Comparison of SIM and MRM for the Quantitative Confirmation of Pesticide Residues in Food

This application note aims to compare the results from SIM and MRM using the Waters Micromass Quattro micro GC Tandem Quadrupole Mass Spectrometer for the quantification and confirmation of pesticide residues in simple through to complex food matrices.

The inappropriate or unlawful use of pesticides on agricultural produce can result in unacceptably high levels of their residues in produce destined for human consumption. Food produce that is to be used for this purpose must contain less than the statutory maximum residue limit (MRL) of any given residue. 

Worldwide, there are over 800 compounds currently in use to control pests such as insects, weeds, rodents and fungi. The legal enforcement of regulations governing pesticide use requires the regular monitoring of foodstuffs. Given the large number of residues that may be found it is often advantageous to extract and determine as many of them as possible during a single analysis. An extraction, with acetonitrile, followed by dispersive solid phase extraction (SPE) clean up was reported for the analysis of a wide range of pesticides in fruits and vegetables¹ and fatty samples.² As the number of target analytes is increased, the selectivity of the extraction method must be compromised, resulting in a more complex sample matrix. The potential for analytical interference from co-extracted substances is high and the analytical selectivity of such a method must be provided by the determinative step. 

Mass spectrometry is a highly selective analytical technique that can be used to monitor the masses of specific ions generated from the analytes of interest. 

The use of selected ion monitoring (SIM) provides a greater level of selectivity than other traditional detection methods, e.g. flame ionization detection (FID) or electron capture detection (ECD). However, when the analysis of multiresidue pesticides is required, in a variety of produce, the low selectivity of the clean-up stage means that even the use of SIM does not eliminate the potential for matrix interference. 

Multiple reaction monitoring (MRM) is a tandem mass spectrometric technique that allows the monitoring of specific collision induced dissociation (CID) reactions. The nature of CID reactions depends on molecular structure as well as mass and, as a result, significant improvements in analytical selectivity may be achieved using MRM. 

The aim of this work was to compare the results from SIM and MRM using the Waters Micromass Quattro micro GC Tandem Quadrupole Mass Spectrometer for the quantification and confirmation of pesticide residues in simple through to complex food matrices.

Extraction Method

10 g frozen sample was weighed in a centrifuge tube. Acetonitrile (10 mL) was added along with 100 μL of internal standard solution (PCB 138 and TPP) and the tube was shaken for 1 min. MgSO₄ (4g) and NaCl (1g) were added and the solution buffered to pH 5.0-5.5 with citrate buffer. After shaking and centrifugation, an aliquot was transferred to a vial containing PSA sorbent and anhydrous MgSO₄. After undergoing further shaking and centrifugation, the extract was acidified to pH 5 to protect base-sensitive residues. The extract was analyzed by GC-MS and GC-MS/MS.

GC-MS/MS Method

The Waters Micromass Quattro micro GC tandem quadrupole mass spectrometer was used in electron impact (EI+) mode. The ion source was operated at 180 °C with an electron energy of 70 eV and a trap current of 200 μA. Two modes of acquisition were employed; SIM and MRM with an argon collision gas pressure of 3.0 x 10^-3 mBar.

The Quattro micro GC was tuned so that the precursor and product ions were resolved with a peak width at half height of less than 0.7 Da. The list of pesticide residues, the SIM masses and the MRM transitions, along with the collision energies for the method are listed in Table 1. The dwell, inter-channel and inter-scan times were unchanged between the methods.

Acquisition and Processing Methods

The data were acquired using Waters MassLynx Software and processed using the Waters TargetLynx Application Manager.

In SIM mode, three masses were acquired for each residue while in MRM mode it was two transitions. These were used so that quantification and confirmation could be performed with a single injection assuming that the ion ratio between the masses or transitions was consistent for standards and samples. The confirmation criteria chosen was in accordance with the European Union (EU) guidance document Quality Control Procedures for Pesticide Residue Analysis (SANCO/10476/2003).³

Five food matrices were compared using SIM and MRM ranging from simple through to complex; cucumber, sweet pepper, grapefruit, wheat flour and curry powder. 0.005 mg/kg was chosen to be the reporting level as this is half of the target MRL, for active substances in products for which no specific MRL is set, as specified in EU regulation (EC) NO 396/2005⁴ of 23 February 2005.

