Multiresidue Methods for Pesticide Residue Analysis

Multiresidue Methods for Pesticide Residue Analysis Michelangelo Anastassiades CVUA Stuttgart Stuttgart Regional Chemical and Veterinary Control Laboratory

Introduction Aims of Pesticide Residue Analysis in Food Check Compliance of products w. Regulations MRLs/GAP, Organic Framing Production Rules, Misuse Assess Long-Term Pesticide Intake by Consumer Coordinated Monitoring Detect Cases of Acute Risks ARfD, Communication through RASFF Strengthen Consumer Confidence in Food How many Pesticides are we dealing with? EU: > 740 pesticides still allowed to be used (shortly < 450) World: > 1100

Introduction How many of them are Sought and Found? Sought: ~100-450 Pesticides/Sample Found: ~360 Pesticides and Metab. (results of many labs 2003-05) Source: www.pesticides-online.com Why are so Many Pesticides not Targeted? Many Pesticides are not covered by the MRMs employed Limited Capacity of Instruments for Determ. Analysis Limited Personnel Available Limited Budget per Sample Remember: Its not only about including pesticides in the target spectrum but also about fulfilling all the validation requirements

Introduction There is Room for Improvement.. In the way HOW WE PERFOM ANALYSIS More Efficient Sample Preparation Methods More Efficient Determinative Instrumental Analysis More Efficient Data Processing In WHICH PESTICIDES WE TARGET Knowledge-Based decisions Consider Data about Pesticide Usage, Findings, Toxicity etc. www.pesticides-online.com

Multiresidue Methods (MRMs): Aim: Cover as many Pesticides as possible from a single sample portion employing a single sample preparation procedure Still more than one determ. analysis run required to cover the target pesticides with the required selectivity and sensitivity The broader the spectrum covered by the MRM, the less additional methods required to cover the analytes of Interest Wide Scope MRMs are more Efficient and Economical (less time, personnel, materials...)

MRM Evolution:

Early Simple but of narrow scope (OCs) MRM Evolution... Intermediate Expanded scope (to cover polar OPs) Very Complex since Determ. Analysis Instr. of poor selectivity and specificity Novel Simplicity, Streamlining, Cost Reduction Miniaturization, Automation 1960 1970 1980 1990 2000 2010

Factors that pushed the developm. of New Approaches Environmental, Health-Related and Economic Factors Need to Monitor Pesticide Intake & Environmental Pollution Need to Improve Sample Throughput & Reduce TAT and Costs Need to Reduce Solvent Consumpt. Advancements in Instrumentation (Electronics, Robotics, IT...) GC/ITD, MSD, MS-MS, TOF, PTV LC/MS, MS-MS, TOF Selective detectors ECD, NPD, FPD 1960 1970 1980 1990 2000 2010

Typical Classical MRM Weigh sample (e.g. 50 g) Add acetone and blend Filter by suction Add non-polar solvent (and salts), perform (multiple) partitioning Dry and filter organic phase Evaporate Reconstitute, perform GPC cleanup Evaporate Perform fractionated cleanup on silica Evaporate Transfer fractions in GC-Vials Analysis by GC-ECD, FPD, NPD

Pesticides and Co-extractives... -5 Amino acids -5 - -1 (ph dependent) Streptomycin -7.5 Sugars -5 - -2 Glyphosate -4 Quats -4.5 - -2.8 GPC/Silica Flavonoids/Anthocyanes Caffeine 0-6 -0,1 Monoterpenes 2.5-5.5 Acrylamide -0.7 Polarity range covered by trad. MRMs Pyrethroids (~45) 3.8-8.3 Acidic Pesticides (~40) GC OCs (~20) ph dependent 3.5-7.0 Ureas (~ 30) GC 1.6-5.9 OPs (~95) -0.9-5.7 Carbamates (~30) GC -0.4-5.5 GPC Basic Pesticides ph dependent GPC GPC Fatty Acids Phytosterols 6-8.5 8.5-11.5 GPC Vit. E GPC 11.5 PAHs PBDEs 3.3 6.8 6.2-9.5 Phthalates PCBs 2.5-6 5 8.5-4 -3-2 -1 0 1 2 3 4 5 6 7 8 9 10 GPC Carotenoids 11-18 GPC Chlorophyll 17.2 GPC TGs 20-24 LogKow 11

