Analysis of Multi-Pesticide Residues in Vegetables, Food ... · Analysis of Multi-Pesticide Residues in Vegetables, Food, and Fruits by ... critical step for both broad-spectrum screening

SUPELCO, 595 North Harrison Road, Bellefonte, PA 16823

Analysis of Multi-Pesticide Residues in Vegetables, Food, and Fruits by SPE/GC-MS

Michael Ye, Yuhui Yang, and An Trinh

T405020HSV

IntroductionCleanup of matrices is the first, the most important, and the most critical step for both broad-spectrum screening and accurate determination of pesticides and their metabolites in vegetables,meats, drinks and fruits. In most cases, solid phase extraction (SPE) methods are used to clean up the extracts before the GC-MS analysis. The ideal SPE material(s) should remove the greatest number of food matrix interferences and offer truly high recoveries for a broad-spectrum of pesticides.

Glass beads, polymeric sorbents, florisil, alumina, silica, C18 and charcoal, although useful for particular classes of pesticides, were found to be inadequate for trapping diverse pesticides.

Luke method, using strong and weak anion-exchange SPE materials, requires the use of a large amount of chlorinated organic solvents, and has inability for the samples for high amount of fatty acids. ENVI-Carb/NH2 dual layer SPE, although useful for extensive pesticide screening, were found to be also inadequate for removal of fatty acids.

Introduction (contd.)

Primary Secondary Amine (PSA) has been found as the most effective sorbent for removal of various matrices and significantly reducing matrix- enhancement effect. On the other hand, graphitized carbon black (GCB) is very useful for removal of coloring substances (i.e. pigments) and sterols. Therefore, PSA itself and/or combination of GCB/PSA dual layer should be most effective for sample cleanup.

Si-CH2CH2CH2NHCH2CH2NH2

Two pKa: at 10.1 and 10.9

Problems with Current PSA and Carbon/PSA SPE Products

• Low capacity and phase bleeding.

• Poor lot-to-lot reproducibility for removal of color.

• Low recoveries on difficult pesticide, such as acephate.

• Several different elution protocols, particularly by acetone:hexane (1:1) or acetonitrile:toluene (3:1), has been extensively used. But their merits and disadvantages have been not revealed clearly.

Experiments and ResultsProperties of Improved PSA and ENVI-Carbon

PSA• PSA bonded on spherical

silica (70Å, 60 µm)

• Ion exchange capacity: 0.96 mmole/g

• PSA bleed: 0 – 1.2 µg/g

• Removal of oleic acid: 224 mg/g

ENVI-Carbon• Non porous

• Granular graphitized carbon 125 µm

• Surface area: 100 m2/g

• Density: 1.8 g/mL

Comparison of PSA from Several Vendors

Items Vendor A Vendor B Vendor C SupelcleanTM

Ion-exchange capacity (mmol/g) 0.65 0.92 0.72 0.96

PSA bleed (µg/g) (1) 2.7 - 5.8 0 - 5.4 2.4 - 7.2 0 - 1.2

Removal ofoleic acid 148 198 202 224(mg/g)

(1) Salicylaldehyde was used as a probe to detect the PSA silane bleeding from SPE cartridges.

PSA Plays a Vital Role for Removal of Fatty AcidsUsing acetonitrile:toluene (3:1) as elution solvent, PSA (500 mg) removes 57.8 mg oleic acid while the same amount of ENVI-Carbremoves only 1.6 mg oleic acid.

Removal of Oleic Acid by 500 mg PSA and 500 mg ENVI-Carb

0

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PSA ENVI-CarbAmou

nt o

f ole

ic a

cid

rem

oved

(mg)

Removal of oleic acid

ENVI-Carb Plays a Vital Role in Color Removal

0: Spinach extracted by acetonitrile without SPE cleanup

1: Typical results with ENVI-Carb/PSA (500 mg/400 mg) cleanup

2: Cleaned by one lot of carbon/PSA from one vendor

3: Cleaned by another lot of carbon/PSA from the competitor

GC-MS Background of PSA SPE CartridgesAn ideal SPE cartridge should not bring additional impurities in the final elution.

Experimental: 500 mg/6 cc PSA tube was conditioned with 5 mLacetonitrile and then eluted with 14 mL acetonitrile. The elution was collected, concentrated and re-constituted into 1.0 mLacetone:hexane for GC-MS total ion scanning.

Vendor B

SupelcleanTM PSA

12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.000

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Abundance

TIC: 1001002.DTIC: 1001004.D (*)

GC-MS Background of Carbon/PSA Dual Layer SPE Products

Experimental:Carbon/PSA dual-layer SPE cartridges were conditioned with 5mL acetontrile:toluene (3:1) and eluted with 20mL of the same solvent. The eluatewas dried and re-constituted into1.0 mL acetone:hexane (1:1) for GC-MS total ion scanning. The bump between 24 min and 26 min may be from column bleeding rather than SPE materials themselves.

