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Matrix-Matched Pesticide Standard Curve Preparation

Tutorial Method

Food testing, LCMS sample prep

Published by

Waters Corporation

Version 1, 19 May 2024 at 8:17 AM

Pesticide analysis is critical to ensure agricultural commodities comply with legal limits. Matrix effects are a common concern, as they can compromise detection and quantification quality. Here, we present a fully automated protocol for matrix-matched standard curve preparation ready for a typical LC-MS/MS method.

Use in OneLab

Please note that this is a “Tutorial Method”

This basic method provides the core methodology for translating a workflow into OneLab-executable script(s) as an attempt to fully or semi-automate a specific procedure. It demonstrates the benefits of automation and highlights OneLab capabilities and best practices to promote solution adoption, helping transition from manual to a more automated approach. It can be used alone or serves as a building block for a more complex workflow and is easily adaptable to users' requirements.

Overview

Pesticide Analysis in Agricultural Commodities

Matrix effects are a common concern in pesticide analysis, which can compromise detection and quantification quality. To improve the efficiency of traditional methods, Anastassiades et al. introduced a sample preparation approach known as QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe). This procedure uses a single extraction in acetonitrile and requires a small amount of sample (10-15 g). The resulting acetonitrile extract is typically analyzed directly by GC-MS/MS or LC-MS/MS after proper dilution.

Challenges in Sample Preparation for Matrix-Matched Standard Curve Generation

The initial mobile phase conditions for a typical LC-MS/MS analysis is water/acetonitrile (9:1). Sample solvent with stronger elution strength than initial conditions of the mobile phase causes peak distortion, peak broadening, and earlier elution due to less effective retention. A proper dilution after QuEChERS extraction is essential to ensure successful and reproducible LC-MS/MS analysis. Moreover, it is also recommended to pre-wet the tips when handling volatile solvents like acetonitrile, methanol, isopropanol, etc., to reduce dripping. All these considerations further increase the number of pipetting tasks required in a typical workflow.

While often not difficult, preparing these samples is repetitive, time-consuming, and requiring consistency to ensure the reliable performance of the analytical method. This makes it ideal for incorporating lab automation, thus allows the analyst to do other tasks, streamlines the sample preparation process, reduces the potential for human error, and ensures consistent analytical method performance.

Figure 1: Typical calibration graphs generated from the analysis of matrix-matched standards prepared in duplicate

Assay notes

This protocol was developed to generate seven calibration levels plus a blank. Column 1 consists of the standard stock solutions at ten times the concentration. Columns 2 and 3 are duplicates of matrix-matched calibration standards. Columns 4 and 5 are duplicates of calibration standards prepared in solvent. By preparing two formats of calibration curve (matrix vs. solvent), it allows the user to investigate and understand the matrix effect.

The standard stock solutions are first prepared in Column 1 to yield the concentration at 10, 50, 100, 250, 500, 750 and 1000 ppb. Matrix-matched calibration curve is prepared by adding 10 μL standards + 10 μL matrix + 80 μL water to give a total of 100 μL volume. 10 μL of matrix is replaced with acetonitrile when preparing calibration standards in solvent.

Figure 2: Labware and solutions required for matrix-matched calibration curve preparation

Protocol options

Protocol 1 - Matrix-matched calibration curve only

This option allows the user to build a matrix-matched calibration curve with the proper dilution suitable for typical LC-MS/MS analysis.

Protocol 2 - Matrix-matched calibration curve + calibration standards prepared in solvent

This option allows the user to investigate and understand the matrix effect.

Figure 3: Andrew+ OneLab deck set up for the calibration standards preparation. Samples and reagents locations on Andrew+ deck: [1] Tip Insertion System Domino, [2] Deepwell Microplate Domino, [3] Microtube Domino, [4] 8-Channel Reservoir Domino

Protocol specifications

Apple Matrix Pesticide Calibration - Protocol 1:

Estimated time of execution: 17m 4s
Hands-on time: 0s
Tip consumption:
41× 10-300 μL tips

Apple Matrix Pesticide Calibration - Protocol 2:

Estimated time of execution: 18m 37s
Hands-on time: 0s
Tip consumption:
66× 10-300 μL tips

Ordering information

Andrew+ System Components: Dominos, Devices, Electronic Pipettes & Tips

Andrew+ Pipetting Robot
OneLab software
Tip Insertion Domino | p/n 186009612
Deepwell Microplate Domino | p/n 186009597
Microtube Domino | p/n 186009601
8-Channel Pipette Reservoir Domino | p/n 186009613
Andrew Alliance Bluetooth Electronic Pipette, 1-ch 300 μL | p/n 186009606
Andrew Alliance Bluetooth Electronic Pipette, 8-ch 300 μL | p/n 186009607

Recommended consumables

Waters 20 Pesticide Mix Standard | p/n 186006348

References

Waters Application Note 720007433EN - Automating Preparation of Matrix-Matched Standards for Pesticide Residue Analysis Using the Andrew+ Pipetting Robot (https://www.waters.com/content/dam/waters/en/app-notes/2021/720007433/720007433-en.pdf)

This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.