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EMR – Lipid:Enhanced MatrixRemoval for FattySamplesJoan Stevens, Ph.D.Senior Applications ScientistAgilent Technologies

Today’s AgendaIntroductionEMR-Lipid - Enhanced Matrix Removal Background, workflows, and resultsApplications Pesticides in avocado Veterinary drugs in beef liver PAHs in salmonSummary and Conclusions2

The challenges and goals of most sample analyses:Accurate, consistent dataKeep the instruments runningProcess as many samples for as littlecost as possibleRemove interferences3

Remove interferences To acquire desired sensitivity/selectivity To reduce contamination/carryover issues Use of sensitive and expensive instruments: Protect yourinvestment!!!Pesticides in Avocado without SPPesticides in Avocado with SP4

Lipids5

Matrices and Approximate Total Lipid Content 2%4-12% 12%MedLowSpinach (0%)Soy Milk (4%)Strawberry (0%)Beef Liver (4%)Onion (0%)Pork Liver (4%)Corn (4%)Paprika (1%)Cumin (2%)Rice (2%)Hops (2%)Tilapia (2%)Sea Bass (3%)Wheat (3%)HighNut Butters (16%)Avocado (21%)Pork (8%)Salmon (27%)Trout (8%)Chocolate (8%)Canned Pet Food ( 10%)Cow’s Milk (5%)Soy Oil (100%)Avocado Oil (100%)Canola Oil (100%)Catfish (12%)Carp (12%) Coconut Oil (100%)Plasma or whole blood(10 – 20 %)6

Current Procedures for Lipid RemovalMethodHow Lipids are removedWeaknessDilute and ShootNo lipid removal, only dilutionNo lipid removalProtein PrecipitationPPT followed by centrifugationInsufficient lipid removalPPT followed by filtration withor without sorbentInsufficient lipid removal; low analyterecoveryPSA/C18 sorbent (dSPE)Not selective; insufficient lipidremoval; analyte lossZr-containing sorbentLow total lipid capacity; analyterecoveryFreeze sampleTime needed; loss of analyteSPE/SLELoad and eluteTime needed; solvent usage;extensive method developmentSEC/GPCChromatographic separationUses copious amounts of solventand time; capital expenseQuEChERS7

EMR-LipidEnhanced Matrix RemovalEMR: As easy to use as QuEChERS; as clean as SPE8

EMR Product offeringExtraction TubeEMR-Polish (p/n 5982-0101)EMR-Lipid (p/n 5982-1010)EMR fits into current sample preparation workflows9

EMR Sorbent - What is it?When “activated” by water -The materials selectivehydrophobic interactions increase.-Suspension of nano particles (highsurface area).-Rapidly interacts with straightchain, “lipid-like” functional groups.1.0 g EMR in 15 mL tubeCentrifugation preferably used toseparate precipitate from solution (notfiltration).EMR-Lipid Mechanism – Size exclusion and hydrophobic interaction.10

and what does it do?EMR sorbent removes LipidsWhat are Lipids?A class of naturally occurring hydrocarbon containing compoundscommonly known as fats and oilsFree Fatty AcidsTriglyceridesCholesterolPhospholipids11

What Does EMR NOT Interact With?EMR does NOT remove analytes of interestExceptions?Compounds containing long aliphatic functional groups (e.g.prostaglandins)FluoroquinolonesOrganochlorine PesticidesTetracyclinesPAHsImidazole pesticidesVitamin D12

EMR Protocols for Applications13

EMR Fits into Existing WorkflowsQuEChERS (Quick, Easy, Cheap, Effective, Rugged,Safe) Easy-to-use sample preparation for food testing, solidsamples (e.g. vegetables, fruits, meat, seafood, etc.)EMR Applications: - Pesticide Residues in Avocado,Modified Liquid Extraction (Protein Precipitation) Proteins are removed by a “crash” step prior to injectionor cleanup (e.g. milk, meat, seafood, etc.)EMR Applications: - PAHs in Salmon,- Veterinary Drugs in Bovine Liver14

Improving dSPE in QuEChERS1. ExtractionPros Fast and inexpensive Takes minimal experience Doesn’t require special equipment Accommodates multiple matrices Accommodates large analyte groupsCons Large amount of coextractives2. Dispersive SPEPros Same as extractionCons Minimal cleanup provided Can remove analytes Lipids are challenging to removeselectively15

