Efficient large-scale pesticide residue analysis based on gas chromatography-high resolution Orbitrap mass spectrometry

Efficient large-scale pesticide residue analysis based on gas chromatography – high resolution Orbitrap mass spectrometry

Dominic Roberts, 1 Hans Mol, 2 Marc Tienstra, 2 Cristian Cojocariu, 1 and Paul Silcock1

1Thermo Fisher Scientific, Runcorn, UK

2RIKILT – Wageningen UR, Wageningen, The Netherlands

Key words

Accurate mass; complex matrix; GC Orbitrap mass spectrometry; pesticide residue analysis; QuEChERS; screening; TraceFinder software

Foreword

Pesticides are used throughout the world to increase crop yields, and the use of pesticides is critical to ensuring adequate global food supply. However, for consumers and government agencies working on food safety, such widespread use of pesticides and their potential to remain in the final product is a concern.

Therefore, laws and regulations guarantee that consumers will not be exposed to food contaminated with pesticides. These laws and regulations require the monitoring of the types and quantities of pesticides present in food and the maximum allowable residual value (MRL) of each pesticide for a specific commodity category. Due to the large number of combinations of compounds and commodity types, it poses significant challenges for accurate and reliable routine monitoring.

The laboratory has been facing increasing analytical pressures and must achieve broad-spectrum pesticide screening in a single sample analysis at high speed and competitive cost. Most laboratories rely on gas chromatography or liquid chromatography coupled with mass spectrometry and target analysis strategies. Such methods do indeed cover a wide range of compound species to be monitored and achieve the desired sensitivity and selectivity. However, they are limited to the compounds in the target list, which are usually selected according to the residue regulations and the requirements of laws and regulations, mainly to prove that the food samples tested are suitable for consumption. These target analysis techniques typically require careful prior optimization of acquisition parameters and acquisition time windows for each compound to ensure efficient detection. In order to expand the field of view of the analysis, compound screening methods using high-resolution full-sweep mass spectrometry have begun to be favored in recent years.

These screening methods use a non-targeted analysis strategy, running an ordinary full-scan acquisition first, and then performing target data processing based on the database used. Although data analysis is based on a list of target compounds, unknown compounds can still be analyzed in data retrospective analysis, even if these unknown compounds are not specifically screened at the time of data collection.

In order for this method to be applied to routine analysis, the data screening processing software must be fast enough and accurate to meet the requirements described in the EU Directive1 to achieve detection of low concentrations of pesticide residues at acceptable low false negative rate levels.

Although there are no specific requirements for false positive rates, routine laboratories need to ensure that this number is as low as possible to minimize the time required for further analysis.

Most of the samples tested in the laboratory are in compliance with laws and regulations, so it is important to be able to quickly distinguish between qualified samples and potentially contaminated samples for efficiency. After the initial screening, the possible positive samples are reanalyzed using another set of validation methods (eg, GC-MS/MS) to confirm positive results and accurately determine the concentration of pesticide residues. Confirmatory analysis involves the steps of preparing a standard curve in a suitable matrix and is not included in the screening methods described herein.

In this study, we evaluated the performance of the Thermo ScientificTM Q ExactiveTM GC Combined Quadrupole-Orbitrap Mass Spectrometer (MS) for accurate screening of pesticides suitable for GC analysis. The Q Exactive GC Orbitrap MS delivers mass resolutions up to 120,000 (m/z 200, full peak width at half the peak height, FWHM), enabling high-accuracy mass measurements to be effectively replicated in complex matrices The analyte is separated from impurities such as co-eluting mass-interfering substances. The high dynamic range (> 5000) within high sweep and single-target analyte scans also helps detect trace compounds from complex matrices.

Experimental condition

Sample preparation

Food and feed samples were first treated with acetate buffer and an extraction procedure based on the QuEChERS method. Briefly, 10 mL of acidified (1% acetic acid) acetonitrile was added to 5 g (breakfast cereal or feed) or 10 g (fruits, vegetables, etc.) homogenized samples. Add mixed salts, shake and centrifuge. A mixture of 55 pesticides was added to the final extract (0.5 or 1 g/mL sample in acetonitrile) at a concentration range of 0.5–100 ng/g (ppb). We analyzed a variety of difficult matrix samples, including wheat, leeks, and horse feed.

