• Application Note

A Rapid Method for the Screening and Confirmation of Over 400 Pesticide Residues in Food

A Rapid Method for the Screening and Confirmation of Over 400 Pesticide Residues in Food

  • James Morphet
  • Peter Hancock
  • Waters Corporation

Abstract

To utilize the power of UltraPerformance Liquid Chromatography (UPLC) combined with fast MS acquisition rates, to give a rapid method for the screening of 402 pesticide residues in a single 10 minute run. A second injection, for confirmatory purposes, will meet SANCO Analytical Quality Control procedures for pesticide residue analysis (SANCO/2007/31311).

Introduction

Pesticides are widely used in the production of foodstuffs to meet consumer demand for plentiful food at reasonable prices, all year round. However, continued growth in the use of pesticides, poor agricultural practices, and illegal use can pose significant risks to human health through the presence of pesticide and metabolite residues in food products. Most countries have strict regulations governing pesticides. Legislation imposes Maximum Residue Limits2 (MRLs) for pesticide residues in food products requiring analytical techniques that are sensitive, selective, and robust.

Multi-residue pesticide analysis is challenging due to the low levels present, the wide variety of pesticides, and the very different chemical classes they represent. As there are currently well over 1,000 pesticides in use, laboratories are under increasing pressure to broaden the range of pesticides determined in a single analysis over a shortened run time.

The need to meet mandated detection limits, develop generic sample preparation techniques for complex matrices, and the desire to increase sample throughput are the main challenges facing food safety testing laboratories today. The use of a single multi-residue method per instrument can dramatically improve return on investment by removing the need to change method parameters. This is often the case when analyzing a wide variety of commodities with differing lists of legislated pesticides.

Advances in chromatographic separation and detection technologies have enabled analysts to increase the number of analytes determined in a single run. Tandem quadrupole mass spectrometry offers a highly specific and selective detection technique that has become the technique of choice within the laboratory.3

The following method describes a solution for the rapid analysis of pesticides in mango, avocado, and fruit-based baby food extracts that is able to exceed current worldwide legislation.

Experimental

Dispersive SPE, commonly referred to as “QuEChERS”, is a simple and straightforward sample preparation technique suitable for multi-residue pesticide analysis in a wide variety of food and agricultural products.4 The homogenized food samples were extracted with organic solvent using Waters DisQuE dispersive sample preparation tubes. Once mixed, the pesticide residues were partitioned into the organic solvent, which was then subjected to further clean-up. The supernatant was collected, diluted, and injected onto the LC-MS/MS system as described below:

Extraction Procedure4:

  1. Add 15 g homogenized sample to a 50-mL DisQuE extraction tube containing 1.5 g sodium acetate and 6 g magnesium sulfate. Add 15 mL 1% acetic acid in acetonitrile.
  2. Add any pre-extraction internal standards.
  3. Shake vigorously for one minute and centrifuge > 1500 rcf for one minute.
  4. Transfer 1 mL of the acetonitrile extract in to the 2-mL DisQuE extraction tube containing 50 mg PSA and 150 mg of magnesium sulphate.
  5. Shake for 30 seconds and centrifuge >1500 rcf for one minute.
  6. Transfer 100 μL of final extract into an autosampler vial. Add any post-extraction internal standards. Dilute with 900 μL water.

 

Chromatographic conditions

LC system:

Waters ACQUITY UPLC System

Column:

ACQUITY UPLC BEH C18 2.1 x 100 mm, 1.7 μm

Column temp:

40 ˚C

Sample temp:

4 ˚C

Flow rate:

0.450 mL/min.

Mobile phase A:

98:2 water: methanol + 0.1% formic acid

Mobile phase B:

Methanol + 0.1% formic acid

Weak needle wash:

98:2 water: methanol + 0.1% formic acid

Strong needle wash:

Methanol + 0.1% formic acid

Total run time:

10 min

Injection volume:

20 μL, full loop injection

Gradient:

Time(min)

Composition

0.00

95% A

0.25

95% A

7.75

0% A

8.50

0% A

8.51

95% A

MS conditions

MS system:

Waters ACQUITY TQ Detector

Ionization mode:

ESI positive polarity

Capillary voltage:

1 kV

Desolvation gas:

Nitrogen, 800 L/Hr, 400 ˚C

Cone gas:

Nitrogen, 5 L/Hr

Source temp:

120 ˚C

Acquisition:

Multiple Reaction Monitoring (MRM)

Collision gas:

Argon at 3.5 x 10-3 mBar

Acquisition and Processing methods

The data were acquired using Waters MassLynx Software, v. 4.1. Incorporated into MassLynx, the IntelliStart technology automates optimization of MS parameters for the sample and also monitors the health of the MS system, reducing the time for operator-intensive troubleshooting and upkeep.