For both methods matrix-matched calibration curves were linear over the range 0.005 – 0.500 mg/kg with correlation coefficients greater than 0.990 for all analytes in all matrices.

The difference in matrix interference from simple and complex foodstuffs is illustrated in Figure 1. In the case of pirimiphos-methyl from cucumber, there are few peaks from matrix interference whereas from curry powder there are many more.

In SIM mode, for our confirmation criteria to be passed, any concentrations in the extracts must have ion ratios of 0.934 (± 30%) and 0.497 (± 30%). In this example, the ion ratios are 0.956 and 0.479 in cucumber, and 0.771 and 0.461 in curry powder which pass our confirmation criteria. For pirimiphosmethyl, quantification and confirmation by SIM is possible in both matrices.

Changing the mode of acquisition from SIM to MRM significantly increases the selectivity of the determinative step. Pirimiphos-methyl in curry powder with no matrix interference is illustrated in Figure 2. The lack of matrix interference peaks allows routine, automatic integration to be performed.

For our confirmation criteria to be passed in MRM mode, any concentrations in the extracts must have an ion ratio of 0.747 (± 30%). In this example, the ion ratio is 0.644 in curry powder, which passes our confirmation criteria. For pirimiphos-methyl, quantification and confirmation by MRM is possible in curry powder.

Other pesticide residues yield similar results. Biphenyl, vinclozolin and fludioxonil are illustrated in Figures 3, 4 and 5, respectively. Screening would be possible using SIM while confirmation is likely to be problematic if not impossible. However, moving to MRM significantly improves the selectivity in all cases, increasing the probability of being able to perform routine, automatic integration.

The results for all of the pesticide residues in all of the matrices are summarized in Table 2. They show that both SIM and MRM can be used successfully for the screening of the majority residues. However, there are significant differences between the percentage of residues confirmed by MRM compared to SIM, particularly in the more complex matrices.

The ion ratios are important as they provide the basis of confirmation. The ion ratio statistics presented in Table 3 are calculated for the 25 matrix injections across all five matrices. The low % RSDs indicate the good repeatability of the MRM method.

The MRM method was also applied to “real” samples containing incurred pesticide residues. 

Five different matrices were supplied to further test the method; orange, cherry tomato, grapes, kiwi and strawberry. The cucumber matrix-matched calibration curve was used for quantification purposes. The TargetLynx Application Manager was used to provide automatic quantification and confirmation with two MRM transitions acquired for each pesticide residue. For illustration purposes, the reporting level was chosen to be 0.005 mg/kg.

An example TargetLynx browser containing 0.031 mg/kg fludioxonil in grapes is illustrated in Figure 6. A summary of all the incurred pesticide residues detected and confirmed above the reporting level is listed in Table 4.

Selected ion monitoring (SIM) is suitable for the screening and confirmation of pesticide residues in relatively simple matrices.

Multiple reaction monitoring (MRM) offers significant improvements for the screening and confirmation of pesticide residues, especially in more complex matrices.

The combination of QuEChERS extraction, the PTV injector in solvent vent mode, the Quattro micro GC in MRM mode and TargetLynx allow routine pesticide monitoring to easily be achieved.

The described MRM method was tested on “real” samples where incurred pesticide residues were confirmed above the reporting level.

1. M. Anastassiades, S. Lehotay, D. Stajnbaher, F. Schenck, J. AOAC Int. 86 (2) (2003) 412.
2. S.J. Lehotay, K. Maštovska, S.J. Yun, J. AOAC Int. 88 (2) (2005) 630.
3. http://europa.eu.int/comm/food/plant/protection/resources/qualcontrol_en.pdf
4. Regulation (EC) NO 396/2005 of 23 February 2005 on Maximum Residue Levels of Pesticides in or on Food and Feed of Plant and Animal Origin and Amending Council Directive 91/414/EEC, Off. J. of the European Communities No. L70/1.