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Table of ContentsIntroductionExperimental GC and Autosampler Parameters MS ParametersMRM Method SetupResults and DiscussionConclusionsAbout Bruker
There are around one thousand pesticides used in modern agricultural practice, and the residues of pesticides in the food from plant origins have been an increasing concern for consumers worldwide. Analytical methodologies based on mentoring programs are established to make sure that pesticide levels in plant foods comply with national and international laws. However, the range of pesticides and complex food sample matrix present a number of challenges for chemists to meet the high stringent requirements for sensitivity, precision and throughput.
A suitable technique for multi-residue analysis in complex matrix samples is gas chromatography coupled to a triple quadruple mass spectrometer (GC-MS/MS) operated in multiple reaction monitoring (MRM) mode. In MRM mode, a precursor ion of a target residue is isolated in the first quadrupole (Q1), followed by collision induced dissociation (CID) in the second quadrupole (Q2), and one or two selected product ions are filtered through the third quadrupole (Q3) to reach the detector. MRM can significantly improve the sensitivity through increased specificity and selectivity by minimizing the background signals from matrix. Also, multiple MRMs from co-eluted residues can be monitored simultaneously.
In this application note, a GC-MS/MS method for detection of 258 pesticides in a vegetable matrix after extraction using the QuEChERS (Quick Easy Cheap Effective Rugged and Safe) method is described. The ease of method setup, and the sensitivity, linearity and precision of the GC-MS/MS system are demonstrated.
The 258 pesticide standards were prepared from five commercial customer-made pesticide standard stocks (10 ppm in 1:1 hexane:acetone), each containing a group of 50-60 pesticides.
The vegetable matrix was prepared by performing QuEChERS extraction on the mixed color peppers purchased from a local supermarket according to the established procedure. The final acetonitrile extract was reconstituted and air-dried in 9:1 hexane:acetone before spiking pesticide standards for matrix- matched calibration experiments.
The GC-MS/MS analysis was performed on a Bruker Scion TQ triple quadrupole mass spectrometer equipped with a Bruker 451 GC and CP 8400 Autosampler using the following conditions:
GC and Autosampler Parameters
- Injector: Pulsed Splitless (psi, 0.2 min); 260°C, 2 µL,
- Column: Bruker BR-5ms, 30 m × 0.25 mm ID and 0.25 µm film thickness
- Carrier Gas: He, 1.5 mL/min
- Oven Temp: 90°C (3 min), 20°C /min to 150°C, 5°C /min to 300°C (4 min)
- Total Run Time: 40 min
- MS Transfer Line Temp: 300°C
- Ionization: EI, -70 eV
- Source Temperature: 250 °C
- Emission Current: 80 µA
- Manifold Temp: 40°C (fixed)
- Active Focusing Q0: 135°C (fixed) with Helium
- Q2 Collision Gas: Argon (3 psi)
MRM Method Setup
An innovative new compound-based scanning workflow for setting up the multi-residue MRM method is deployed by the Bruker MS-Workstation software. Along with the software, a factory installed MRM library containing more than 2500 MRM transitions of more than 900 most common contaminants including pesticides is provided and compounds with MRMs can be selected from the MRM library to add into the method editor with ease. After several initial runs to locate the retention time window for each compound, the optimal scan time (dwell time) for each MRM can be calculated based on the average peak width and automatically set by the software accordingly after considering all the overlapped retention time windows, thus enabling the elimination of the use of segments as shown in Figure 1. Also, with the CBS, MRM information for each compound in acquisition method is data processing method therefore there is no need for setting up the processing method separately.
Figure 1. Setup of MRM method table with Compound Based Scanning (CBS)
Results and Discussion
Figure 2 shows TIC chromatograms of a 40-min run of 258 pesticides belonging to several classes, which include organochlorine, organophosphate and pyrethroids. The pesticide peaks were eluted between 5.95 and 34.7 min. With the use of CBS for method development, the scan time for each compound (each with 2 MRMs) has been easily optimized (from 14 to 250 ms) to satisfy both the sensitivity and sufficient data points (>15) across each peak. The sensitivity for most of the compounds can reach 0.1-0.5 ppb (LOQ) and with good linearity up to 50 or 100 ppb (compound dependent). Examples of chromatograms and calibration curves at 0.1 ppb levels are shown in Figure 3.
It is important to note that initial study involving comparison of pesticide analysis in solvent and matrix shows matrix effect of either enhancement or suppression of the signals in matrix, suggesting matrix matched spikes be used for method development as reported by others. Hence, the subsequent calibrations were all carried out with pesticides spiked in the QuEChERS matrix. A summary of the representative results is shown in Table 1.
The average factors over the complete calibration range have RSD ≤ 12%, demonstrating high method precision for analyzing these pesticides in the complex sample matrix. As needed by regulation, to confirm the presence of any individual pesticide in the sample, both MRMs (primary for quantification, secondary for qualification) must be observed, and their ratio (of peak areas) often used as a confirmation criteria also. Figure 4 shows both the quantifier and qualifier product ions of 0.5 ppb phosmet in matrix. The ion ratio (quantifier/qualifier) was consistent at 0.65±0.02 over the entire calibration range from 0.5 to 100 ppb.
Figure 2. TIC Chromatogram of 258 pesticides spiked at 20 ppb in QuChERS extract of mixed color peppers
Table 1. Representative results of calibration of pesticides spike in vegetable matrix.
||% Response Factor RSD|
Figure 3. Examples of matrix matched calibration curves for Trifuratin, HCB and Lindane from 0.1 to 100 ppb and their MRM chromatograms at 0.1 ppb.
Figure 4. MRM chromatograms of Quantifier (m/z 160>133, red) and Qualifier (m/z 160>105, green) ions for 0.5 ppb of phosmet in QuEChERS extraction matrix of peppers. The peak area ratio (quantifier/qualifier) is 0.66.
The method development for multi-residue analysis on Scion TQ triple quadrupole mass spectrometer for enhanced productivity has been greatly simplified by compound-based scanning. The method has shown excellent sensitivity, linear calibration range and robustness in matrix.
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