The QuEChERS Method –

Background Information
and Recent Developments

Michelangelo Anastassiades
CVUA Stuttgart

CRL-SRM
1
Community Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

Outlook
v Classical Multiresidue Methods (MRMs)
 Evolution
 Limitations and Expectations
v Original QuEChERS-Method
 Strategy of Method Development (Background Info)
v Recent Developments in QuEChERS Methodology
 pH-Adjustment (during extraction, in final extracts)
 Improved Selectivity (extraction, cleanup)
 Expanded Matrix Spectrum (dry food, fatty food)
v Experiences of its Implementation in the Lab
v Method Validation
 EU-Proficiency Tests (incurred and fortified residues)
CRL-SRM
2
 Inter-laboratory Ring Tests
Community Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

Pesticide Residue Analysis:
Sample Processing

Sample Preparation
Multi- and Single-Residue Methods

Measurement

Data Processing
CRL-SRM
3
Community Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

Multiresidue Methods (MRMs):
Aim of MRMs:
Cover as many pesticides as possible from a single sample
portion employing a single sample preparation procedure

But, still
more than one determinative analysis run
is required to cover all analytes of interest 80000

70000

w ith su fficie n t se le ctivity a n d se n sitivity…
60000

50000

40000

30000

20000

10000

0

4 5 6 7 8 9 mi n

The broader the spectrum of analytes covered by the MRM,
 The less additional methods are required to cover all analytes
 The more efficient and economical the analysis
CRL-SRM
4  Less time, personnel, materials...
Community Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

of poor selectivity and specificity Novel Simplicity.Stuttgart. analysis instr. automation CRL-SRM 5 Community Reference Laboratory Pesticide Residues 1960 1st 1970 Joint CRL-Workshop ...Early MRM Evolution. cost reduction miniaturization. 06/12/2006 1980 1990 2000 2010 using Single Residue Methods . streamlining. Simple but of narrow scope (OCs) Intermediate Expanded scope (to cover polar OPs) Very complex since determ.

Stuttgart. 06/12/2006 . MRM Evolution: “Technical Development always follows the way from the Primitive via the Complicated to the Simple … ” Antoine de Saint-Exupéry (1900-1944) CRL-SRM 6 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

TOF „S elective“ d etecto rs E C D . TOF. PTV LC/MS.. MS-MS.. 06/12/2006 1980 1990 2000 2010 using Single Residue Methods . IT. Advancements in Instrumentation (Electronics. Robotics. MSD. Health-Related and Economic Factors Need to Assess Risks for Humans and Environment Need to Improve Productivity and Sample Throughput & to Reduce TAT and Costs Need to Reduce Solvent Consumpt.) GC/ITD. N P D . F P D CRL-SRM 7 Community Reference Laboratory Pesticide Residues 1960 1st 1970 Joint CRL-Workshop .Stuttgart. MS-MS. of New Approaches Environmental.Factors that pushed the Developm.

NPD Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .g.Stuttgart. 06/12/2006 . perform GPC cleanup Evaporate Perform fractionated cleanup on silica Evaporate Analysis Transfer fractions in GC-Vials CRL-SRM 8 by GC-ECD. CommunityFPD. Typical Classical MRM Weigh sample (e. 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.

Stuttgart. 5 .5Community Reference CRL-SRM 9 Laboratory Pesticide Residues -5 -4 -3 -2 -1 0 1st Joint CRL-Workshop .5-5..2 -0. -0.-.7 Glyphosate Carbamates (~30) .5.9.5 TGs 4 PAHs PBDEs 20-24 Basic Pesticides 3.7.5 .0 Strepto. LogKow 4 5 2 8 2..5 11 .6 .2 .-1 Polarity 0-6 range covered .3 – 6.5 . Pyrethroids (~45) 5 2 3.9 Chlorophyll -7.9 . Ureas (~ 30) mycin 1. 06/12/2006 1 2 3 4 5 6 7 8 9 10 11 using Single Residue Methods . trad.Pesticides and Co-extractives.5 MRMs 6 8 5 (pH dependent) Monoterpenes Vit..5.5-11.8 6.6 5 – 8.8. -.4 .by .5.5 OPs (~95) 17. .8 .5 Quats PCBs pH dependent Phthalates .3 Acidic Pesticides (~40) OCs (~20) Carotenoids pH dependent 11-18 3. E Sugars 2.8. Amino acids Flavonoids/Anthocyanes Fatty Acids Phytosterols -5 .

Scope and performance of classical MRMs CRL-SRM 10 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .Stuttgart.

Typical inefficiencies of classical MRMs Main Drawbacks Consequences Wasteful: Large Sample-Sizes Solvent & Material “M a cro -A p p ro a ch ” Critical for Limited Scope Environment (p o la rs… ) & Health Analysts Exposure Time-Consuming to Solvents Troublesome Expensive Unpopular Limited LC Too Many Amenability Error-Prone Complicated Too many Steps Additional Methods required CRL-SRM 11 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .Stuttgart.

andtSingle-Residue ck Methods of Pesticide Residue Analysis Measurement Data Processing CRL-SRM 12 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . Sample Processing Sample Preparation has traditionally been Sample Preparation the tleneBo Multi.Stuttgart. 06/12/2006 .

