac z Add 15 mL of ethyl acetate. Vortex 30 seconds z Freeze the sample at – 80°C for 25 minutes z Decant the upper layer to a 15 mL centrifuge tube z Reduce the extract to near dryness. Make up to 4 mL with 50% ACN, vortex 10 seconds z Filter 1.5 mL of sample with 0.2 micron GHP Acrodisc filter into an autosampler vial z Note: no SPE is used in the sample prep, which saves a lot of time. Up to 32 samples can be processed in one day by one chemist InternalInternal StandardStandard z Doramectin was used as an internal standard. z Doramectin is also a member of the avermectins family but is seldom used in applications, which makes it ideal as an internal standard. z Amount of doramectin spiked = 0.1 ppm

z Good signal response

z Good S/N z All samples, blanks, and standards were spiked with IS at the beginning of the analysis. AgilentAgilent 11001100 HPLCHPLC ParematersParematers

Mobile Phase A H2O + 0.1% formic acid Mobile Phase B ACN + 0.1% formic acid Flow Rate 0.25 mL/min Column Temperature 30oC LC Column Zorbax SB-C8, 2.1 x 100 mm, 3.5μm Guard Column Javelin Basic C8, 2.1 X 10 mm, 5μm Injection Volume 60 μL

Gradient Time 0 2.5 2.6 6.0 6.1 10 (min) %B (ACN+0.1% 5 909595 5 5 formic acid ) AvermectinAvermectin IonsIons

Compound M+1 Na adduct Emamectin 886.3 908.3 Eprinomectin 913.3 936.3 Doramectin (IS) 898.3 921.3 Ivermectin 874.3 897.3 Abamectin 872.3 895.3 Moxidectin 639.4 662.4 QuattroQuattro IIII MSMS ParametersParameters (SIR)(SIR)

Ion Mode ESI + Capillary 3.00 kV Cone Voltage 50–80 V * Ion Energy 2 eV Source Temperature 120oC Desolvation Temperature 400oC Cone Gas 100 L/hr Desolvation Gas 500 L/hr

* Compound specific BatchBatch AnalysisAnalysis forfor ValidationValidation andand forfor SampleSample AnalysisAnalysis z 6 matrix standards at various levels ranging from 10 ppb to 400 ppb z 3 QC spikes at 50 ppb z 3 sample blanks z 20 real world samples in each batch of samples run

By eliminating SPE, method robustness is improved, and it is possible to process 20 real world samples plus QA/QC samples in one batch. LowerLower LimitLimit ofof QuantitationQuantitation,, PrecisionPrecision,, RecoveryRecovery andand ReproducibilityReproducibility

z Lower limit of quantitation (LLOQ) for all five avermectins was 10 ppb.

z The recovery of the avermectins was between 60 –140 %

z The relative standard deviation of the pre- validation run was within 20%. ChromatographyChromatography ofof AvermectinsAvermectins

060412_EMA-24 SIR of 6 Channels ES+ 100 7.45 936.3 4.95e6

% Eprinomectin 0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 060412_EMA-24 SIR of 6 Channels ES+ 7.99 921.3 100 Doramectin (IS) 4.95e6 %

0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 060412_EMA-24 SIR of 6 Channels ES+ 8.63 897.3 100 8.50e6

% Ivermectin 0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 060412_EMA-24 SIR of 6 Channels ES+ 7.60 895.3 100 Abamectin 1.54e6 %

0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 060412_EMA-24 SIR of 6 Channels ES+ 6.74 886.3 100 Emamectin benzoate 4.27e7 %

0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 060412_EMA-24 SIR of 6 Channels ES+ 662.4 100 Moxidectin 1.03e6 %

0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Standard mix at 1 ppm SummarySummary z A multi-residue LC/MS method was developed for the quantitative determination of 5 avermectins in fish tissues. z Traditional liquid-liquid extraction technique compared favourably with some of the modern instrument based techniques. z A systematic approach for optimization of extraction procedure was used, resulting in a simple and rapid method and allowing up to 20 real world samples to be processed by one chemist on a daily basis. z Unlike most other similar methods, this method does not require solid phase extraction. In this way, a potential source of cross contamination is eliminated, sample turn out becomes much faster and more stringent quality control can be applied. z A systematic method development strategy was implemented, significantly shortening the time for optimization of LC/MS parameters. FutureFuture WorkWork z Further improve the LOD and LOQ of the method by using LC/MS/MS. z Use Lithium in the mobile phase to produce Li adducts; preliminary results show Li adducts are stable, and that significant stable fragments can be produced. z Extend the validation from salmon tissues to other animal tissues, vegetables, milk and fruits. z Participate in proficiency tests for analysis of endectocides in animal tissue, organized by the CFIA. z Discuss with stakeholders (CFIA, Kitasoo First Nation, and BCAFN) to decide on a sampling plan for a fish survey. AcknowledgementsAcknowledgements

We gratefully acknowledge the invaluable support and input of our colleagues at the Western Region Laboratory of Health Products and Food Branch, Health Canada. NextNext GenerationGeneration ofof ChemistsChemists

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