Typical inefficiencies of classical MRMs Main Drawbacks Large Sample-Sizes Macro-Approach Limited Scope (polars ) Too Many, Analytical Steps Complicated Tasks Limited LC Amenability Consequences Wasteful: Solvent & Material Analyst Exposure to Solvents Too many Additional Methods required Complicated Time-Consuming, Troublesome End Result Critical for Environment & Health Expensive Error-Prone

Desirable Characteristics of MRMs Fast (as Few Steps as Possible) Easy to Perform Inexpensive Low Solvent Consumption Safe for Personnel and Environment Selective Rugged and precise Achieve Good Recoveries for a Broad Analyte Spectrum Thus Reducing the No. of Single (-Group) Residue Methods to Perform

Novel Sample Preparation Techniques used for Pesticide Multiresidue Analysis Focusing on Automation SFE PLE Focusing on Automation and/or Miniaturization SPME/SBSE MSPD Focusing at Simplification of Classical Methods SPE of diluted Extracts QuEChERS

SFE (Supercritical Fluid Extraction) CO 2 pump Extraction Cell Solvent pump Oven Restrictor Modifier pump CO 2 gas Trap Solv. 1 Solv. 2 Liquid CO 2 Modif. Vial

SFE (Supercritical Fluid Extraction) Advantages Selective Extraction Automated extraction, cleanup Low consumption of org. solvent Simple and fast sample preparation Little glassware needed Drawbacks Limited scope, CO 2 too nonpolar Modifier-addition makes difficulties Sequential extractions long exposition times/ degradation Poor reliability of instruments High instrument costs Small sample sizes (subsampling variability)

PLE (Pressurized Liquid Extraction) S o lv e n t P u m p V a lv e O v e n E x tr a c t io n c e ll V a lv e N itr o g e n V ia l

PLE (Pressurized Liquid Extraction) Advantages Automated extraction Broad polarity spectrum Lower consumption of org. solvent Simple and fast sample preparation Little glassware needed Faster extraction kinetics (for strongly bound pesticides) Drawbacks Sequential extractions long exposition times/ degradation Frequent frit pluggings instruments High instrument costs Degradation of thermolabile analytes Poor selectivity (cleanup needed)

Micro-Extraction Techniques Involving LLP SPME (Solid Phase Microextraction) SBSE (Stir Bar Sorptive Extraction) SPDE (Solid Phase Dynamic Extraction) In-tube SPME Employ small amounts of Gum-Like Extraction Media (mostly PDMS) immobilized on devices that can be automatically introduced or connected to GC-Systems for thermal desorption HPLC connection requires liquid desorption but has not been widely applied yet

SPME (Solid Phase Micro-Extraction) Adsorption/Extraction step Thermodesorption step I II III IV V VI SPMEsyringe Sample vial SPME-fiber GCinlet GC- Column

SBSE (Stir Bar Sorbtive Extraction) PDMS-Coating Magnet Glass

Other Microextraction Techniques Involving LLP Inner walls coated with extractant e.g. PDMS Advantage: mechanical protection E.g. GC Capillary In-tube SPME, Open Tubular Trap (OTT) Syringe Solid Phase Dynamic Extraction (SPDE) Coating

Micro-Extraction Techniques Involving LLP Extraction follows the Rules of LL-Partitioning Recoveries at Equilibrium Conditions depend on: Polarity of the Analytes (Partitioning Coefficients Kow) Volume Ratio between the two Phases (unfavourable) Composition of the Sample Phase Equilibration Times often several hours! Thus non-equilibrium sampling (e.g. 20-60 min) Quantitative analysis: Keep all the parameters affecting partitioning constant (Stiring Velocity, Temp., Time, Sample Composition )

SBSE vs. SPME Theoretical Recovery [%] from 10 ml water 100 80 60 40 20 0 Ethiofencarb log kow ~2.05 Buprofezin log Kow 4.3-2 0 2 4 6 8 SBSE 126µL SPME 0,5 µl Log Kow By Achieving more Complete Extractions SBSE more Robust

SBSE / SPME Theoretical recovery does not occur in practice Non equilibrium conditions Competition of other lipophilic particles in the sample Solubility based precipitation of most lipophilic Pesticides (Log Kow>5) Direct sampling not suited for Multiresidue Approach!