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 need to run Single (-Group) Residue Methods CRL-SRM 13 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart. 06/12/2006 .

Stuttgart.Some Novel Sample Preparation Techniques  Focusing on Automation SFE PLE  Focusing on Automation and/or Miniaturization SPME/SBSE MSPD  Focusing at Simplification of Classical Methods SPE of water-diluted extracts Partitioning on Macroporous Sorbents QuEChERS CRL-SRM 14 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .

Original-Method Weigh 10 g of Sample (50 mL Teflon-Tube) Add 10 mL Acetonitrile Shake Vigorously 1 min Add 4 g MgSO4 and 1 g NaCl Shake Vigorously 1 min Add ISTD-Solution Shake 30 s and Centrifuge Take Aliquot and Add MgSO4 and Sorbent(s) Shake 30 s and Centrifuge Anastassiades et al. (A d d “A n alyte P ro tectan ts” . 86 (2003) 412-431 GC-MS (and LC-MS) CRL-SRM 15 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . QuEChERS .Stuttgart. ad ju st p H ) JAOAC Int. 06/12/2006 .

Procedure in Pictures – 1. 06/12/2006 .Stuttgart. Initial Extraction Step Weigh 10 g Sample Add 10 mL MeCN Shake Intensively for 1 min CRL-SRM 16 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

Procedure in Pictures – 2.Stuttgart. 06/12/2006 . Extraction/Partitioning Step (Pre-)Weigh 4 g MgSO4 + 1 g NaCl Add to the Tube Shake Intensively for 1 min CRL-SRM 17 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

Stuttgart. 06/12/2006 . Procedure in Pictures – 3. Addition of ISTD and Centrifugation Add ISTD Shake for 30 s Centrifuge (ca. 5 min) Separated Raw Extract CRL-SRM 18 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

30 s Centrifuge (ca. 2 min) Cleaned up Extract CRL-SRM 19 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . Dispersive SPE Step (Pre-) Weigh MgSO4 and PSA Add Extract to Tube and Shake ca. Procedure in Pictures – 4. 06/12/2006 .Stuttgart.

06/12/2006 . • Convenient to perform • Low Material. Columns (SPE. Sensitive Instr. Simplifications Introduced in the Method Time Consuming. Complicated or Simplified Alternatives Error Prone Steps of traditional MRMs Use of Ultra-Turrax during Initial Extraction Shaking Filtration Centrifugation Multiple LL-Partitioning Steps S in g le P artitio n in g (“O n -Line-A p p ro ach ”) and Isolation of Entire Extract Take Aliquots (Use ISTD) Use of a Lot of Glassware Extraction/Partitioning in Single Vessel Evaporation/Reconstitution Large Volume Injection.and Solvent consumption CRL-SRM 20 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart. Cleanup w. GPC) Dispersive SPE Sample Processing/Homogenization No Way Around this!! a Goal achieved: Simple and Streamlined MRM • Few working steps. Trad.

.. ECONOMICAL... Strategies in the Development of the QuEChERS-Method STREAMLINED AND SIMPLE. FIT FOR PURPOSE..Stuttgart.. CRL-SRM 21 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .

of Extracts. Degradation)  Type and Amount of Salts Used to Induce Phase Separation  Selectivity (Gravimetric Anal. 06/12/2006 . GC-Interferences) Instrumental Analysis  Matrix Effects (Influence of Cleanup) CRL-SRM 22  U se o f “A n a lyte P ro te cta n ts” Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . Method Development .Stuttgart. Anal. of Extracts. (Ionization.Aspects Considered Initial Extraction & Extraction/Partitioning Step  Choice of Extraction Solvent and Sample/Solvent Ratio  Sample Amount  Blending Vs. Shaking (Incurred Residues)  Influence of Sample pH on Recov. GC-Interferences)  Use of ISTD (Check that Recovery-Correction is minimal) Cleanup (Dispersive SPE)  Type and Amount of Sorbent and MgSO4  Selectivity (Gravim.

from Water-Phase by Salt-Add. (No Non-Polar Solv. Needed)  Easier to Remove Water (with MgSO4) than from Acetone Q Difficult to Evaporate But PTV with Solvent Venting Q High Expansion Volume could be used Q Not Compatible With NPD Q Not Compatible with GPC (But. consistent at same Lipid/solvent ratio)  Accessibility problems of pesticides enclosed in Lipid particles (Ultra Turrax) Q Rel. Lipid-Co-Extraction is Low) Q Low Lipid Solubility  Losses of non-polar pesticides (Recov. Method Performed in a Closed Vessel. thus minimal exposure) CRL-SRM 23 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .and SPE-Applications  Not Chlorinated  Miscible with Water (Good for Initial Extraction)  Separ. Method Development – Choice of Acetonitrile as Solvent  Selective (Few Co-Extractives but still broad pesticide Spectrum covered)  Compatible with LC.Stuttgart. 06/12/2006 . Toxic (But.

) CRL-SRM For More Details and Comparison with EtAc .50 1.00 Mix of Fruits and vegetables 2. Method Development – Acetonitrile vs. other Solvents Residual co-extracted matrix components in mg/mL 3.50 using QuEChERS 2. AcetoneCommunity 24 Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .50 - c N ne SA A SA SA eC Et to rP rP rP M ce te te te A af af af c N ne A eC Et to M ce A (see AOAC publ.00 0.00 1.Stuttgart.50 Extracts of a 3. 06/12/2006 .