Traditional QuEChERS versus QuEChERS EMR-LipidStandard QuEChERSEMR QuEChERSExtraction/Partition (6 steps)Extraction/Partition (6 steps) Mix and CentrifugeMix and Centrifuge Transfer Sample to dSPE(sorbents) Transfer Sample to dSPE(sorbents & H20) Mix and CentrifugeMix and Centrifuge Evaporate and reconstitute or dilutePolish Tube Filter out precipitateEvaporate and reconstitute or diluteTransfer to A/S VialTransfer to A/S Vial16

Improving Liquid Extraction Workflows1. Add crash solvent.(ACN)2. Add food sample.SaltProteinLipidAnalyte3. Mix to precipitate proteins.Centrifuge4a. EMR dSPE and Polish.4b. SPE and/or GPC.Transfer for analysis17

Traditional versus EMR-Lipid Liquid ExtractionLiquid Extraction with SPE/GPCEMR Liquid ExtractionAdd Sample and AcetonitrileAdd Sample and Acetonitrile Mix and CentrifugeMix and Centrifuge Sample PretreatmentTransfer Sample and H2O to EMR dSPE SPE and/or GPCMix and Centrifuge Evaporate and reconstitute or dilutePolish Tube Filter out precipitateEvaporate and reconstitute or diluteTransfer to A/S VialTransfer to A/S Vial18

EMR – Lipid – dSPE CleanupAA.B.C.D.E.BCDQuEChERS or Liquid ExtractAdd H2O to EMR tube (“activation”)Transfer extractVortex and centrifugeSupernatant (1:1; extract: H2O)E19

EMR – Polish – ACN/H2O Phase SeparationAA.B.C.D.E.BAdd supernatant to EMR – Polish tubeVortex immediatelyPhase separation after centrifugeTransfer upper layer for analysisFinal samples split for GC and LC analysisCDE20

Tips for Success: EMR-Lipid ProtocolsExtraction Solvent *Acetonitrile – preferred for broad extraction and water miscibility. Acetone – also amenable to EMR workflow. Do not use - Water immiscible solvents (e.g. hexane, ethyl acetate, DCM) – nointeraction without initial miscibility.-Alcohols – can dissolve salts, EMR, and give poor partitioning.Agilent Confidential10/20/201521

Tips for Success: EMR-Lipid ProtocolsdSPE “Activation” Addition of water for dSPE- Increases hydrophobic interaction between EMR-Lipid and matrixcomponents. “Activation Strength”- 1 to 1; extract/water (recommended) – Excellent matrix removaland analyte recovery.-1 to 1; extract/water – marginal increase in matrix removal, lowerhydrophobic analyte recovery.-1 to 1; extract/water – decrease matrix removal, improvehydrophobic analyte recovery.Agilent Confidential10/20/201522

Tips for Success: EMR-Lipid ProtocolsPolishing Step (Partition) Contains 2.0 g NaCl/MgSO4 (1:4; same as QuEChERS) Applied AFTER EMR-Lipid dSPE for phase separationPurpose Remove unwanted water from “activation” Removes dissolved solids Improves recoveries for non-polar compoundsAdditional Information Use the Polish salts after EMR-Lipid Mix immediately – avoid clumping Customer may chose bulk saltsSeparate Product but HIGHLY RECOMMENDED for optimal resultsAgilent Confidential10/20/201523

Tips for Success with EMR-LipidRecommend Centrifugation NOT Filtration Filter membranes put EMR Samples at Risk for AnalyteRetention Analytes with properties that risk retention on hydrophobic membranes Less matrix leaves analytes “susceptible” for retention If filtration must be used, choose a hydrophilic membrane such asregenerated cellulose.Recommend Immediate Mixing Avoid Clumping; Immediately vortex extract/water in the EMR dSPEtube and EMR Polish tube.Agilent Confidential10/20/201524

GC-MS Fullscan AvocadoAvocado – Untreated25

GC-MS Fullscan AvocadoAvocado – UntreatedAvocado – EMR Treated26

GC-MS Fullscan Pet FoodPet Food – Untreated27

GC-MS Fullscan Pet FoodPet Food – UntreatedPet Food – EMR Treated28

GC-MS Full Scan– Avocado OilAvocado Oil Crude ExtractEMR – Lipid Extract29

GC-MS Spinach FullscanAbundance50000004500000Spinach matrix blank w/ Q-Universal dSPE cleanup40000003500000Spinach matrix blank w/ Q-Pigment dSPE cleanup30000002500000Spinach matrix blank w/o cleanup200000015000001000000Spinach matrix blank w/ EMR me-- 30

