Authors

Fred Foster & Virgil Settle,
GERSTEL, Inc., Linthicum, MD U.S.A.

Paul Roberts,
Anatune, Ltd., Cambridge, UK

Peter Stone,
Agilent Technologies, Santa Clara,CA, U.S.A.

Joan Stevens,
Agilent Technologies, Wilmington, DE, U.S.A.

Jon Wong, Kai Zhang,
US FDA, College Park, MD, U.S.A

Figure 1. Representative Mass Chromatograms for low QC sample.
Figures 2 through 4 show representative overlay mass chromatograms of neat, hop matrix matched, and ginseng matrix matched calibration standards respectively at 10 ppb. The standards were prepared automatically by the MPS XL.
Figure 3. Representative overlay mass chromatogram for a 10 ppb hop matrix matched standard.
Figure 4. Representative overlay mass chromatogram for a 10 ppb ginseng matrix matched standard.
Figure 5. shows a representative calibration curve resulting from automated preparation of neat standards. The calibration curves were shown to be linear from at least 1.00 to 200 ppb for the pesticides monitored, using a linear, 1/x regression method.

Automated QuEChERS extract
clean-up for LC-MS/MS

So long, troublemakers II

LC/MS and GC/MS systems are increasingly confronted with QuEChERS extracts that have to be cleaned prior to determination of pesticide residues in order to avoid build-up of matrix residue in the analysis system. Automated QuEChERS extract clean-up, including vortexing, centrifugation, and filtration directly followed by LC-MS/MS analysis of the cleaned extract is demonstrated in this article.

The QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample extraction method offers food safety laboratories a novel method that is a genuine step forward. QuEChERS is now the basis for efficient monitoring of pesticides in an evergrowing range of foods. Still, the method is quite labor intensive with several manual steps such as shaking, centrifugation, and dispersive SPE. If the dispersive SPE clean-up step could be automated, laboratory productivity could be improved significantly. When using the automated QuEChERS clean-up procedure for challenging botanical samples, it can be difficult to reach the low limits of detection required in order to meet acceptance criteria for reporting the maximum residue levels (MRLs) as established by regulatory agencies. Automated QuEChERS clean-up of fruit and vegetable extracts combined with LC-MS/MS determination of pesticides has been reported previously.

This study focuses on using a similar system to automate the second step of the QuEChERS procedure and introduce the cleaned extract directly to an LC-MS/MS system. The aim is to provide high throughput analysis for the confirmation of pesticide residues in botanical matrices. Automated QuEChERS extractions are performed using a QuEChERS dispersive SPE sorbent blend for fatty matrices.

Experimental

Stock solutions containing the pesticide compounds listed in Table 1 in acetonitrile were prepared and provided by the FDA. Calibration standards and matrix matched standards were prepared by making appropriate dilutions of the pesticide stock solutions using mobile phase, blank hop extract, or blank ginseng extract resulting in the following concentrations: 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL. Crude acetonitrile extracts of pesticide-fortified samples, incurred samples, and blank matrix samples based on both hops and ginseng root were prepared and provided by the FDA. These samples were generated using QuEChERS extraction salts for the DIN EN 15662 Method and the recommended sample preparation method supplied with the salts. All automated PrepSequences were performed using a MultiPurpose Sampler (MPS XL Dual Head) configured for QuEChERS-LC- MS/MS analysis.

QuEChERS extract pretreatment:

  • Pipette 1 mL of the acetonitrile extract obtained following the 1st centrifugation step of the QuEChERS sample preparation method into a 2 mL glass autosampler vial containing a sorbent from a dispersive SPE kit for fatty samples, AOAC.
  • Place the sample onto a tray on the GERSTEL MPS XL Dual Head.

Automated QuEChERS extract clean-up:

  • Agitate the sample vial for 1 minute using the Anatune CF-100 centrifuge.
  • Centrifuge the sample vial at 575 g for 3 minutes using the Anatune CF-100 centrifuge.
  • Filter 500 μL of the resulting supernatant through a 0.45 μm GERSTEL format syringe filter.
  • Combine 100 μL of the resulting filtrate with 400 μL of mobile phase A in a clean 2 mL vial. 
  • Agitate the sample vial using the Anatune CF-100 centrifuge for 30 seconds. Inject 2 μL into the LC-MS/MS system.