Method Development –
Sample Amount and Sample/Solvent Ratio
Sample Amount: 10 g
Miniaturization improves efficiency
 Less material consumption
 Reduced costs
Important:
 Good homogenization is needed (e.g. use of dry ice)
Studies: Acceptable variations for 10 g subsamples using cryogenic milling

Sample:Solvent Ratio: 1:1 (w/vol)  gives 1g/mL
Still good recoveries of incurred residues (polar and non-polar)
N o e va p o ra tio n o f fin a l e xtra ct n e ce ssa ry…
 1g/mL is enough when using modern instruments (PTV in GC is better)

CRL-SRM
25
Community Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

5500000
Method Development -
MgSO4 / NaCl 4:1 for the Partitioning
5000000
Step
4500000
Many Salts tested
4000000 + 2 g NaCl
MgSO4 gave best salting-out of ACN
3500000 Methamidophos
and Best Overall Recoveries + 1 g NaCl Rec. 80-85 %
(especially for polar pesticides) 3000000

2500000 + 0.5 g NaCl
However: too much water in ACN-
phase and too many Polar Co- 2000000
HMF
extractives (e.g. Sugars) 1500000 GC-Degradant of Fructose
used as indicator
NaCl Addition increases Selectivity 1 0 0 0 0 0 0
Methamidophos
Less Water (and Sugar) in ACN-Phase 4g MgSO4 Rec. 95-100 %
500000
 NaCl is used to Control Selectivity + 0 g NaCl
0
6 .0 0 8 .0 0 1 0 .0 01 2 .0 01 4 .0 01 6 .0 01 8 .0 02 0 .0 02 2 .0 02 4 .0 02 6 .0 02 8 .0 03 0 .0 03 2 .0 03 4 .
T im e -->
CRL-SRM
26
Community Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

Method Development -
MgSO4 / NaCl 4:1 for the Partitioning Step

Extraction of Honey (5g/10 mL MeCN)
mg co-extractives/mL extract

3,8
4,0
3,0
1,9 1,7
2,0

1,0 0,6

0,0
Partitioning with 4g MgSO 4 Partitioning with 4g MgSO 4 + 1g NaCl
(Original QuEChERS)

Raw Extract After PSA

 NaCl reduces the amount of co-extracted matrix

CRL-SRM
27
Community Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

Method Development –
Shaking vs. Blending
Advantages
 No Exposure to Metal Surfaces
 Can Be Done by Hand and in Parallel
 No Cleaning of Jar and Blender Between Samples
 No Carry Over Between Samples
 Only One Container necessary
 Safer (Closed Vessel)
 Less Noisy than Blending
 No Frictional Heat
- May be less reproducible that blending
Pesticides from Fruits + Veg.: Ultra-Turrax usually not necessary
CRL-SRM

Community
28Checked with Incurred Residues (Cryogenic milling)
Reference Laboratory
Pesticide Residues
using Single Residue Methods
1st Joint CRL-Workshop - Stuttgart, 06/12/2006

 Faster and Cheaper. Vessels for Eluent Collection.  No Dilution of Extract  No Evaporation.  No Conditioning. Channeling.Method Development - Dispersive SPE for Cleanup Advantages over classical SPE with Cartridges  No SPE Manifold. Vacuum/Pressure.  No Experience Needed. W h e n “Chemical Filtration” is n e e d e d CRL-SRM  “Dispersive SPE” is a se rio u s o p tio n 29 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .  No Add. Drying-Out. Flow Control.  No problems w.  No Elution Step Needed.Stuttgart. 06/12/2006 .  Less Sorbent Needed.

9 8 pH 8.0 1.Stuttgart.5 1 0.Dispersive SPE –Removal of Co-extractives PSA Cleanup and effect on pH 3. 06/12/2006 .0 3 2 0.0 mg/mL 5 1.5 pH 5.9 9 3.4 1.4 2.0 pH 8.0 0 No PSA PSA 25 mg/mL PSA 50 mg/mL Amount of Co-extractives in the extract (mg/kg) pH of Acetonitril Extract Drawbacks: Solutions:  pH goes up (degradation risk)  Addition of Acids (see later)  Matrix-Induced Analyte CRL-SRM 30 Protection in GC reduced Community Reference Laboratory Pesticide Residues  Addition of Analyte Protectants using Single Residue Methods 1st Joint CRL-Workshop .5 7 6 pH value 2.3 4 1.5 10 2.

Impact of Matrix-Effects „M atrix-In d u ced P eak E n h an cem en t“ CRL-SRM 31 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .Stuttgart.

active Community Reference Laboratory Pesticide Residues sites and protect analytes using Single Residue Methods 1st Joint  32 CRL-Workshop 06/12/2006 .Stuttgart.„M atrix-In d u ced P eak E n h an cem en t E ffect“ GC-Liner GC-Capillary Active Sites (on Surface of GC-Liner & Column ) (Siloxanes & deposited non-volatile matrix-co-extractives) Analytes (in teract w ith A ctive S ites w h ich cau ses… )  Unwanted Retention/Tailing  Quasi-catalysed degradation (susceptible compounds) Matrix-Components (in Excess) CRL-SRM Bloc.