This data was processed using TargetLynx Application Manager. This quantification package enables automated data acquisition, processing, and reporting for quantitative data, incorporating a range of confirmatory checks that identify samples that fall outside user-specified or regulatory thresholds.

Results and Discussion

The analysis of 402 pesticide residues (Appendix 1) in mango, avocado, and fruit-based baby food was achieved using ACQUITY TQD: liquid chromatography combined with tandem quadrupole mass spectrometry (UPLC-MS/MS) operated in Multiple Reaction Monitoring (MRM) mode.

The rapid determination and confirmation method was achieved in two parts. Part one was a single injection with one MRM transition per pesticide, ideal for screening purposes. Part 2, where compounds of interest can then be confirmed, was achieved by two separate injections with two MRM transitions per pesticide.

Figure 1 shows all 402 pesticide residues in one 10 minute run, fully utilizing the enhanced speed and resolution of UPLC.

Figure 1. Chromatogram showing all 402 pesticide residues in one 10 minute run in injection solvent.

For all injections, the same UPLC conditions were used saving analytical time and costs, thus maximizing return on investment. This single setup will allow analysts with less experience to run the method as the need for changes to be made in between batches is removed.

The IntelliStart technology provides simple instrument setup and MS method development and therefore easy access even for the most inexperienced MS user.

Part 2, where compounds of interest can then be confirmed, was achieved by two separate injections with two MRM transitions per pesticide. Figures 2 and 3 show the separation of 201 pesticide residues across two run times of 10 minutes each.

Figure 2. Chromatogram showing first 201 pesticide residues at 10 μg/kg in injection solvent.
Figure 3. Chromatogram showing second 201 pesticide residues at 10 μg/kg in injection solvent.

The selectivity given using a tandem quadrupole mass spectrometer (ACQUITY TQD) shows an advantage over a single quadrupole instrument as it allows co-eluting compounds to be identified and quantified with confidence.

The enhanced speed and resolution of UPLC enabled all peaks to elute within eight minutes. Dwell times of 5 ms were used to achieve at least 12 data points across each peak for both quantification and confirmatory ions.

A calibration curve was prepared in the injection solvent (water:methanol, 90:10 v/v) and injected. Excellent linearity was achieved using a weighting factor of 1/x with a high coefficient of determination achieved. This is shown in Figure 4.

Figure 4. TargetLynx view showing a solvent standard calibration curve over a linear range from 0.02-2 pg/μL. The highlighted chromatogram is at 0.02 pg/μL.

The 402 pesticide mix was spiked into the three matrices and the extracts analyzed. Figures 5, 6, and 7 show pesticides at 10 μg/kg, equivalent to the lowest worldwide (EU) legislation, in mango, avocado, and fruit-based baby food extracts respectively.

Figure 5. Five pesticides in mango extract at 10 μg/kg.
Figure 6. Five pesticides in avocado extract at 10 μg/kg.
Figure 7. Five pesticides in fruit-based baby food extract at 10 μg/kg.

The advantage of using ACQUITY TQD is that ion ratio confirmation is also possible. This is used to confirm the identity of any pesticide that was presumptive positive from the screening method. Within the EU, ion ratio confirmation is important for pesticide analysis as documented in SANCO/2007/31311.

In Part 2, the confirmatory runs, all 402 pesticides were chromatographed with both primary (for quantitation) and secondary (for confirmation) MRM transitions present. Figure 8 shows three more compounds in the three matrices with both MRM transitions.

Figure 8. Confirmation through secondary MRM transition using ACQUITY TQD
at 10 μg/kg in matrix. The ion ratios for carbendazim, primiphos ethyl, and
flufenacet are 0.16, 0.18 and 0.61 respectively.

Conclusion

A rapid multi-residue method was developed for the screening of over 400 pesticides in one 10 minute run with one MRM transition per pesticide. For confirmation, two 10 minute runs were required with two MRM transitions per pesticide. The analysis of pesticides in mango, avocado and fruit-based baby food extracts was able to exceed current worldwide legislated limits.

Improved efficiency and increased sample throughput were realized through the combination of powerful UPLC and fast MS acquisition technologies. The Waters ACQUITY TQD as shown in Figure 9 offers:

  • Enhanced chromatographic resolution and short analysis times
  • Incorporation of confirmatory MRM traces
  • Compliance with legislative regulations such as SANCO
  • IntelliStart technology is designed to reduce the burden of complicated operation, training new users, time-intensive troubleshooting, and upkeep
  • The small footprint of the ACQUITY TQD will give any laboratory an advantage as it removes the need for larger instrumentation. 
Figure 9. ACQUITY TQD.

