Comparison of Automated vs. Manual QuEChERS Extraction … Notes/AutoMate-Q40 vs... · 2017-01-06 · tandem mass spectrometry (LC-MS/MS). Introduction QuEChERS has proven to be the

Comparison of Automated vs. Manual QuEChERS Extraction

Application Note Tyler Trent, Applications Sales Specialist; Teledyne Tekmar P a g e | 1

AutoMate-Q40 vs Manual QuEChERS Extraction.docx; 6-Jun-16

Sales/Support: 800-874-2004 • Main: 513-229-7000 4736 Socialville Foster Rd., Mason, OH 45040

www.teledynetekmar.com

Abstract Since the development of the QuEChERS method in 2003, the quantity of samples requiring extraction, and pesticides analyzed, has grown. The analysis of pesticide residues has become a challenging task for many laboratories amounting to 500 samples per day and identification of 1000 pesticides per extraction. In this study, a fully automated QuEChERS system was used to extract 204 different pesticides from various commodities. The matrices used for this evaluation were cabbage, valencia orange, raisin and purple corn flour.

The performance of the Teledyne Tekmar AutoMate-Q40 Automated QuEChERS Platform was evaluated in comparison to a manual sample preparation workflow. Both preparations employed the AOAC 2007.01 unbuffered QuEChERS methodology, where all the samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Introduction QuEChERS has proven to be the extraction method of choice for numerous laboratories due to its ease of use and robustness. These analyses typically involve the use of multi-residue methods (both GC-MS/MS and LC-MS/MS) to test for pesticide residues. As the demand to process more samples in less time increases, automation of the QuEChERS manual sample preparation steps has become an area of particular interest to pesticide residue laboratories. 1,2,3

Teledyne Tekmar’s AutoMate-Q40 is a robotic system designed to optimize and automate the QuEChERS sample preparation workflow. This unique system automates the following sample extraction requirements: solvent, internal standard and matrix spike addition, vial shaking, vortex mixing, addition of solid reagents (salts, buffers), centrifugation, identifying liquid levels, pipetting, dSPE cleanup, and preservation of the final extract.

The United States Food and Drug Agency (USFDA) validated the extraction performance of the AutoMate-Q40 against the manual process, comparing the automated and manual QuEChERS extraction using the AOAC 2007.01 unbuffered method.4 All samples in this validation were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Experimental Instrument Conditions

Four commodities (cabbage, valencia orange, raisin and purple corn flour) were chosen to represent a wide range of sample choices for this validation. The appropriate sample weights of the homogenates for cabbage and valencia orange were 10.0 g, while 5.0 g was chosen for the raisin and purple corn flour. Samples were then transferred into 50.0 mL polypropylene centrifuge tubes. The analyst matrix spiked the samples according to the validation guidelines, waited 15 minutes to allow for proper matrix interaction, and then placed the tubes inside the AutoMate-Q40. Once placed into the system, the AutoMate-Q40 performed the AOAC 2007.01 unbuffered QuEChERS extraction method (Figure 1).

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Sales/Support: 800-874-2004 · Main: 513-229-7000 4736 Socialville Foster Rd., Mason, OH 45040


Figure 1 Overall AOAC 2007.01 sample preparation for both manual and AutoMate-Q40 QuEChERS Extractions

Table I AutoMate-Q40 Method Parameters

AOAC 2007.01 Unbuffered

Extraction Salts Weight (g)

Unbuffered Salts 5.0

Extraction Solvents Volume (mL)

Acetonitrile 10.0 Solvent 2 0.0

Water 0.0

Extraction Mix/Vortex Duration (min)

50 mL Sample Mix 1.0 15 mL dSPE Mix 1.0

Sample Vortex Before Extraction Salts 0.5

Centrifuge Duration (min)

50 mL Sample Centrifuge Time 1.0 15 mL dSPE Centrifuge Time 1.0

Extraction Volumes Volume (mL)

dSPE Volume 5.0 Final Extract Volume 2.0

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Figure 1 shows the steps performed by the AutoMate-Q40 to extract the pesticide residues from the commodities. For this analysis, the AutoMate-Q40 used the unbuffered QuEChERS extraction salts: 4.0 g Magnesium Sulfate (MgSO4), 1.0 g sodium chloride (NaCl). Replicates of residue free samples were spiked with a mixture of 204 different pesticides (Restek® LC Multiresidue Standard Kit) at concentrations of 10.0, 50.0 and 250.0 ng/mL to validate this study. In the second part of the QuEChERS method, the samples were cleaned up using a mixture of PSA and C18.

UHPLC-MS/MS Analysis A Waters’ Acquity® UHPLC was interfaced to an AB Sciex™ QTrap® 5500 triple quadrupole (UHPLC-MS/MS). For separation, a Restek® Ultra-Aqueous C18 column (100 × 2.1 mm, 3 um) separated the pesticides for detection and quantification by the MS/MS. Table II gives UHPLC-MS/MS analysis parameters, and Figure 2 shows the MRM chromatogram for a solvent calibrant standard.