05 9.10 9.90 8.20 9. Matrix: Strawberry RT= 8.70 8.00 9.80 min 30000 WITH Matrix co-extractives 25000 (Strawberry-Extract) 20000 WITHOUT Matrix co-extractives (in pure solvent) 15000 e.40 9.80 8.35 9.Analyte: Atrazine . 06/12/2006 .30 9.25 9.95 9.g.Stuttgart. Calibration standard 10000 RT= 8.5:1 a OVERESTIMATION OF RESULTS!! • Peak-Heights: ~ 4:1 CRL-SRM 33• Peak-Width (at half height): ~ 1:3 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .45 Ratios: Matrix-Induced Peak Enhancement • Peak-Areas: ~ 1.92 min  Stronger Tailing 5000  Apex-Shift towards longer RTs! 0 8.75 8.15 9.85 8.

Analyte Protectants Principle „ Protection“ Cleaned-up Extract + AP Standard + AP Raw Extract Addition of „A n alyte PSA P rotectan ts“ cleanup (AP) Cleaned up Extract Standard in Pure Solvent Analyte Protectants Reduce: CRL-SRM 34 Analyte Interactions with Active Community Reference Laboratory Sites Pesticide Residues and thus 1st Joint CRL-Workshop Errors . Related to Matrix-Induced Peak Enhancement 06/12/2006 in GC using Single Residue Methods .Stuttgart.

50 2.50 3.00 no AP AP Without withAP With AP AP was added to both : CRL-SRM Sample 35 Extract and Calibration Standard (in pure Community Reference Laboratory Pesticide Residues Solvent) using Single Residue Methods 1st Joint CRL-Workshop .Analyte Protectants- Reduction of Matrix Induced Enhancement Errors Errors eliminated if: Response in Matrix/Response in Solvent ~ 1 coumaphos azinphos-methyl imazalil endosulfan I cyprodinil Response in Matrix fenthion Response in Solvent dichlofluanid carbaryl chlorpyriphos Overestimations metalaxyl 1.00 2. 06/12/2006 .00 1.Stuttgart.00 0.50 1.0 when using vinclozolin Standards in Solvent omethoate acephate mevinphos methamidophos 0.

06/12/2006 .Analyte Protectants – Examples  V a rio u s C o m p o u n d s T e ste d fo r “P ro te ctive P o te n tia l”. ~derivatives) Examples: H O O HO OH OH OH Ethylglycerol O HO O HO OH OH OH HO OH Sorbitol δ-Gulonolactone Give broadly eluting peaks  protection over a wide volatility range CRL-SRM 36 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart.  Best Protection : Polyhydroxy-Compounds (sugars.

06/12/2006 .Analyte Protectants- Desirable properties v Strong interactions with active sites (H-Bond activity) v Simmilar volatility to analytes to be protected (so that protection extents during entire run) v Soluble in sample extract v Not accumulating in GC-system v Not reactive with analytes (not inducing their degradation) v Minimal interference with analyte detection (small m/z) v Not deteriorating GC-column separation performance v Cheap and not hazardous CRL-SRM 37 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart.

Stuttgart. 06/12/2006 .CRL-SRM 38 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

Stuttgart. 06/12/2006 .CRL-SRM 39 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

QuEChERS New Developments CRL-SRM 40 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart. 06/12/2006 .

Stuttgart. Carotenoids • Expanding Matrix Spectrum OIL  Fatty Commodities CRL-SRM 41  Dry Commodities Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . Sugars Chlorophyll. 06/12/2006 . QuEChERS – Further Improvements Some Issues Addressed • pH-issue  Stability of pH-labile Compounds  Recoveries of Ionizable Compounds • Selectivity Issue  Of Extraction/partitioning  Of Cleanup Lipids.

The pH Issue  Recoveries of Ionizable Compounds  Stability of pH-labile Compounds  Selectivity of Extraction (see later) CRL-SRM 42 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .Stuttgart.

5 – 7 CRL-SRM 43 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart. pH-issue – Ionization of Pesticides Some pesticides get ionized at low or high pH-values Acids: HX  H+ + X- Bases: B + H+  BH+  Ionic form prefers to stay in the water phase pH-Range of agricultural samples: ~2. 06/12/2006 .

3 deprotonized form Dicamba 1.7 which compounds lay Cyromazine 5.7 Clopyralid 2.4-DB Acids 4.5 in protonized form Fenpropimorph 7 Propamocarb 9.2 pKa = pH below Thiabendazole 4.5 In traditional methods.Pka-Values of Acidic and Basic Pesticides OPP 9. Community pH-adjustment CRL-SRM 1-2 Reference Laboratory 44 units > or < PKa is recommended for quantitative recoveries Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . using non-polar solvents.9 Nitenpyram 3.Stuttgart.1 compounds lay in 2.1 Prochloraz 3.4-D 2.2 Imazalil 6.8 pKa = pH above which MCPA 3.8 Bases Carbendazim 4.8 Ioxynil 4 MCPP 3. 06/12/2006 .9 2.