Vendor C

ENVI-Carb/PSA

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.000

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Abundance

TIC: 1001007.DTIC: 1001009.D (*)

ENVI-Carb/PSA on Orange Juice Matrices Clean-Up

Extraction and Cleanup of Florida Orange Juice:

6.00 8.0010.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.000

1000000

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1e+07

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TIC: OJ_DS_SC.DTIC: OJ_GL_SC.D (*)

Vendor C

ENVI-Carb/PSA

1. 50 mL of the orange juice was extracted with 100 mLacetonitrile by PMRA method.

2. Carbon/PSA cartridges were first conditioned with 5 mLacetonitrile:toluene (3:1). Then 10 mL acetonitrile extract was loaded, followed by 20mL acetone:hexane elution (1:1). The elution was concentrated and re-constituted into 1.0 mLacetone:hexane (1:1) for GC-MS analysis.

Recoveries of Metamidophos from PSAExperimental:Supelclean PSA SPE tube: 500 mg, 6 mL; condition and elution with acetonitrile; sample: 1 mL (1 ppm) in acetonitrile.

Metamidophos recovery as the function of acetonitrile elution volume

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0 5 10 15 20

Elution volume (mL)

Met

amid

opho

s re

cove

ry (%

)

Supelco PSAVendor AVendor B

Metamidophos has a full recovery from Supelclean PSA. On the other hand, its recovery is only 67.2% on Vendor A, and 46.2% on Vendor B.

CH3O P

O

SCH3

NH2

Recoveries of Acephate from PSA

Experimental:SPE tube: 500 mg, 6 mL cartridge; condition and elution with acetonitrile; sample: 1 mL (1 ppm) in acetonitrile.

Acephate recovery as the function of acetonitrile elution volume

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Elution volume (mL)

Ace

phat

e re

cove

ry (%

)

SupelcoVendor AVendor B

Less volume of acetonitrile is required to fully desorb acephate from SupelcleanPSA.

CH3S P NHCCH3

O

OCH3

O

Matrix-Induced Effect on the Desorptionof Acephate from ENVI-Carb/PSAAlthough it is recommended to collect 25 mL acetonitrile:toluene (3:1) elution, matrices compete with acephate for active sites on PSA and ENVI-Carb and therefore generally enhance the desorption of acephate and other pesticides.

Acephate recovery as the function of acetonitrile:toluene (3:1) elution volume

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Elution volume (mL)

Reco

very

(%)

Without matrixOrange juiceBacon

Generic Elution Protocol with ENVI-Carb/PSA Dual Layer SPE Cartridges

1. Pre-condition the cartridge 5 mL acetonitrile:toluene (3:1)2. Load the sample3. Elute with 25 mL acetontrile:toluene (3:1)4. Collect the eluant5. Concentrate the eluant down to 1 mL

Why Not to Choose Acetone:Hexane?

1. Acetone:hexane eluate has much higher GC-MS background. Acetone may react with PSA.

2. PSA’s capacity for removal of fatty acids is severely damaged.

3. Dichlorvos, quintozene, chlorothalonil, tolyfluanid and captafolhave lower recoveries.

Solvent Effect on PSA Capacity of Fatty Acids

Supelclean PSA 500 mg/6 cc 1. Conditioned and eluted with

acetonitrile.

2. Conditioned and eluted with acetonitrile:toluene (3:1).

3. Conditioned and eluted with acetone:hexane (1:1).

4. Preconditioned by acetonitrile:toluene:acetic acid (74:25:1), then conditioned and eluted by acetonitrile:toluene (3:1).

Solvent Effect on PSA Capacity

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1 2 3 4Amou

nt o

f ole

ic a

cid

rem

oved

by

500

mg/

6cc

PS

A ca

rtrid

ge

(mg)

Removal of oleic acid by PSA

Recoveries of Sixteen Pesticides from Milk

Methyl parathionMethyl primophos

Cypermethrin-3Difenoconazole-1Difenoconazole-2Imibenconazole

132160142

9114893

132

104

827564808333777288

104103114107

107100107104

91997999

Compound

DichlorvosAcephate

Pesticide Recovery fromENVI-Carb/PSA (%)

Pesticide Recovery fromCompetitor (%)

988394

Metamidophos

DDE

TrifluralinDiazinon

DimethipinVinclozoline

Triadimenol-1

Chlorothalonil

A Typical GC-MS Selective Ion Chromatogram

Conclusions

• The improved PSA showed clean GC-MS background, low bleeding, high capacity for fatty acids, and powerful for removal of food matrices.

• The PSA ensures full recoveries for almost all the difficult pesticides reported in the literatures.

• The ENVI-Carb/PSA dual-layer SPE cartridges can be operated under gravity flow.

• Acetonitrile:toluene (3:1) is recommended as the generic protocol

• The improved ENVI-Carb/PSA dual-layer cartridges are a reliable SPE product for cleanup of food matrices and determination of multi-residue pesticides.

Acknowledgement

We thank Ms. Katherine Stevenson and Mr. Chuck Germek of Supelco for GS-MS analyses. We also thank Dr. Steven J. Lehotay and Dr. Katerina Mastovska from US Department of Agriculture, Eastern Regional Research Center for fruitful discussions.