Preparation of all standards was automated using the MPS XL Dual Head as follows:

  • Transfer 100 μL of previously extracted matrix blank or 100 % acetonitrile to an empty 2 mL autosampler vial.
  • Transfer 250 μL of mobile phase A to the vial.
  • Transfer 150 μL of the respective standard stock solution to the vial.
  • Agitate the vial using the Anatune CF-100 and centrifuge for 30 seconds.

All analyses were performed using an Agilent 1290 HPLC, an Agilent 6460 Triple Quadrupole Mass Spectrometer with electrospray source and Jet Stream Option and a GERSTEL MPS XL autosampler configured with Active Wash Station. Sample injections were made using a 6 port (0.25 mm) Cheminert C2V injection valve fitted with a 2 μL stainless steel sample loop. The mass spectrometer acquisition parameters and respective quantifier/ qualifier ion transitions were chosen using the pesticide database option available for the MassHunter B.03.01 software. Table 1 provides a list of the more than 200 pesticides that were monitored using this single LC-MS/MS method. A retention time window value of 0.5 minute was used for each positive ion transition being monitored during the course of the dynamic MRM experiment.

Analysis conditions LC
Mobile Phase:
A - 5 mM ammonium formate in water
with 0.01 % formic acid
B – 0.01 % formic acid in acetonitrile
Gradient: Initial 94 % A / 6 % B
0.3 min 94 % A / 6 % B
14 min 5 % A / 95 % B
17 min 5 % A / 95 % B
Pressure: 600 bar
Flowrate: 500 μL/min
Runtime: 17 min
Post time: 2.5 min
Column: 2.1 mm x 100 mm, 1.8 μm,
Zorbax Eclipse+ C18 RRHT (Agilent)
Oven: 55°C
Injection volume: 2 μL
Analysis conditions MS
Operation: ESI+ mode (Jet Stream)
Time Filter Width: 0.04 min
Scan Type: Dynamic MRM
Delta EMV: 0 V
Cycle Time: 660 ms
Gas Temperature: 225 °C
Gas Flow (N2): 10 L/min
Nebulizer pressure: 25 psi
Sheath Gas (N2): 350 °C
11 L/min
Capillary voltage: 4500 V
Nozzle Voltage: 500 V

Results and discussion

Figures 1 - 4 show representative overlay mass chromatograms resulting from QuEChERS extracts of pesticide-fortified samples. More than 200 different pesticides were successfully determined in botanical matrices using the automated QuEChERS-LC-MS/MS method.

The total time required per sample to perform the QuEChERS extract clean-up was 15 minutes. This was shorter than the LC-MS/MS analysis run, enabling the MPS system to complete preparation of the next sample during the LC-MS/MS run for maximum sample throughput.

Conclusion

The study has demonstrated:

  • Successful monitoring of more than 200 pesticides in botanical matrix samples using automated QuEChERS extract clean-up coupled with LC-MS/MS analysis using the Agilent 6460 Triple Quadrapole Mass Spectrometer.
  • Automation of both the QuEChERS extract clean-up and the preparation of standards using the GERSTEL MPS XL Dual Head robotic sampler.
  • The “just-in-time” sample preparation capability included in the MAESTRO software enables highly efficient QuEChERS extract clean-up and analysis.