9 Carbendazim pKa of corresp. acid 7.5 Prochloraz pKa of corresp.9 Spiroxamine pKa of corresp. Level: 0. 06/12/2006 ESI (+) using Single Residue Methods . acid 4.8 Thiabendazole pKa of corresp. acid 3.2 Fenpropimorph pKa of corresp. Stuttgart. acid 6. Community Reference Laboratory Pesticide Residues Analysis: 1st Joint CRL-Workshop -LC-MS/MS. Basic Pesticides – Not affected! pKa = pH below which the compound lays primarily in its protonized form Propamocarb pKa of corresp.7 pH 2. acid 9. acid 6.5 Imazalil pKa of corresp.0 0 20 40 60 80 100 120 Commodity: Apple.1 mg/kg. Recovery % CRL-SRM 45 Fortif. acid 4.

3 with H2SO4 EtAc QuEChERS EtAc QuEChERS pH 3 54 90 51 92 pH 4 85 90 73 94 pH 5 96 84 84 86 pH 6 104 90 94 90 Despite theoretically unfavourable pH. pKa Basic Pesticides and pH pH below which the compound lays predominantly in its Effect of pH on Recoveries (%) protonized form Apple juice. Thiabendazole Imazalil pH adjusted pKa = 4. Possible Reason: CRL-SRM After 46 Community Reference Laboratory partitioning ACN still contains a considerable amount of water Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .7 pKa = 6. the basic pesticides still prefer to partition into the MeCN phase.Stuttgart.

ESI (-). 06/12/2006 .5-TP Propyzamid 2.4-D Fluoxypyr 2.5 40 20 Clopyralid Imazapyr Picloram Benazolin Imazethapyr 4-CPA Naphthylacetic acid Dicamba Imazaquin MCPA 2.4-DP Bentazon Bromoxynil Ioxynil 0 Fluazifop Bromacil 2. pH 5.5-T Mecoprop Triclopyr 2. No PSA Cleanup 120 100 Recovery % 80 60 max.Stuttgart.4-DB MCPB Lower pKa-Values General Trend Higher pKa Values CRL-SRM 47 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .4.4.Acidic Pesticides – Recovery-Drop at pH 6 LC-MS/MS.

pH-Issue . adjust pH SPE with PSA  Extract pH > 8 CRL-SRM 48 Community Reference Laboratory Pesticide Residues 1st Joint CRL-Workshop .Stuttgart.Labile Compounds Some Pesticides degrade at high or low pH-values! • In the sample (processing. storage)  Keep low temperature During sample preparation  Work fast. adjust pH  Use frozen samples for analysis MgSO4 + Water  Heat • In the extract during storage (1 week common)  Keep low temperatures. 06/12/2006 using Single Residue Methods .

carbosulfan. dioxacarb.. Extraction Step d ico fo l.5 ) Step • Still good protection for Base-Sensitives tolylfluanid. captan.Stuttgart. CRL-SRM 49 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . pymetrozine. p yrid a te … + Extract Storage •Still good protection for Acid-Sensitives sulfonylureas.. 4-C P A . folpet. 06/12/2006 . Optimal pH for QuEChERS ? Goals: Relevance: • Still good recoveries for the Strongest Acids Extraction dicamba. clo p yra lid … (p H < 5 . dichlofluanid.

pH Adjustment in Extraction Step Various Buffers tested Compromise: Citrate Buffer at pH 5 to 5.  1 g Trisodium citrate dihydrate and  0.)  Acceptable recoveries for base.5 g Disodium hydrogencitrate sesquihydrate  Good recoveries even for most acidic pesticides (dicamba .and acid-sensitive pesticides  Impoved Selectivity (less co-extractives from acidic samples) No negative effect on PSA cleanup (unlike Acetate CRL-SRM Community Buffer) Reference Laboratory Pesticide Residues 50 1st Joint CRL-Workshop . 06/12/2006 using Single Residue Methods .5  4 g Magnesium sulphate anhydrous...Stuttgart..  1 g Sodium chloride (still kept for better selectivity).

Problem: T ed io u s W eig h in g o f S alts… Solution: R ap id & E asy P o rtio n in g b y “S am p le D ivid ers” Also suitable to portion the sorbents for d isp ersive S P E …  Some companies offer ready-to-use mixturesCommunity forPesticide QuEChERS CRL-SRM Reference Laboratory Residues 51 Partitioning 1st Joint Salts and Dispersive SPE Mixtures CRL-Workshop .Stuttgart. 06/12/2006 using Single Residue Methods .

Stuttgart. 40 after PSA dicofol. 60 50 pH folpet. 06/12/2006 . Rec.Need to Adjust pH in Final Extracts Goal: Avoid degradation of Base-labile compounds in final extract. 30 20 pyridate 10 0 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 MeCN Measured pH in extract Addition of formic acid (5% in ACN): CRL-SRM  10 µL p er m L extract b rin g s “p H ” to ~ 5 52 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 80 13 days 70 captan. % Tolylfluanid stability in extract 100 Also the case for: 90 7 days dichlofluanid.

in % Primisulfuron-Methyl Sulfonylureas. 120 7 Days Storage Carbosulfan 100 80 13 Days Storage acid labile. in % Carbosulfan 120 7 Days Storage If these 100 13 Days Storage compounds are 80 included in the 60 target spectrum 40 use an aliquot of 20 the final extract 0 pH 4 pH 5 pH 6 pH 7Community pH 8 Reference MeCN pH 9 Laboratory CRL-SRM before 53 acidifying Pure Pesticide Residues Measured pH of Extract using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart. 60 40 20 0 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 MeCN Pure Measured pH of Extract Rec. 06/12/2006 ... Rec.

