QuEChERS Extracts to Give Improved Matrix Removal for Pesticide - - PowerPoint PPT Presentation

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QuEChERS Extracts to Give Improved Matrix Removal for Pesticide - - PowerPoint PPT Presentation

May 18, 2023 37 likes •265 views

Use of a Robotic Solid Phase Extraction Clean-up of QuEChERS Extracts to Give Improved Matrix Removal for Pesticide Residue Analyses by GC-MS/MS and LC-MS/MS. Bruce Morris*, Richard Schriner*, Rick Youngblood and Kim Gamble *RJ Hill

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Use of a Robotic Solid Phase Extraction Clean-up of QuEChERS Extracts to Give Improved Matrix Removal for Pesticide Residue Analyses by GC-MS/MS and LC-MS/MS.

Bruce Morris*, Richard Schriner*, Rick Youngblood† and Kim Gamble† *RJ Hill Laboratories, Hamilton, New Zealand.

†ITSP Solutions Inc., Hartwell, GA, U.S.A. www.itspsolutions.com

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Introduction

 Why did we look at a robotic column SPE alternative to commonly used dispersive SPE?  In our lab, QuEChERS for LC-MS/MS was successful, but less so for GC.

  • dSPE gave extracts that were poor for GC, with the samples we wanted to

analyse.

 Bench cartridge SPE gave better clean-up, but seemed impractical for 100s

  • f samples per day.

 With some experience in the use of CTC autosamplers, it seemed a robotic method could be the answer.  ITSP SPE cartridges became available for CTCs, and we started a collaboration developing these for QuEChERS.

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Robotic SPE clean-up Cartridges.

 A miniaturised SPE cartridge invented by ITSP Solutions.  Stationary phase mixtures co-developed and trialled for QuEChERS at Hill Labs.

  • 30 – 45mg of stationary phase, depending on mixture.

Cap/septum Stationary phase bed

3.5 cm

Needle guide

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Robotic SPE clean-up

  • CTC autosampler

 Robotized on a CTC autosampler

  • Hence ITSP “Instrument Top Sample Prep”, although it can also be done stand-alone
  • n a bench top.
  • Well-plate dimensions, although 2-mL vials used rather than wells.

Raw sample extracts Cartridge pre- rinse tray Load and elute tray Glass syringe

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Robotic SPE clean-up

  • Load and Elute tray

 Developed specifically for QuEChERS-ITSP.  Samples eluted into 2-mL vials in a 54-well tray, allowing volume for LC-buffer addition.  Tray cover aligns cartridges and allows them to be removed from vials after elution, for instrument injection

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ITSP-QuEChERS Procedure

QuEChERS extraction (20g for fresh fruit) LC-MS/MS GC-MS/MS LC-ITSP cartridge Aliquot taken off into 2-mL vial, ISTD added

Analytes eluted Extract loaded

Diluted in aqueous buffer for LC-MS/MS analysis. GC-ITSP cartridge Aliquot taken off into 2-mL vial, ISTD added Analyte protectants added for GC-MS/MS injection. Takes place on Load and Elute tray

Extract loaded Analytes eluted

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Cartridge Development

 LC-MS/MS cartridges. The aim was to remove non-polar extract matrix (oils/waxes).

  • Drop out of solution when diluted in aqueous buffer for injection.
  • Are retained on the LC column which becomes fouled.
  • Cause matrix suppression in high organic end of chromatogram, or in the next

injection.

Polar sugars/acids washed out at the start of the LC gradient, are not such a big problem.  GC-MS/MS cartridges. Wanted to remove extracted sugars, fatty acids, sterols and pigments.

  • Foul the inlet liner/pre-column, creating active sites.
  • Cause interferences or suppression in the chromatogram.

GC can cope with some higher MW oils/waxes, especially with backflushing.

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LC-MS/MS Cartridge Sorbent Trials.

 Various cartridge stationary phase mixtures were trialled with Avocado extract (oily).

  • 5g fruit/20mL acetonitrile, 150µL of extract loaded onto cartridges (slightly overloaded),

eluted with 150µL of acetonitrile. MgSO4/PSA/C-18/ CarbonX 45mg Z-Sep/C-18/ CarbonX 30mg Z-Sep+ 30mg Z-Sep 10mg Z-Sep/HLB/ CarbonX 20mg Raw extract PSA/C-18 /CarbonX Z-Sep/C-18 /CarbonX Z-Sep+ Z-Sep Z-Sep/HLB/ CarbonX

0.41 0.23 0.15 0.07 0.18 0.20

Dry weight Recovered (mg) Best oil removal, but lose many pesticides 2nd best oil removal, but better pesticide recoveries

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Recoveries off Trial Cartridges

Recovery of Triazoles (acetonitrile elution, solvent-only standards)

10 20 30 40 50 60 70 80 90 100

PSA/C18/Cx ZSep/C18/Cx Z-Sep+ Z-Sep HLB/ZSep/Cx cartridge packing % recovery

Azaconazole Cyproconazole Difenoconazole Epoxyconazole Flusilazole Flutriafol Hexaconazole Metconazole Myclobutanil Paclobutrazole Penconazole Propiconazole Tebuconazole Tetraconazole Triadimefon Triadimenol Uniconazole

Hexaconazole

  • highly retained

Z-Sep+

Tetraconazole

  • lesser retention

pKa=2.3

Pesticide spike on Avocado extract.