GC-MS Red Pepper Fullscan55000005000000450000040000003500000Red Pepper matrix blank w/ Q-Universal dSPE cleanup3000000Red Pepper matrix blank w/ Q-General dSPE cleanup25000002000000Red Pepper matrix blank w/o cleanup15000001000000Red Pepper matrix blank w/ EMR cleanup50000005.0010.0015.0020.0025.0030.0035.0031

Suggested Workflow for Multi-Residue Analysis Sample Preparation: EMR-Lipid with QuEChERS/ModifiedLiquid Extraction workflow System: Agilent LC or GC MS/MS (6400, 6500, 5977, 7000,7010, 7200 series) Columns:-LC: Agilent Poroshell 120 EC-C18, 2.7µmLC: ZORBAX RRHD Eclipse Plus C18, 1.8 µmGC: Agilent J&W DB-5ms Ultra Inert Agilent MassHunter Software Experimental setup: Matrix-matched CalibrationConfidentiality LabelOctober 20, 201532

Matrix Matched CalibrationWhat is matrix matched calibration? A sample blank is added to final calibration standards to correct any ionenhancement/suppression due to co-extracted matrix.-Example: 950 µL blank extract 25 µL working standard 25 µLinternal standard 1000 µL matrix matched calibration standardMatrix Matched Calibration and EMR-Lipid We HIGHLY RECOMMEND the use of matrix matched calibrationstandards for complex food samples.-Small amounts of matrix can cause poor results.Agilent Confidential10/20/201533

Benefits to Instrumental Flowpath34

Comparison of Analytes Response Consistency over MultipleAvocado Sample InjectionsTPP Peak Area over 100 Injections20000EMR cleanup1800016000Z-Sep cleanupTPPCommonlyused IS14000TPP Peak AreaRT: 18.3 min12000C18 dSPE cleanup10000Linear (EMR cleanup)8000Response drops 4% after100 avocado sample injections6000Linear (Z-Sep cleanup)4000Response drops 50% after100 avocado sample injections2000Linear (C18 dSPE cleanup)0510 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100Response increases 35% after100 avocado sample injectionsInjections of Avocado Samples on GC-QQQ35

Analytes Responses Reproducibility on GC/MS/MS over 100 Injectionsof Avocado Samples36

MS Source Critical Tuning ParametersMS (GC/MS) Performance Monitoring over Multiple Avocado Sample Injections1301600EM voltage, higher than 1400 indicated needs to replace120140010012009010008070Rel Abundance for Ion 219, Minimum of 70 needed6050800EM VolatageRelative Abundance for Tuning Ion (%)110600404003020Rel Abundance for Ion 502, minimum of 5 needed200100050010001500200002500Injections of Avocado Samples with EMR cleanup on GC/MS37

Pesticides in Avocado38

72 Pesticides Analysis in Avocado by LC and GC-QQQAgilent Confidential10/20/201539

QuEChERS-EMR Protocol for Multi-residue Analysis of Pesticides in AvocadoAccurately weigh 15 g comminuted Avocado sample in 50 mL centrifuge tubeAdd 15 mL of 1% Acetic Acid in Acetonitrile, and AOAC QuEChERS extraction kitCap and shake vigorously on mechanical shaker for 2 minCentrifuge @ 5000 rpm for 5 minTransfer 5 mL of upper ACN extract and 5 mL of water to EMR dSPE 15 mL tubesVortex and CentrifugeTransfer 5 mL supernatant to EMR polish tubeVortex, centrifuge, and transfer the upper ACN layer to another vialCombine 200 µL of ACN extract and 800 µL water, vortex, and centrifuge if neededSamples are ready for GC-QQQ analysisSamples are ready for LC-QQQ analysis40

Comparison of Avocado Co-extractives by WeightCleanupAmount of coextractives(mg, n 3)Amount of coextractivesremoved bycleanup (mg, n 3)No further cleanup14.7--C18/PSA Cleanup9.55.235.4EMR-Lipid Cleanup4.210.571.4Zirconia sorbentCleanup7.07.752.4% Matrix Co-extractives Removed by Cleanup % of matrix coextractivesremoved ���𝐴 𝑜𝑜𝑜𝑜 𝐶𝐶𝐶𝐶 ��𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 ��𝑅 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝐴 𝑜𝑜𝑜𝑜 𝐶𝐶𝐶𝐶 ��𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 �� ��𝐶 100%The use of EMR material cleanup removes extra 20-30% of Avocado co-extractives incomparison to traditional QuEChERS and/or competitor’s cleanup41

Comparison of GC/MS Full-scan Chromatogram for Matrix BackgroundThe use of EMR material cleanup provides significantly cleanup chromatographicsample background.42