SBSE/SPME Strategy: 1) Initial Extraction with solvent (e.g. methanol) 2) Dilution with water (e.g. to 10 % methanol 3) Sampling Problem: Partitioning becomes more unfavorable, polar compounds impossible to sufficiently extract (log Kow<1) Example: Pirimicarb (log kow 1.7) SBSE with 24 µl PDMS / 20 ml Sample Vol. Theoretical Recovery from Water (equilibrium): 5.7% Real Recovery at 10% Methanol: 0.74% (/60 min)

Multiresidue SBSE-Procedure Weigh 25 g of Homogenized Sample Add 100 ml Methanol Ultra-sonic Extraction 20 min Centrifuge (~ 80% Methanol) Ochiai et al. 2005 GERSTEL Appl. Note 10 ml Extract + 10 ml Water ~40% Methanol (20 ml) SBSE (60 min) 4 ml Extract + 16 ml Water ~20% Methanol (20 ml) SBSE (60 min) GC-MS (using TDU and PTV)

SPME/SBSE Advantages Drawbacks Broad applicability (Solids, liquids, gases) Potentially Solvent free extraction but risk too loose most lipophilic compounds Compact device Low cost Simple sample preparation Automated online operation possible Selectivity (no cleanup needed) Little glassware needed Limited range of polarity very small amount of extr. phase non-polar extr. phase Too long equilibration times thus non-equilibrium sampling Quantitative analysis requires standard addition approach Sequential extractions long exposition times/ degradation

MSPD (Matrix Solid Phase Dispersion) Sorbent Hom ogenized sam ple Glass m ortar Column

MSPD (Matrix Solid Phase Dispersion) Advantages Simple and fast sample preparation to perform Incorporates Extraction & Cleanup Low solvent consumption (e.g. 10mL) Little glassware and Instrumentation needed Drawbacks Small sample sizes e.g. 0.5-1 g (subsampling variability) ODS costs Backpressure

SPE of Diluted Extracts Weigh 10 g of Homogenized Sample Add 20 ml Acetone Extraction by shaking Stajnbaher et al. 2003 Centrifuge (~ 66% Acetone) 10 ml Aliquot + 80 ml Water ~14% Acetone Enrichment on PS-DVB Elute with Ethylacetate/Acetone Cleanup with DEA and evaporation GC-MS

123453678 92 2 89 5 Weigh 10 g of Frozen Sample 80000 70000 Add 10 ml Acetonitrile 60000 Excellent Results Anastassiades et al. JAOAC 86 (2003) 412-431 in 4Int. consecutive (Modified Version) EU Profficiency tests 50000 40000 Add ISTD-Solution Shake 30000 20000 10000 Add 4 g MgSO4 / 1 g NaCl / Buffered to ph 5-5.5 with Citrate Buffer Shake & Centrifuge 0 Tomatoes 2004 Grapes 2003 2005 Lettuce Oranges 2002 4 5 6 7 8 9 Optionally: Acidic Pesticides LC-MS Mix an Aliquot with MgSO4 & SPE Sorbent Shake & Centrifuge Acidify extract to ph ~5 to protect base-sensitive pesticides 2nd Symposium on Recent Advances in Food Analysis Optionally: Add other Analyte Protectants Multiresidue Analysis by GC-MS, LC-MS... min

Time Consuming, Complicated or Error Prone Steps of traditional MRMs Sample Processing/Homogenization Use of Ultra-Turrax during initial extraction Filtration Multiple LL-Partitioning Steps Phase Separation/Transfer of Entire Extract Use of a Lot of Glassware Simplified Alternatives Evaporation/Reconstitution Classical with Columns (SPE etc.) Main Goal: Method should be as Simple and Streamlined as Possible Avoiding troublesome steps...

