Targeted And Non-Targeted Pesticide Screening In Food Using Elevated Resolution GC-Tof/MS 

In this application note, a method will be introduced for the targeted screening of 93 pesticide residues with 106 components in pear, lettuce, and fruit-based baby food to the legislated concentration of 0.01 mg/kg. The method will also be extended to include non-target screening of real samples using automatic peak detection, deconvolution, and library searching with exact mass scoring. 

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. 

In the European Union (EU) and Japan, legislation has recently been established for setting and controlling MRLs in food.^1,2 One key feature of the legislation is that a Uniform Limit or default MRL of 0.01 mg/kg will apply to those commodities where no MRL is set. 

Given that there are approximately 1000 compounds registered worldwide to control pests it is often advantageous to extract and determine as many of them as possible during a single analysis. An extraction method, with acetonitrile, followed by dispersive solid phase extraction (SPE) clean up has been reported for the analysis of a wide range of pesticides in fruits and vegetables³ and fatty samples.⁴

Targeted detection such as selected ion recording (SIR) or multiple reaction monitoring (MRM) allow for the screening of a finite number of compounds to be achieved. However, much of the chemical information is discarded so full spectrum techniques are still required in non-target screening environments. 

To establish a suitable non-target screening technique the method must be 

• Sensitive to achieve the default MRL of 0.01mg/kg. 
• Selective to reduce or eliminate matrix interferences. 
• Multi-residue so multiple targets can be analyzed in a single run. 
• Rugged so complex samples can be analyzed with reduced or no sample clean-up.

However, the method must also be generic so that ‘food scares’ or any changes in legislation do not require updating of methods and re-analysis of samples while non-target components can be detected post acquisition or retrospectively.

Exact mass time of flight mass spectrometry (Tof/MS) is a full spectrum technique capable of both the targeted and the non-targeted approaches. An exact mass Tof/MS method was recently introduced in Waters Application Note 720001607EN⁵ and the results described here build on that initial work.

In this application note, a method will be introduced for the targeted screening of 93 pesticide residues with 106 components in pear, lettuce and fruit-based baby food to the legislated concentration of 0.01 mg/kg. The method will also be extended to include non-target screening of real samples using automatic peak detection, deconvolution and library searching with exact mass scoring.

Extraction Method

10 g sample was weighed in a centrifuge tube. Acetonitrile (9.9 mL), acetic acid (0.1 mL), anhydrous MgSO₄ (4 g) and sodium acetate (1.66 g) were added and the tube was shaken immediately. After centrifugation at 4300 g for 5 min, an aliquot (1 mL) of the supernatant was transferred to a vial. The aliquot was blown down gently with nitrogen so that 0.2 mL toluene could be added. The 1 mL extract was transferred to a microcentrifuge vial containing 50 mg primary secondary amine (PSA) sorbent, 50 mg carbon sorbent and 150 mg anhydrous MgSO₄. The contents were vortex mixed for 30 s and centrifuged at 5000 g for 1 min. The supernatant was transferred to a vial and analysed by GC-Tof/MS. 

GC-Tof/MS Method

The Waters GCT Premier orthogonal acceleration time of flight (oa-Tof) mass spectrometer was used in electron ionisation (EI+) mode. The source was operated at 200 °C with an electron energy of 70 eV and a trap current of 200 μA. The temperature of the transfer line was held at 280 °C during the run. Spectra were acquired between 50 and 500 Da in a time of 0.24 s and a delay of 0.01s (4 spectra/s). Exact mass spectra were obtained using a singlepoint lock mass (Tris[trifluoromethyl]triazine, m/z = 284.9949) infused into the ion source continuously during the run. The GCT Premier was tuned so that the resolution was greater than 7000 full width half maximum (FWHM). The pesticide residues analysed are listed in Table 1. 

Acquisition and Processing Methods

The data were acquired using Waters MassLynx software version 4.1 and processed using either the TargetLynx or ChromaLynx Application Managers.

Target Screening Results

Three food commodities were screened; pear, lettuce, and fruit-based baby food. To test the extraction method described, five recovery experiments were performed in fruit-based baby food, spiked at 0.01 mg/kg. The mean recovery and relative standard deviation (% RSD) in parenthesis of each analyte are listed in Table 1. 

Satisfactory recoveries were obtained for the majority of the pesticides in fruit-based baby food with 81% of the residues recovered in the accepted 70–110% range as specified in the AQC guidelines.⁶ The % RSDs were less than 30% for all residues, with 87% residues giving % RSDs less than 10%. Consistent low recoveries for a few (quintozene, pyrimethanil, chlorothalonil, pentachloroaniline, fenazaquin, and pyrazophos) are attributed to losses during the dispersive SPE clean-up step.

Methidathion at a concentration of 0.01 mg/kg in fruit-based baby food was chosen to illustrate the improved selectivity and signal-to-noise (S/N) ratio of exact mass chromatograms. The nominal mass chromatogram (1 Da, m/z 145), illustrated in Figure 2, has a S/N ratio of 6:1, close to the limit of detection (LOD). In the exact mass chromatogram (20 mDa, m/z 145.0072) the S/N ratio is now 48:1 just by reducing the mass window by which the chromatogram is drawn. Improved selectivity and S/N reduce the likelihood of interference when using automatic integration packages. 