▪ For Z-Sep+, 68 out of 266 pesticides lost

(< 70% recovery).

▪ Z-Sep/C-18: 40 analytes < 70% recovery. ▪ As show by 17 triazole fungicide recoveries.

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Z-Sep/C-18 ITSP : Effect of Buffer Elution.

LC-ITSP: Triazole Recoveries (Lemon). Cyproconazole Cyproconazole Flutriafol Flutriafol Hexaconazole Hexaconazole 20 40 60 80 100 acetonitrile elution buffer elution % recovery

 Z-Sep/C-18/Cx was chosen as the best compromise between retention of oils and recovery of analytes.  However 40 compounds were still “lost”, so we tried elution with formate buffer.  This improved recoveries of e.g. triazole fungicides, macrocyclic lactones, fenhexamid.  However, buffer can push some matrix oils off, so have to be careful with elution volumes.

LC-ITSP: Macrocyclic Lactone Recoveries (Lemon) Emamectin B1a Milbemectin Spinetoram Spinosad Abamectin Abamectin Emamectin B1a Milbemectin Spinetoram Spinosad 20 40 60 80 100 acetonitrile elution buffer elution % recovery

Fenhexamid (anilide fungicide) Phenolic –OH interacting with Z-Sep?

LC-ITSP: Fenhexamid Recovery (Lemon) 20 40 60 80 100 acetonitrile elution buffer elution % recovery

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Spike recoveries - LC-ITSP compared with Z-Sep/C-18 dSPE (buffer elution, solvent only standards)

20 40 60 80 100 120 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

retention time (min) % recovery

LC-ITSP dSPE

Z-Sep/C-18 : dSPE vs. LC-ITSP - Avocado

 LC-ITSP with buffer elution, only 4 out of 272 analytes have < 50% recovery.

  • e.g. Pymetrozine with poor extraction recovery. Solutions are clear.

Pymetrozine Halfenprox Flumethrin

Drop-out zone  dSPE, with buffer added, significant amounts of matrix oils are pushed off, giving cloudy solutions, resulting in drop-out of non-polar analytes.

  • Out of 272 analytes, 19 with retention times > 12 minutes (logPow≳ 5) have < 50% recovery.

dSPE

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LC-ITSP Citrus Percent Spike Recoveries vs. Percent Standard Deviation (solvent-only standards)

10 20 30 40 50 60 70 80 20 40 60 80 100 120 140 160 180 200 % spike recovery % standard deviation

LC-ITSP spike recoveries for Citrus.

 282 Pesticides (incl. metabolites/homologues) analysed by LC-MS/MS, shown in the plot below.

DDAC Sulfentrazone Thiabendazole Anilazine Forchlorfenuron

 16 falling outside 70-120% recovery and 40% standard deviation (N = 10).

  • Low recoveries - during extraction, and/or some retention on Z-Sep/C-18/CarbonX, however highly

reproducible with ITSP (e.g. Anilazine, Forchlorfenuron, Thiabendazole).

  • High recoveries (e.g. DDAC, Sulfentrazone), due to losses in solvent-only calibration standards.

Carbendazim 77%, 5% SD

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Cartridge Development

 LC-MS/MS cartridges. The aim was to remove non-polar extract matrix (oils/waxes).

  • Drop out of solution when diluted in aqueous buffer for injection.
  • Are retained on the LC column which becomes fouled.
  • Cause matrix suppression in high organic end of chromatogram, or in the next

injection.

Polar sugars/acids washed out at the start of the LC gradient, are not such a big problem.  GC-MS/MS cartridges. Wanted to remove extracted chlorophyll/pigments, sugars, fatty acids, sterols, HCs.

  • Foul the inlet liner/pre-column, creating active sites.
  • Cause interferences or suppression in the chromatogram.

GC can cope with some higher MW oils/waxes, especially with backflushing.

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GC-ITSP - Chorophyll removal

 GC-ITSP stationary phase mixture uses the same sorbents as standard QuEChERS dSPE.

  • PSA/C-18/MgSO4 with CarbonX for chorophyll removal.

Spinach Extract

Raw extract GC-ITSP cartridge dSPE -contents

  • f GC-ITSP

cartridge used Extract after dSPE Extract after GC-ITSP Chlorophyll retention seen on top of bed

 CarbonX appears to be more effective in ITSP format than in dSPE.