 

3-Hydroxycarbofuran Acephate Acetamiprid Acibenzolar-S-methyl Alanycarb
Aldicarb Aldicarbsulfone Aldicarb sulfoxid Aspon Avermectin B1a
Avermectin B1b Azadirachtin Azoxystrobin Benalaxyl Bendiocarb
Benfuracarb Benoxacor Benthiavalicarb Benzoximate Bifenazate
Bifenthrin Bitertanol Boscalid Bromuconazole-1 Bromuconazole-2
Bupirimate Buprofezin Butafenacil Butocarboxym Butoxycarboxim
Cadusafos Carbaryl Carbendazim Carbetamid Carbofuran
Carboxine Carfentrazone-ethyl Chlordimeform Chlorfenvinphos-beta Chlorfluazuron
Chlorotoluron Chloroxuron Clethodim Clofentezine Clothianidin
Coumaphos Cumyluron Cyanazine Cyanophos Cyazofamid
Cycluron Cymoxanil Cyproconazole Cyprodinil Cyromazine
d10-Diazinon d6-Dichlorvos d6-Dimethoate d6-Diuron d6-Linuron
d6-Malathion Daimuron Dazomet Deltamethrin Diazinon
Dichlorvos Dicrotophos Diethofencarb Difenoconazol Diflubenzuron
Dimethenamid Dimethoat Dimethomorph A Dimethomorph B Dimoxystrobin
Diniconazole Dinotefuran Dioxacarb Disulfoton Dithiopyr
Diuron Dodemorph 1 Dodemorph 2 E-Fenpyroximate Emamectin B1a
Emamectin B1b Epoxiconazole Eprinomectin B1a EPTC Esprocarb
Ethidimuron Ethiofencarb Ethion Ethiprole Ethirimol
Ethofumesate Ethoprop Etobenzanid Etofenprox Etoxazole
Famoxadone Fenamidone Fenarimol Fenazaquin Fenbuconazol
Fenhexamid Fenoxanil Fenoxycarb Fenpropathrin Fenpropimorph
Fenuron Flonicamid Flucarbazone Fludioxinil Flufenacet
Flufenoxuron Flumetsulam Flumioxazin Fluometuron Fluquinconazole
Flusilazol Fluthiacet-methyl Flutolanil Flutriafol Forchlorfenuron
Formetanate Fuberidazole Furalaxyl Furathiocarb Heptenophos
Hexaconazol Hexaflumuron Hexythiazox Hydramethylnon Imazalil
Imazapyr Imibenconazole Imidacloprid Indanofan Indoxacarb
Ipconazole Iprovalicarb Isocarbamid Isofenfos Isopropalin
Isoproturon Isoxaben Isoxaflutole Kresoxim-methyl Lactofen
Leptophos Linuron Lufenuron Mandipropamid Mefenazet
Mepanipyrim Mepronil Metalaxyl Metconazole Methabenzthiazuron
Methamidophos Methiocarb Methomyl Methoprotryne Methoxifenozid
Metobromuron Metribuzin Mevinphos Mexacarbate Molinate
Monocrotophos Monolinuron Moxidectin Myclobutanil Neburon
Nitenpyram Norflurazon Novaluron Nuarimol Omethoate
Oxadixyl Oxamyl Paclobutrazol Penconazole Pencycuron
Phenmedipham Picoxystrobin Piperonyl butoxide Pirimicarb Prochloraz
Promecarb Prometon Prometryn Propachlor Propamocarb
Propargite Propazine Propham Propiconazole Propoxur
Pymetrozine Pyracarbolid Pyraclostrobin Pyridaben Pyrimethanil
Pyriproxyfen Quinoxyfen Rotenone Sebuthylazine Secbumeton
Siduron Simazine Simetryn Spinosyn A Spinosyn D
Spirodiclofen Spiromesifen Spiroxamin Sulfentrazone Tebuconazole
Tebufenozide Tebufenpyrad Tebuthiuron Teflubenzuron Temephos
Terbumeton Terbutryn Terbutylazine Tetraconazole Tetramethrin cis
Thiabendazole Thiacloprid Thiametoxam Thiazopyr Thidiazuron
Thiobencarb Thiofanox Thiophanate-methyl Triadimefon Triadimenol
Trichlamide Trichlorfon Tricyclazole Trifloxystrobin Triflumizole
Table 1. 200+ pesticides monitored using automated QuEChERS extract clean-up.

 

Literature:

AppNote-2010-04
Automated QuEChERS Extraction for the Confirmation of Pesticide Residues in Foods using LC/MS/MS