The benefits of this Waters UPLC-MS/MS solution for a revenue conscious laboratory can be realized through increased efficiency through analytical time savings and decreased need for sample retesting, resulting in increased lab productivity. Cost savings can be made by lowering the use of lab consumables with the environmental impact of solvent usage also being reduced.

The sensitivity achieved for a large number of pesticide residues in real food matrices indicates this UPLC-MS/MS method is an ideal basis for the rapid analysis of pesticides in a wide range of food samples.

References

  1. Website: http://ec.europa.eu/food/plant/protection/resources/qualcontrol_en.pdf
  2. Commission of the European Communities EC 396/2005, OJ 2005; L70:1.
  3. Leandro C.C., Hancock P., Fussell R.J., Keely B.J., J. Chrom A 2007; 1144:161.
  4. Lehotay, J.AOAC Int. 90(2) 2007, 485-520.

Acknowledgements

The authors would like to thank Central Science Laboratory (CSL), Sand Hutton, York, UK and VWA, Amsterdam, The Netherlands for kindly supplying MRM transitions and standard solution mixes that were analyzed in this project. Furthermore, University of Jaume I Castellon, Spain, Waters UK, Waters US and Nihon Waters, Japan demonstration laboratories are all thanked for their contributions in supplying MRM transitions.

Appendix: List of the 402 pesticides analyzed

3,4,5-Trimethacarb”