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Recovery of Planar Aromatics off ITSP

GC-ITSP Spike Recoveries of Planar Pesticides (Persimmon)

20 40 60 80 100 120

c h

  • r
  • t

h a l

  • n

i l d e l t a m e t h r i n h e x a c h l

  • r
  • b

e n z e n e c y p r

  • d

i n i l c

  • u

m a p h

  • s

% recovery

 Using CarbonX, recovery of planar aromatic pesticides has been acceptable, without toluene eluent.

LC-ITSP Spike Recoveries of Planar Pesticides (Persimmon)

20 40 60 80 100

t h i a b e n d a z

  • l

e d e l t a m e t h r i n c y p r

  • d

i n i l c a r b e n d a z i m c

  • u

m a p h

  • s

% recovery

Chlorothalonil Hexachlorobenzene Cyprodinil Carbendazim Coumaphos

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 10g fruit/20mL acetonitrile, GC-ITSP compared with PSA/C-18 dSPE.

  • Full-scan GC-MS chromatograms show improved removal of oils/tocopherols/sterols

compared with dSPE (same sorbent mixture and loading).

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 2000000 4000000 6000000 8000000 1e+07

1.2e+07

1.4e+07

Sitosterol Campesterol Tocopherols Squalene Eicosanoic acid (C20) Ferulic acid Quinic acid Allose 2-methoxy-5-vinylphenol Catechol PSA/C-18 ITSP PSA/C-18 dSPE Raw extract

GC-ITSP clean-up of Blueberry extract.

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 2000000 4000000 6000000 8000000 1e+07

1.2e+07

1.4e+07

Sitosterol Campesterol Tocopherols Squalene Eicosanoic acid (C20) Ferulic acid Quinic acid Allose 2-methoxy-5-vinylphenol Catechol PSA/C-18 ITSP PSA/C-18 dSPE Raw extract

 10g fruit/20mL acetonitrile, GC-ITSP compared with PSA/C-18 dSPE.

  • Full-scan GC-MS chromatograms show improved removal of oils/tocopherols/sterols

compared with dSPE (same sorbent mixture and loading).

contamination

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www.itspsolutions.com GC-ITSP Citrus Percent Spike Recoveries vs. Percent Standard Deviations (solvent-only standards)

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 20 40 60 80 100 120 140 160 % spike recovery % standard deviation

GC-ITSP spike recoveries for Citrus.

 228 Pesticides analysed by GC-MS/MS, in plot below.

Fenpropimorph Imazalil Etridiazole Naled

 10 falling outside 70-120% recovery and 40% standard deviation (N = 10).

  • Those with some retention on the cartridge (e.g. imazalil), show highly reproducible recoveries.
  • Poor reproducibility is due to variable breakdown or losses (e.g. naled, etridiazole).
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Summary

 Robotic clean-up means high-throughput, low labor input, and high reproducibility.  Column SPE format means improved clean-up compared with dispersive SPE.

  • Desirable for more difficult samples, reducing instrument fouling (esp. for GC).
  • Can generally use solvent-only calibration standards, due to fewer matrix effects.
  • Reduces MS/MS interferences and oil drop-out in-vial (for LC).
  • Avoids dSPE stationary phase transfer into analysis vials (ends up in LC injector or in-

line filter).

 Careful choice of stationary phase mixtures and elution solvents allows an extended suite of pesticides to be recovered, while retaining effective matrix removal.

  • Chlorophyll, pigments, sugars, sterols, oils/fatty acid removal, keeps columns and

inlets clean!

 SPE integrated into the instrument cycle and takes place between injections.

  • Uses idle autosampler time (takes ~7 minutes per clean-up).
  • Gives freshly cleaned up extracts, with reduced potential for analyte breakdown,

especially for LC with the addition of aqueous buffer.

 Has proven to be reliable and robust in a commercial laboratory environment.

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References

 AOAC Official Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate.  EN15662:2008, Foods of plant origin – Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and cleanup by dispersive SPE – QuEChERS-method.  Increased Removal of Fat and Pigment from Avocado Extracts Prior to GC-MS Analysis of Pesticide and Metabolite Residues. Katherine K. Stenerson and Jennifer Claus, Reporter US Volume 31.2.  EURL-FV(2013-M11). Determination of pesticide residues in avocado and almond by liquid and gas chromatography tandem mass spectrometry (http://www.eurl-pesticides.eu/userfiles/ file/EURL-FV%20(2013-M11)Determination%20of%20pesticide%20residues%20in%20high% 20oil%20vegetal%20commodities.pdf).  Novel Porous Carbon Sorbent Materials for Use in Sample Preparation. Dwight Stoll, David C. Harmes, Jon Thompson, Doug Fryer, Conor Smith, and Bill Barber. EAS poster presentation,

  • 2012. (http://uniscicorp.com/wp-content/uploads/2012/12/2012-Quechers-EAS-Presentation-

v61.pdf)

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