Instrument and method settings

All analyses were performed using a Thermo ScientificTM Q ExactiveTM GC combined quadrupole-Orbitrap mass spectrometer. The Thermo ScientificTM TriPlusTM RSH autosampler was used for the injection. The chromatographic separation was performed using a Thermo ScientificTM TRACETM 1310 GC with a Thermo ScientificTM TraceGOLDTM TG-5SilMS 15 m × 0.25 mm ID × 0.25 μm thin film capillary column (P/N: 26096). -1301).

See the table below for more instrument parameters and details.

The Q Exactive GC system operates in EI full sweep mode with a resolution of 60,000 (FWHM, m/z 200). In addition, other experiments were performed at different resolutions such as 15K, 30K and 120K. Chromatographic data acquisition ensures no less than 11 points per peak to ensure accurate peak area integration.

data processing

Data acquisition and processing was performed using the Thermo ScientificTM TraceFinderTM software. The software integrates functions such as instrument control and method development, as well as different workflows such as qualitative screening and quantitative analysis.

Results and discussion

The goal of this study was to screen a wide range of pesticides in a wide range of sample matrices with the highest confidence. The analysis was conducted to screen whether the pesticide residue in the sample exceeded the MRL level (usually 10 ppb). The assessment method was to determine the minimum detectable concentration of the standard added under the conditions by screening different spiked concentrations of fortified wheat, horse feed, and leek extract. We chose these matrix samples because they are known for their complexity and challengingness in pesticide disability analysis, as evidenced by the total ion current spectrum shown in Figure 1.

The sample extraction method used in conventional pesticide residue analysis is very common (for example, QuEChERS), and the resulting extract is very complicated and variable. Due to the lack of selectivity in the sample preparation stage, it must be compensated for during instrumental analysis. The way to improve selectivity can be to analyze with high quality resolution and high quality accuracy. As the complexity of the sample increases, the resolution of the mass spectrometer is critical for reliable pesticide detection. The importance of resolving power has been reflected in the analysis of pesticide residues suitable for LC2. In addition, high-resolution full-scan analysis is also attractive for expanding the field of view of the analysis without the need to optimize data acquisition parameters.

Figure 1. Total ion chromatogram (TIC) of wheat, horse feed, and leek extracts with 55 pesticide standards added. The complexity of the sample is shown.

Sample throughput

Sample throughput is an important factor to consider in pesticide residue analysis. Therefore, we used a flash chromatography method to test the performance of the system under normal conditions. This chromatographic method completes a complete analysis within 17 minutes (between two injections), enabling 84 analyses to be completed in less than 24 hours. Although this is a fast GC method, the high sweep speed of the mass spectrometer ensures that at least 11 points are taken for each peak. Figure 2 shows the diazinon peaks with a peak width of 1.8 seconds, showing a total of 11 data points.

Figure 2. Extracted ion chromatogram (XIC) of a wheat sample analyzed with 10 ng/mL diazinon (m/z 179.11789 ± 5 ppm mass window) with full scan containing 11 scans (peak width 1.8 sec). Data acquisition was performed in full sweep mode with a resolution of 60,000 FWHM (m/z 200). As shown, the mass accuracy and mass difference (ppm) for each scan are ideal, with an average mass difference of 0.3 ppm for the full peak.

Screening

After performing a full sweep analysis at 60,000 mass resolution, the TraceFinder software was used for data processing. Sample screening was performed using a database containing 183 pesticide formulas, accurate mass, retention time, isotope peak distribution (calculated by molecular formula of diagnostic ions), and fragment ions. Although all of the above parameters can be used for identification, the key basis for positive detection of software use is: the extracted ion chromatogram of the main identified ions must have a chromatographic peak within the expected peak time ± 20 seconds window, and the measured ions The exact mass must be within ± 2 ppm of the theoretical value. Isotope peak distribution and retention time and accurate mass of fragment ions. The introduction of these parameters can increase the credibility of the analysis results and reduce the false positive rate.