Carotenoids CRL-SRM 54 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . Sugars v Chlorophyll. Improving Selectivity  At Extraction/partitioning Step v pH v Salts  At Cleanup Step v Lipids.Stuttgart. 06/12/2006 .

Role of pH in the Selectivity of Extraction/Partitioning Influence of pH in the Amount of Co-Extractives Red Currant (pH adjusted with NaOH) 9 8.1 8 Raw Extracts 7 before Cleanup Co-extracted Matrix 6 [mg/ml] 5 4.5 2.Stuttgart.2 2 1 0 Natural pH pH 4 pH 5 pH 6 pH-Value CRL-SRM The higher the pH the less co-extractives… 55 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .2 4 3 2. 06/12/2006 .

4 6 8 6 8 6 p H = 5 . 8 9 12 S a m p le p H 5 .2 5 5 5 6 6 6 4 4 4 p H = 3 .8 1 Red Currant 0 .0 p H = 1 .8 pH m g /m L pH 8 8 8 10 1 1 . 1 9 p H = 8 . 06/12/2006 using Single Residue Methods .B u f f e r e d A c e ta te -B u ffe re d 12 S a m p le p H 2 . 1 9 12 S a m p le p H 5 .6 2 2 2 2 2 2 1 .0 p H = 5 .1 p H = 5 .2 10 10 7 7 7 8 p H = 5 .9 2 .2 p H = 3 .7 3 4 3 4 3 2 .5 4 3 .Stuttgart.5 1 1 0 0 0 0 0 0 R a w E x tra c t P S A 2 5 m g /m L P S A 50 m g /m L (aR adifferent w E x tra c t P S A 2 5 m g /m L one) P S A 50 m g /m L R a w E x tra c t P S A 2 5 m g /m L P S A 50 m g /m L m g c o -e x tr a c tiv e s/m L E x tr a c t m g c o -e x tr a c tiv e s/m L E x tr a c t m g c o -e x tr a c tiv e s/m L E x tr a c t pH pH pH  Buffering to pH ~5 reduces amount of acidic co-extractives CRL-SRM Community Reference Laboratory Pesticide Residues  Acetate buffer negatively affects PSA cleanup efficiency 56 1st Joint CRL-Workshop .6 0 .3 m g /m L pH m g /m L p H = 7 .5 3 . Role of pH in the Selectivity of Extraction/Partitioning Step Comparison of QuEChERS-Modifications O r ig in a l Q u E C h E R S C it r a t e .4 2 .

Selectivity of Cleanup More than 50 SPE Sorbents and freezing-out tested! Mainly removed: v Amino-Sorbents.Stuttgart. Chlorophyll. Sterols planar pesticides v Reversed-Phase Sorbents: No losses • Lipids and Waxes observed v Freeze-out: No losses • Lipids and Waxes CRL-SRM Community Reference Laboratory observed Pesticide Residues 1st • 57 Sugars Joint CRL-Workshop . 06/12/2006 using Single Residue Methods . Alumina: • Acids (including fatty acids) Losses of • Sugars acidic pesticides • Pigments (Anthocyanes. some Chlorophyll) v Carbon-based Sorbents: Losses of Carotinoids.

06/12/2006 using Single Residue Methods . GCB (Graphitized Carbon Black) was best in handling .Use of Carbon Sorbents PSA  PSA not satisfying when high contents of carotinoids or chlorophyll C Carbon Sorbents more Effective Many tested.Cleanup time (shaking) extended from 30 s to 2 min  Small GCB amounts are d ifficu lt to h a n d le … C CRL-SRM Pre-mixtures GCB/MgSO 4 (powder) facilitate weighing Community Reference Laboratory Pesticide Residues 58 1st Joint CRL-Workshop .Used in combination with PSA at small amounts .Stuttgart.

Stuttgart. 06/12/2006 . Recoveries > 70% will indicate that no unacceptable CRL-SRM losses of pesticides have occurred. thiabendazole But chlorophyll has higher affinity than all pesticides C Final extract should remain slightly coloured!! Anthracene may be used as surrogate QC standard. chlorothalonil.Problems with GCB: Planar pesticides have a high affinity towards GCB e. 59 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . hexachlorobenzene.g.

Stuttgart.10 1 0 CRL-SRM 60 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .23 1.33 1.15 1.20 OIL EXTRACTS 4.25 1. Removal of co-extracted lipids by C18 or freezing out 5 4. 06/12/2006 .03 4 Extractives [mg/ml] 3 2 1.48 1.

06/12/2006 . Removal of co-extractives from Whole-Wheat flour CleanupofW hole-WheatFlour Q uEChERS-Extracts 7 6 5 4 Co-extractives/mLextract 3 2 1 0 RawExtract C18 Freeze-out C18+PSA Freeze-out +PSA CRL-SRM 61 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart.

Scope and Pereformance of QuEChERS CRL-SRM 62 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .Stuttgart.