The selectivity and S/N ratio improvement obtained from using exact mass chromatograms depends on the complexity of the final sample extract. This will ultimately depend on the complexity of the original food commodity and the sample preparation method used. Figure 3 illustrates that when nominal mass windows (1 Da, m/z 248) are used, the detection of endosulfan sulfate at 0.01 mg/kg is affected by matrix interference but this differs from matrix to matrix. However, when exact mass windows (20 mDa, m/z 248.0397) are used, the likelihood of matrix interference is significantly reduced. The two peaks that can be seen preceding endosulfan sulfate in the exact mass chromatogram are endosulfan I and II. 

The sensitivity of the method is illustrated in Figure 4, showing that propyzamide can be screened to a level below 0.01 mg/kg in pear. Increasing the number of ions, as in the case of confirmation or increasing the number of residues, on a scanning instrument will decrease the overall sensitivity (peak area and S/N) in SIR mode due to the reduced duty cycle. With Tof increasing the number does not decrease the sensitivity, as can be seen by the two peak areas (29) when moving from one ion acquired to three ions acquired. Therefore, if there is interference for one mass, processing can be moved to a different mass without re-injection. The number of confirmation ions or residues can be increased without effect. 

The TargetLynx browser for chlozolinate at a spiked concentration of 0.01 mg/kg in fruit-based baby food is illustrated in Figure 5. 93 residues with 106 components could be screened using this method in pear, lettuce, and fruit-based baby food to a concentration of 0.01 mg/kg. This number is not absolute because more residues could be added as there will no effect on sensitivity. The results show that the GCT Premier can be used in a targeted screening environment. 

The method was also applied to a blind study of 11 pear samples and 12 lettuce samples containing incurred residues. Matrix-matched calibration standards was used for quantification purposes. The TargetLynx Application Manager was used to provide automatic quantification with two exact mass chromatograms (0.02 Da) processed for each residue. For illustration purposes, the reporting level was chosen to be 0.005 mg/kg while the maximum residue limit was chosen to be 0.01 mg/kg. 

A TargetLynx browser showing three exact mass chromatograms for incurred chlorpyrifos is illustrated in Figure 6. In this example, the results indicate a screened concentration of 0.023 mg/kg in pear, not exceeding the UK/EU MRL of 0.500 mg/kg. The correlation between the elevated resolution GC-Tof/MS method and the established single quadrupole SIR method was good.

ChromaLynx automatically plots the reconstructed ion chromatograms (RICs) of up to the eight most intense ions at any point in the chromatogram. If a peak is found to satisfy user-defined parameters the software will display its deconvoluted mass spectrum. The spectrum can then be submitted to an automatic library search routine with the ability to confirm by exact mass scoring. 

Deconvolution is important because as the number of residues and/or matrix peaks increase, the probability of peaks co-eluting also increases. In target screening, this is not important unless the co-eluting peaks have the same exact masses but in non-target screening being able to obtain a “clean” spectrum for each component is the basis of any result. The importance of deconvolution for non-target screening can be observed in Figure 7. Given the data shown an analyst is likely to conclude there are five components in this section of the chromatogram at 17.61, 17.73, 17.78, 17.87, and 17.97 min. 

However, submitting the same section of the chromatogram to ChromaLynx results in the example browser displayed in Figure 8. Here eight components, indicated by a pink triangle, were found with three peaks co-eluting under the peak at 17.87 min and two peaks co-eluting under the peak at 17.97 min. 

Figure 9 illustrates each component under the peak at 17.87 min as an exact mass chromatogram and indicates overlapping peaks with slightly different peak centres. 

ChromaLynx processing of a real lettuce extract located more than 500 components in the chromatogram. Components can be reduced to a list of possible candidates by using the fit factor from the mass library search. ChromaLynx automatically performs exact mass scoring of the library search. The formula from the library hit is submitted to elemental composition and the “n” most intense ions are confirmed/rejected by exact mass.

An example of one of the non-target residues, DCPA or chlorothal-dimethyl, that was found is illustrated in Figure 10. In this example, DCPA was scored with two ions within 0.8mDa of their expected masses. 

Following the non-target screening of DCPA in lettuce, CSL re-extracted the sample and quantified using their established single quadrupole method. 

The validation recoveries at 0.01 mg/kg were 95–103% (n=5). The confirmation of identity was based on three ions (m/z 301, 299, and 332). DCPA was confirmed at 0.07 mg/kg indicating that non-target residues can be screened successfully using the GCT Premier together with ChromaLynx. 

An exact mass GC-Tof/MS method has been developed for the quantification of approximately 100 pesticide residues. 

The residues can be screened to concentration levels of 0.01 mg/kg or less in pear, lettuce, and fruit-based baby foods with the use of exact mass chromatograms. 

Good linearity and satisfactory recoveries for the majority of pesticides were obtained. 

A targeted method using TargetLynx and a nontargeted method using ChromaLynx were then employed for residue screening. 

The targeted method correlated well with previous results obtained from an established single quadrupole MS method. 

This joint approach led successfully to the identification of incurred residues in pear and lettuce samples. 

1. Regulation (EC) NO 396/2005 of 23 February 2005, Off. J. of the European Union No. L70/1. 
2. www.ffcr.or.jp/zaidan/FFCRHOME.nsf/pages/ eng.h-page 
3. M. Anastassiades, S. Lehotay, D. Stajnbaher, F. Schenck, J. AOAC Int. 86 (2003) 412. 
4. S.J. Lehotay, K. Maštovska, S.J. Yun, J. AOAC Int. 88 (2005) 630. 
5. www.waters.com/watersdivision/pdfs/720001607EN.pdf 
6. Quality Control Procedures for Pesticide Residues Analysis of 24 March 2006, Document Number SANCO/10232/2006.