Hill Laboratory NACRW Presentation July 2014

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


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

of 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.


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


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 on 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


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


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.

Ta

ke

s p

lace

on

Lo

ad

an

d E

lute

tra

y

Extract loaded

Analytes eluted


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.


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


Recoveries off Trial Cartridges

Recovery of Triazoles (acetonitrile elution, solvent-only

standards)

0

10

20

30

40

50

60

70

80

90

100

PSA/C18

/Cx

ZSep/C

18/C

x

Z-Sep

+

Z-Sep

HLB

/ZSep

/Cx

cartridge packing

% r

ec

ov

ery

Azaconazole

Cyproconazole

Difenoconazole

Epoxyconazole

Flusilazole

Flutriafol

Hexaconazole

Metconazole

Myclobutanil

Paclobutrazole

Penconazole

Propiconazole

Tebuconazole

Tetraconazole

Triadimefon

Triadimenol

Uniconazole

Hexaconazole -highly retained

Z-S

ep+

Tetraconazole -lesser retention

Fenhexamid

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.


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

LC-ITSP: Triazole

Recoveries (Lemon).

Cypro

conazole

Cypro

conazole

Flu

tria

fol

Flu

tria

fol

Hexaconazole

Hexaconazole

0

20

40

60

80

100

acetonitrile

elution

buffer

elution

% r

eco

very

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)

Em

am

ectin

B1a

Milb

em

ectin

Spin

eto

ram

Spin

osad

Abam

ectin

Abam

ectin

Em

am

ectin

B1a

Milb

em

ectin

Spin

eto

ram

Spin

osad

0

20

40

60

80

100

acetonitrile elution buffer elution

% r

eco

very

Fenhexamid

(anilide fungicide)

Phenolic –OH

interacting with

Z-Sep?

LC-ITSP: Fenhexamid

Recovery (Lemon)

0

20

40

60

80

100

acetonitrile

elution

buffer elution

% r

eco

very


Spike recoveries - LC-ITSP compared with Z-Sep/C-18 dSPE

(buffer elution, solvent only standards)

0

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)

% r

ec

ov

ery

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. dS

PE


LC-ITSP Citrus Percent Spike Recoveries vs. Percent Standard Deviation

(solvent-only standards)

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120 140 160 180 200

% spike recovery

% s

tan

da

rd d

ev

iati

on

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


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.


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

of 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.


Recovery of Planar Aromatics off ITSP

GC-ITSP Spike Recoveries of

Planar Pesticides (Persimmon)

0

20

40

60

80

100

120

chor

othalo

nil

delta

met

hrin

hexa

chloro

benz

ene

cypr

odinil

coum

aphos

% r

ec

ov

ery

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

LC-ITSP Spike Recoveries of

Planar Pesticides (Persimmon)

0

20

40

60

80

100

thiabe

ndaz

ole

delta

met

hrin

cypr

odinil

carb

endaz

im

coum

aphos

% r

eco

very

Chlorothalonil

Hexachlorobenzene

Cyprodinil

Carbendazim

Coumaphos


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

0

2000000

4000000

6000000

8000000

1e+07

1.2e+07

1.4e+07

1.6e+07

1.8e+07

Time-->

Abundance

TIC: BB RAW.D

TIC: BB DSPE.D (*)

TIC: BB ITSPB.D (*)

TIC: BB LCA2.D (*)

Sit

ost

ero

l C

am

pest

ero

l

Tocophero

ls

Squale

ne

Eic

osa

noic

acid

(C

20)

Feru

lic a

cid

Quin

ic a

cid

All

ose

2-m

eth

oxy-5

-vin

ylp

henol

Cate

chol

Z-Sep/C-18 ITSP

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

0

2000000

4000000

6000000

8000000

1e+07

1.2e+07

1.4e+07

1.6e+07

1.8e+07

Time-->

Abundance

TIC: BB RAW.D

TIC: BB DSPE.D (*)

TIC: BB ITSPB.D (*)

TIC: BB LCA2.D (*)

Sit

ost

ero

l C

am

pest

ero

l

Tocophero

ls

Squale

ne

Eic

osa

noic

acid

(C

20)

Feru

lic a

cid

Quin

ic a

cid

All

ose

2-m

eth

oxy-5

-vin

ylp

henol

Cate

chol

Z-Sep/C-18 ITSP

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


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

0 20 40 60 80 100 120 140 160

% spike recovery

% s

tan

da

rd d

ev

iati

on

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).


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.


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)

Hill Laboratory NACRW Presentation July 2014

Food

Transcript of Hill Laboratory NACRW Presentation July 2014