Table II UHPLC-MS/MS Parameters

Agilent™ 1290 Infinity® UHPLC

Column Restek® Ultra-Aqueous C18 column (100 × 2.1 mm, 3 um)

Column Flow 0.50 mL/min

Mobile Phase Mobile Phase A: 10.0 mM NH4HCO2 with 0.1% formic acid (Water) Mobile Phase B: 10.0 mM NH4HCO2 with 0.1% formic acid (Methanol)

Injection Volume 5µL

AB Sciex™ QTrap® 5500 Triple Quadrupole

Source 500˚C

Curtain Gas 30 psi

GS1/GS2 60 psi

Collision Gas HIGH

Ion Spray Voltage 5000 V

Figure 2 MRM Chromatogram for a Solvent Calibrant Standard

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Results The automation of the QuEChERS extraction provided a more reliable and reproducible extraction in comparison to the manual QuEChERS process exhibiting excellent repeatability and consistency between samples. Figure 3 shows the precision and accuracy recoveries of the 204 pesticides spiked onto the cabbage, valencia orange, raisin, and purple corn flour samples. The samples were spiked at 10.0, 50.0 and 250.0 ng/mL. The AutoMate-Q40 system is capable of adding these standards directly to the sample, however in this study; the analyst added the spike directly to the sample manually and allowed 15 minutes of interaction before placing inside the AutoMate-Q40.

Figure 3 Laboratory Spiked Sample Results

Further analysis of the data was performed using the t-test to determine if there was significance difference between the results obtained from the AutoMate-Q40 Automated QuEChERS Platform and the manual technique. Results of the t-test are shown below in Table III. The following equation was used for all 204 pesticides at all three spike levels.5

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Table III Results of T-Test for Statistical Comparison

Cabbage

Similar Different 10 ppb 163 (80%) 41 (20%) 50 ppb 146 (72%) 58 (28%) 250 ppb 174 (85%) 30 (15%)

Avg. ± St. Dev. 79% ± 7% 21% ± 7%

Valencia Orange


Avg. ± St. Dev. 79% ± 8% 21% ± 8%

Raison


Avg. ± St. Dev. 89% ± 14% 11% ±14%

Purple Corn Flour


Avg. ± St. Dev. 87% ± 5% 13% ± 5%

Conclusion This validation demonstrates the AutoMate-Q40’s ability to successfully process cabbage, valencia orange, raisin and purple corn flour. By automating the liquid handling, addition of salt/buffers, sample mixing, pipetting, and liquid level sensing using the VialVision™ feature, the AutoMate-Q40 relieves the scientist from a labor-intensive extraction method and exposure to unhealthy chemicals.

Samples were prepared using the AutoMate-Q40 and were statistically compared to the same samples manually extracted by a novice analyst. Percent recoveries and relative error were determined for all samples using both preparations. Automation of the QuEChERS extraction method was found to produce reliable results for the spiked samples and compared favorably with those from the existing manual procedure.

Acknowledgements Special thanks to the United States Food and Drug Agency (USFDA) CFSAN Group, namely Kelli A. Simon, James B. Wittenberg, Jon W. Wong and Alexander J. Krynitsky. Their exceptional work to validate the AutoMate-Q40 Automated QuEChERS Platform is greatly appreciated. In fact, the data they generated provided the basis of this comparison. This project wouldn’t have been possible without their support and hard work.

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References 1. European Committee for Standardization/Technical Committee CEN/TC275 (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.

2. AOCA Official Method 2007.07 Pesticide Residues in Food by Acetonitrile Extraction and Partitioning with Magnesium Sulfate. Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Tandem Mass Spectrometry, First Action 2007

3. M. Anastassiades: QuEChERS a mini-multiresidue method for the analysis of pesticide residues in low-fat products

4. Kelli A. Simon, James B. Wittenberg, Jon W. Wong, Alexander J. Krynitsky and Gregory O. Noonan PERFORMANCE EVALUATION OF AN AUTOMATED QuEChERS WORKFLOW PLATFORM FOR ANALYSIS OF PESTICIDES IN FRESH PRODUCE [Online] http://www.teledynetekmar.com/resources/Application%20Notes/NACRW%20poster%20Kelli%20Simon%20FDA%20Automate%20Q40.pdf (accessed June 2, 2016)

5. Harris, Daniel C. Quantitative Chemical Analysis, 7th Ed.; W.H. Freeman and Company: New York, 2007; pp 59-64

http://www.teledynetekmar.com/resources/Application%20Notes/NACRW%20poster%20Kelli%20Simon%20FDA%20Automate%20Q40.pdf

http://www.teledynetekmar.com/resources/Application%20Notes/NACRW%20poster%20Kelli%20Simon%20FDA%20Automate%20Q40.pdf

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Comparison of Automated vs. Manual QuEChERS Extraction … Notes/AutoMate-Q40 vs... · 2017-01-06 · tandem mass spectrometry (LC-MS/MS). Introduction QuEChERS has proven to be the