Time Consuming, Complicated or Error Prone Steps of traditional MRMs Sample Processing/Homogenization Use of Ultra-Turrax during initial extraction Filtration Multiple LL-Partitioning Steps Phase Separation/Transfer of Entire Extract Use of a Lot of Glassware Evaporation/Reconstitution Classical with Columns (SPE etc.) Simplified Alternatives No Way Around this!! Shaking Centrifugation Single Partitioning ( On-line -Approach) Take Aliquots (Use ISTD) Extraction/Partitioning in Single Vessel Large Volume Injection; Sensitive Instr. Dispersive SPE Goal achieved: Simple and Streamlined MRM Few working steps, Low Material- and Solvent consumption

MgSO 4 alone gave the Best Overall Recoveries (polar pesticides) However, it resulted in too many Polar Components (e.g. Sugars) in the Extract 2 g NaCl 1 g NaCl 0.5 g NaCl Addition of NaCl Reduced Water (and Sugar) content in the MeCN-Phase HMF GC-Degradant of Fructose used as indicator NaCl is used to Control Selectivity 0 g NaCl 6.008.0010.0012.0014.0016.0018.00 20.0022.0024.0026.0028.0030.0032.00

Advantages No SPE Manifold, Cartridges, Vacuum/Pressure, No Conditioning, Channeling, Flow Control, Drying-Out, No Elution Solvent, No Dilution of Extract, No Collection Tubes, No Evaporation, Less Sorbent Needed, Faster and Cheaper, No Experience Needed.

Pesticides and Co-extractives... -5 Amino acids LLP/ -5 -D-SPE -1 (ph dependent) Sugars LLP/ D-SPE -5 - -2 Streptomycin -7.5 Glyphosate -4 Quats -4.5 - -2.8 D-SPE (PSA) D-SPE (PSA) Flavonoids/Anthocyanes Fatty Acids Phytosterols Caffeine Polarity range covered by trad. 8.5-11.5 MRMs 0-6 6-8.5-0,1 Monoterpenes 2.5-5.5 Acrylamide -0.7 Acidic Pesticides (~40) ph dependent Pyrethroids (~45) 3.8-8.3 OCs (~20) 3.5-7.0 Ureas (~ 30) 1.6-5.9 OPs (~95) -0.9-5.7 Carbamates (~30) -0.4-5.5 Basic Pesticides ph dependent PAHs 3.3 6.8 Phthalates 2.5-6 PCBs 5 8.5 PBDEs 6.2-9.5-4 -3-2 -1 0 1 2 3 4 5 6 7 8 9 10 Vit. E 11.5 D-SPE (GCB) Carotenoids 11-18 D-SPE (GCB) Chlorophyll 17.2 LLP/ D- SPE (C18) TGs 20-24 LogKow 11

Rapid (8 Samples in Less Than 30 min) Simple (No Laborious Steps, Minimal Sources of Errors) Cheap (~1 $ Sample Prep. Materials for 1 ml Extract) Low Solvent Consumption (10 ml Acetonitrile) Practically no Glassware Needed Wide Pesticide Range (Polar, ph-dependent Compounds) Extract in Acetonitrile (GC- and LC-Amenable)

ml Solvent/Sample 655 more pesticides than Becker 535 including basic & acidic pesticides, but low recoveries for very polar ones 325 265 485 215 more pesticides included 330 200 215 10 sum 10 other solv. 65 50 0 0 organochlorine Becker, up to 1990 Becker, Mini, 1990-96 Specht, 1993-96 Anastassiades, 1996-2002 MRM = multiresidue method QuEChERS; since 2002 Solvent-Cost-Savings 15.000 in 1 Year!!

Thank you very much for your Attention!

The CVUA Stuttgart Pesticide Residue Team

The CVUA Stuttgart Pesticide Residue Team Dražen Kostelac Hubert Zipper Nadja Looser Ellen Scherbaum Eberhard Schüle Diane Fügel Carmen Wauschkuhn