Add ISTD-Solution The method Shake will become Add 4 g MgSO4 / 1 g NaCl / Citrate Buffer official CEN method (pH 5-5.5) Shake & Centrifuge Optionally: Acidic Pest. freeze-out Shake & Centrifuge Optionally: SUs by LC-MS/MS Acidify extract to pH ~5 to protect base-sensitive pesticides Multiresidue Analysis CRL-SRM 63 O p tio n ally: A d d o th er “A n alyte P ro tectan ts” Community Reference by GC-MS.Stuttgart.QuEChERS. MgSO4 & Sorbents.. using Single Residue Methods 1st Joint CRL-Workshop .Multiresidue-Method Weigh 10 g of Frozen Sample Changes introduced Add 10 mL Acetonitrile to the method. by LC-MS/MS Mix an Aliquot w.. Laboratory Pesticide Residues LC-MS . 06/12/2006 .

Stuttgart. Broaden matrix spectrum Dry commodities (cereals. dried fruits) Fatty Commodities CRL-SRM 64 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .

g. if necessary. 06/12/2006 . Sample amount is reduced and water is brought to 10 mL Co-extracted fat removed by freezing out or C18.Stuttgart.. dried fruits Water-Addition prior to extraction  to weaken interactions of pesticides with matrix and to ensure adequate partitioning. cereals... CRL-SRM 65 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . Broaden matrix spectrum – Dry Commodities E.

Stuttgart. 06/12/2006 .5 g of homogenate Honey 5g 10 g Spices 2g 10 g CRL-SRM 66 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . Dry Commodities Sample type Weigh Water Annotation Fruit/Vegetables 10 g - (water >80 %) Fruit/Vegetables 10 g Xg X = 10 g – water (water 30-80 %) amount in 10 g sample Cereals 5g 10 g Dried fruits 5g 8.5 g Add water to comminute. weigh 13.

HCB and DDT)  Accessibility of residues may be limited (Ultra Turrax) CRL-SRM 67 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .g. 06/12/2006 .Stuttgart. Broaden matrix spectrum – Fatty commodities Commodities with a high lipid load. such as avocados or plant oils can be employed. Problems:  Co-extracted lipids should be removed prior to GC-analysis  Highly non-polar pesticides may give recoveries < 70% (e.

Recoveries of pesticides in high fat samples Recoveries 100 % 75 50 25 1g 2g 0 3g Hexachlorobenzene Chlorpyriphos-methyl HCH.Stuttgart. 06/12/2006 . for HCB 0.).4 g lipids are still OK (>70% rec. for Endosulfane 5 g (NOTE: In presence of water (ternary system) values are different.g.p' -DDE Fenthion Diazinon Malathion BS138 Carbaryl Dieldrin Trifluralin TPP g Oil / 10 mL ACN Values in absense of water PCB 138 or 153 may be used as surrogate QC standards Rec. less lipid is tolerable) Compromise for Oil samples: 2 g oil + 10 mL ACN • H C B a n d D D E g ive re co ve rie s < 7 0 % … CRL-SRM •1stbut 68 equilibrium is defined and recovery-correction is justified Community Reference Laboratory Pesticide Residues using Single Residue Methods Joint CRL-Workshop . gamma- Pirimiphos-methyl Endosulfan Deltamethrin Chlorpyriphos Cypermethrin p. > 70% will indicate that no unacceptable pesticide losses occurred The tolerable lipid-amount depends on the selection of pesticides to be covered e. for DDE 1 g.

9 from the extract than MgSO4. Thus interctions with the 104 Co-extractives [mg/ml] 25 21. 06/12/2006 .0 become stronger 15  Better Cleanup results 8.2 20 58 sorbent (H-binding.7 2.8 2.7 91 5 3.5 0 Cl2 aCl2 aCl2 PSA xt PSA PSA PSA PSA PSA PSA 50 Ca ectra 100 C 200 C 25 + 25 + 25 + 25 + 50 + 75 + 100 aw Cl2 + aCl2 aCl2 gSO4 gSO4 gSO4 Tea r gSO4 50 Ca 100 C 200 C 150 M 150 M 150 M 150 M CRL-SRM Problem with CaCl2: recoveries of polar pesticides drop 69 Jointif CRL-Workshop polar pesticides Community Reference Laboratory are not of interest CaCl2 / PSA is a serious cleanup Pesticide Residues option using Single Residue Methods 1st .5 0.2 1. Cleanup of Fermented Tea extracts Removal of Black-Tea Co-extractives Recoveries of 200 mg Tea/ml solvent Methamidophos 100 35 31.0 88 CaCl2 removes more water 30 27.Stuttgart.9 10 24 6. ionic) 14.

Stuttgart. 06/12/2006 using Single Residue Methods . Release of covalently bound phenoxy- acids by alkaline cleavage Alkaline Hydrolysis for the release of phenoxy-acid pesticides wheat sample 1400 At 70 °C At Room Temperature 1200 pH 12 pH 10 pH 10 pH 9 Relativ concentration 1000 30 min 60 min 30 min 30 min 800 715 606 619 600 336 400 275 200 135 100 0 in in n n n n 0m m mi mi mi mi l. /30 ro /30 /30 /60 /30 9/3 yd 12 12 10 10 10 oh pH pH pH pH pH pH w/ RS hE EC CRL-SRM Community Reference Laboratory Qu Pesticide Residues 70 1st Joint CRL-Workshop .