Dibrom

Indoxacarb

Propanil

Acephate

Dichlofluanid

Iodosulfuron methyl

Propaquizafop

Acetamiprid

Dichlorvos

Iprobenphos

Propazine

Acibenzolar-S-methyl

Diclobutrazol

Iprovalicarb

Propetamphos

Acitidone

Dicrotophos

Isazophos

Propham

Aldicarb

Diethofencarb

Isocarbamide

Propiconazole

Aldicarb sulfone

Difenoconazole

Isocarbofos

Propoxur

Aldicarb sulfoxide

Difenoxuron

Isofenphos

Propyzamide

Ametryn

Diflubenzuron

Isomethiozin

Prosulfocarb

Amidosulfuron

Dimefuron

Isonoruron

Prosulfuron

Aminocarb

Dimepiperate

Isoprocarb

Pymetrozine

Amitrole

Dimethachlor

Isopropalin

Pyracarbolid

Anilazine

Dimethametryn

Isoproturon

Pyraclostrobin

Anilofos

Dimethenamid

Isoxaben

Pyrazophos

Asulam

Dimethirimol

Kresoxim-methyl

Pyrazosulfuron-ethyl

Atraton

Dimethoate

Lenacil

Pyridaben

Atrazine

Dimethomorph

Linuron

Pyridafol

Atrazine-desethyl

Dimetilan

Malaoxon

Pyridaphenthion

Atrazine-desisopropyl

Dimoxystrobin

Malathion

Pyridate

Azaconazole

Diniconazole

Mecarbam

Pyrifenox

Azamethiphos

Dioxacarb

Mefenacet

Pyrimethanil

Azinphos-ethyl

Diphenamid

Mepanipyrim

Pyriproxifen

Azinphos-methyl

Diphenylamine

Mephosfolan

Pyroquilon

Aziprotryne

Disulfoton

Mepronil

Quinalphos

Azobenzene

Disulfoton-sulfone

Mesosulfuron-methyl

Quinmerac

Azoxystrobin

Disulfoton-sulfoxide

Mesotrione

Quinoxyfen

Benalaxyl

Ditalimfos

Metalaxyl

Quizalofop-ethyl

Benazolin

Dithiopyr

Metamitron

Quizalofop-methyl

Bendiocarb

Diuron

Metazachlor

Rabenzazol

Benfuracarb

DMST

Metconazole

Rotenone

Benfuresate

Dodemorph

Methabenzthiazuron

Sebuthylazin

Bensulfuron methyl

Edifenphos

Methacrifos

Sebuthylazin-desethyl

Bensulide

Epoxiconazole

Methamidophos

Secbumeton

Bentazone

EPTC

Methfuroxam

Sethoxydim

Benzoximate

Esprocarb

Methidathion

Siduron

Benzthiazuron

Ethidimuron

Methiocarb

Simazine

Bifenazate

Ethiofencarb

Methiocarb sulfone

Simeconazole

Bitertanol

Ethiofencarb sulfone

Methiocarb sulfoxide

Simetryn

Boscalid

Ethiofencarbsulfoxide

Methomyl

Spinosad A

Bromacil

Ethirimol

Methoprotryne

Spinosad D

Bromuconazole

Ethofumesate

Methoxyfenozide

Spiromesifen

Bupirimate

Ethoprophos

Metobromuron

Spiroxamine

Buprofezin

Ethoxyquin

Metolachlor

Sulcotrione

Butocarboxim

Ethoxysulfuron

Metolcarb

Sulfallate

Butocarboxim sulfoxide

Etofenprox

Metosulam

Sulfaquinoxaline

Butoxycarboxim

Famphur

Metoxuron

Sulfometuron-methyl

Buturon

Fenamidone

Metrafenone

Sulfosulfuron

Butylate

Fenamiphos

Metribuzin

Sulfotep

Cadusafos

Fenamiphos sulphone

Metsulfuronmethyl

Tebuconazole

Carbaryl

Fenamiphos sulphoxide

Mevinphos

Tebufenozide

Carbendazim

Fenarimol

Molinate

Tebufenpyrad

Carbetamide

Fenazaquin

Monocrotophos

Tebupirimfos

Carbofuran

Fenazox

Monolinuron

Tebutam

Carbofuran-3-hydroxy

Fenbuconazole

Monuron

Tebuthiuron

Carbofuran-3-keto

Fenfuram

Myclobutanil

Temephos

Carbosulfan

Fenhexamid

Napropamide

Tepraloxydim

Carboxin

Fenobucarb

Naptalam

Terbufos

Carfentrazone-ethyl

Fenoxycarb

Neburon

Terbufos-sulfone

Chlorbromuron

Fenpiclonil

Nicosulfuron

Terbufos-sulfoxide

Chlorfenvinphos

Fenpropathrin

Nicotine

Terbumeton

Chlorfluazuron

Fenpropidin

Nitenpyram

Terbumeton-desethyl

Chloridazon

Fenpropimorph

Nitralin

Terbuthylazine

Chloroxuron

Fenpyroximat

Nuarimol

Terbuthylazine-2-hydroxy

Chlorpropham

Fensulfothion

Ofurace

Terbuthylazine-desethyl

Chlorpyrifos

Fenthion

Omethoate

Terbutryn

Chlorpyriphos-methyl

Fenthion-sulfone

Orbencarb

Tetrachlorvinphos

Chlorsulfuron

Fenthion-sulfoxide

Oryzalin

Tetraconazole

Chlorthiophos

Fenuron

Oxamyl

Thiabendazole

Chlortoluron

Flamprop-isopropyl

Oxasulfuron

Thiacloprid

Cinidon-ethyl

Flamprop-methyl

Oxycarboxin

Thiamethoxam

Cinosulfuron

Fluazafop-P-butyl

Oxydemeton-methyl

Thiazafluron

Clethodim

Fluazifop

Paclobutrazol

Thidiazuron

Clodinafop-propargyl

Flucycloxuron

Paraoxon-methyl

Thifensulfuron methyl

Clomazone

Flufenacet

Parathion

Thiodicarb

Clopyralid

Flufenoxuron

Pebulat

Thiofanox

Cloquintocet-mexyl

Fluomethuron

Penconazole

Thiofanox-sulfone

Clothianidin

Fluoxastrobin

Pencycuron

Thiophanate

Coumaphos

Fluroxypyr

Pendimethalin

Thiophanate-methyl

Cruformate

Fluroxypyr-meptyl

Phenmedipham

Tolylfluanid

Cyanazine

Flurtamone

Phenthoate

Topramezone

Cyanofenphos

Flusilazole

Phorate

Tralkoxidym

Cyazofamid

Flutolanil

Phorate sulfone

Triadimefon

Cycloate

Flutriafol

Phorate sulfoxide

Triadimenol

Cycloxydim

Fonofos

Phosalone

Triallate

Cycluron

Foramsulfuron

Phosphamidon

Triasulfuron

Cyflufenamid

Formetanate

Phoxim

Triazophos

Cymoxanil

Fosthiazate

Picloram

Triazoxid

Cyproconazole

Fuberidazole

Picolinafen

Trichlorfon

Cyprodinil

Furathiocarb

Picoxystrobin

Tricyclazole

Cyromazine

Halosulfuron methyl

Piperonyl butoxide

Trietazine

Daminozide

Haloxyfop

Piperophos

Trifloxystrobin

Demeton O

Haloxyfop-2-ethoxyethyl

Pirimicarb

Trifloxysulfuron

Demeton S

Haloxyfop-methyl

Pirimiphos-ethyl

Triflumizole

Demeton-S-methyl

Heptenophos

Pirimiphos-methyl

Triflumuron

Demeton-S-methyl-sulfon

Hexaconazole

Procloraz

Triflusulfuron-methyl

Desmedipham

Hexazinone

Profenofos

Triticonazole

Desmethyl-formamido-pirimicarb

Hexythiazox

Promecarb

Vamidothion

Desmethyl-pirimicarb

Imazalil

Prometon

Vernolat

Desmetryn

Imazapyr

Prometryn

Zoxamide

Dialifos

Imazaquin

Propachlor

Diallate

Imidacloprid

Propamocarb

720002628, May 2008

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