Impact of QuEChERS-Implementation More time for instrumental analysis More time for QA/QC (incl.Stuttgart. 06/12/2006 . validation) Broader analyte spectrum Higher sample throughput and turnaround time Less solvent consumption Less lab space needed (hoods are empty) Sample preparation more pleasant CRL-SRM 71 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

06/12/2006 .Stuttgart. Impact of QuEChERS-Implementation CRL-SRM 72 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

but low recoveries for very polar ones 485 265 Many more pesticides covered 325 215 330 215 10 200 Sum [ml] 10 Other solvents 65 50 0 0 Organochlorine solvents Becker. Mini Specht. S-19 CVUA-Method QuEChERS up to 1990 1990-96 1993-96 1996-2002 since 2002 CRL-SRM 73 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .000 € savings in 1 year just for solvent !! mL Solvent/Sample (for ca.Stuttgart. 06/12/2006 . Reduction of Solvent Consumption 15. S-8 Becker. 2000 samples) more pesticides than Becker 655 535 including basic & acidic pesticides.

Stuttgart. “M ass M ig ratio n ” o f P erso n n el CRL-SRM 74 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .

06/12/2006 . In 2004: 200 Pesticides in Total 149 (CVUA Stuttgart) 121 124 105 100 84 144 69 73 68 121 49 100 83 68 46 40 44 40 Fruits 37 Vegetables 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Nr. YES!! More finding s. of different pesticides detected in fruits and vegetables CRL-SRM 75 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop ..Stuttgart..

Stuttgart. Participation in EU-Proficiency Tests using the QuEChERS-Method CRL-SRM 76 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .

06/12/2006 . Citrate- 160 QuEChERS QuEChERS Buffered Buffered 140 Median of all (100-130) Labs QuEChERS QuEChERS 120 100 80 60 2002 2003 2004 2005 40 20 0 CRL-SRM 77 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Using QuEChERS in EU .Stuttgart.Proficiency Tests 180 Original Original Citrate.

Using QuEChERS in EU . 06/12/2006 .Proficiency Tests Unknown Pesticides and Unknown concentrations Participants: 100-130 EU-Official Labs Results:  All 57 identified (100%)  95% (54/57): within +/-30% from median concentration  82% (47/57): within +/-20%  53% (30/57): within +/-10% On average +8% above the median 2002 2003 2004 2005 CRL-SRM 78 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart.

QuEChERS Inter-Laboratory Validation Studies CRL-SRM 79 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .Stuttgart.

: 101% 100% 105% 102% 100% 100% RSD: 3% 4% 8% 9% 5% 3% LC (+) CRL-SRM LC (-) 80 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop . 06/12/2006 .025 0.25 0.25 0.025 0.Stuttgart.025 Rec.GC-MS and LC-MS/MS Inter-Laboratory Validation Study (GDCh) GC Mean Orange Orange Apple Apple Lettuce Lettuce 0.25 0.

06/12/2006 .1 0.1 0.01 0.: 97% 97% 98% 98% 96% 98% 95% 97% RSD 7% 8% 7% 7% 5% 8% 6% 7% Ethiofencarb was oxidized in cucumber SUs degraded CRL-SRM 81 in acidified extract Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .01 0.Stuttgart.1 0.1 0.01 0. LC-MS/MS Inter-Laboratory Validation Study (BLAPS-Working Group) Mean Cucumber Cucumber Lemon Lemon Wheat Wheat Raisins Raisins 0.01 Rec.

06/12/2006 .1 0.01 0.1 0.: 101% 98% 99% 94% 100% 101% 100% 97% RSD 3% 8% 7% 8% 4% 9% 6% 11% Oxidation Acids. lost in PSA cleanup Degraded in the standard solution provided CRL-SRM 82 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .Stuttgart.01 0.01 0.01 Rec.LC-MS/MS Inter-Laboratory Validation Study (BLAPS-Working Group II) Mean Cucumber Cucumber Lemon Lemon Wheat Wheat Raisins Raisins 0.1 0.1 0.

06/12/2006 .Multiresidue-Method Advantages  Rapid (8 Samples in Less Than 30 min)  Simple (No Laborious Steps. Materials for 1 mL Extract)  Low Solvent Consumption (10 mL Acetonitrile)  Practically no Glassware Needed  Wide Pesticide Range (Polar.Stuttgart.and LC-Amenable) CRL-SRM 83 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .QuEChERS. pH-Dependent Compounds)  Extract in Acetonitrile (GC. Minimal Sources of Errors)  Cheap (~1 € Sample Prep.

www.the modified QuEChERS method including all presented modifications and a lot of background information is available via the internet. 06/12/2006 .com ... as well as the validation data. www..quechers.Stuttgart.com CRL-SRM 84 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .QuEChERS.

06/12/2006 .Stuttgart. Thank you very much for your Attention ! CRL-SRM 85 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .

06/12/2006 . Pesticides Online Internet platform for Pesticide Residue Analysts www.com FEEL FREE TO REGISTER & TEST !! CRL-SRM 86 Community Reference Laboratory Pesticide Residues using Single Residue Methods 1st Joint CRL-Workshop .pesticides-online.Stuttgart.