Screening software

Good data processing software is a key to successful routine screening. TraceFinder software quickly screens data and detects the presence of target pesticides. We used a target compound database to detect and report detected pesticides and indicate which detection criteria were met. Figure 3 shows an example of a TraceFinder browse window showing some pesticides detected in wheat samples with a 10 ng/mL standard added. The detection and confirmation of pesticide p,p'-DDT is based on retention time, accurate mass (0.21 ppm), fragment ion, and isotope distribution information, and is indicated in red in the figure. The data is presented to the user in the form of traffic lights for quick review. More detailed information can be found in the summary and window columns, such as the theoretical and actual isotope distributions of XIC and p,p'-DDT in this example. Even in complex matrices, the system provides unparalleled accurate mass data, making the detection of compounds very reliable. All pesticide screenings are performed with a mass accuracy of < 2 ppm, and as shown in Figure 3, the actual mass difference is usually only at the sub-ppm level. For the main diagnostic and fragment ions, accurate quality testing can achieve this level, which can automatically screen out false positive results or prompt users to quickly assess.

Screening software

Good data processing software is a key to successful routine screening. TraceFinder software quickly screens data and detects the presence of target pesticides. We used a target compound database to detect and report detected pesticides and indicate which detection criteria were met. Figure 3 shows an example of a TraceFinder browse window showing some pesticides detected in wheat samples with a 10 ng/mL standard added. The detection and confirmation of pesticide p,p'-DDT is based on retention time, accurate mass (0.21 ppm), fragment ion, and isotope distribution information, and is indicated in red in the figure. The data is presented to the user in the form of traffic lights for quick review. More detailed information can be found in the summary and window columns, such as the theoretical and actual isotope distributions of XIC and p,p'-DDT in this example. Even in complex matrices, the system provides unparalleled accurate mass data, making the detection of compounds very reliable. All pesticide screenings are performed with a mass accuracy of < 2 ppm, and as shown in Figure 3, the actual mass difference is usually only at the sub-ppm level. For the main diagnostic and fragment ions, accurate quality testing can achieve this level, which can automatically screen out false positive results or prompt users to quickly assess.

Figure 3. Pesticides detected in wheat samples supplemented with a 10 ng/mL standard (using p,p'-DDT as an example) display the interface in the TraceFinder Screening Browser. Confirmation of pesticide detection is based on an agreement between accurate mass identification (± 2 ppm mass window), retention time (RT), isotope peak distribution (IP), and fragment ion (FI) information. The red box indicates the sub-ppm level mass accuracy of the primary ion and confirmatory ion detection.

Screening below MRL levels

In this study, when the spiked concentration was 10 ng/mL, all 55 pesticides were successfully detected in wheat, horse feed, and leek samples. Not only that, the vast majority of pesticides can be detected at lower concentrations. As shown in Figures 4 and 5, in the wheat matrix, 53 pesticides can be detected at a concentration of < 2.5 ng/mL, and when the spike level is reduced to 0.5 ng/mL, there are still 47 pesticides that can be Check out. This unparalleled sensitivity, also achievable in complex matrices, can effectively support reliable screening at or below the MRL-specified concentration level, which is a unique feature of the Q Exactive GC system.

Figure 4. Minimum concentration of standard concentrations of 55 pesticides in wheat. The identification is based on an accurate mass difference < 2 ppm and a retention time difference of no more than 20 seconds. The red circle is at a concentration level of 5 ng/mL.

Figure 5. Minimum concentration of standard concentrations of 55 pesticides in horse feed. The identification is based on an accurate mass difference < 2 ppm and a retention time difference of no more than 20 seconds. The concentration levels of 5 ng/mL and 10 ng/mL are highlighted in the figure.

Avoid false negative results with resolving power

Use of lower mass accuracy tolerance is only possible when the resolution is sufficient to distinguish the target compound from matrix interference and other target compounds. When the two mass profiles are coincident, the measured mass profile is actually the sum of the two, resulting in inaccurate quality determination of the target compound. This is exactly what Figure 6 shows. The leek samples were analyzed at 15K, 30K, 60K and 120K resolutions. A diagnostic ion of chlorpheniramine is visible in the mass spectrum, and a similar background matrix ion interferes with it. At 60K and 120K resolution, the analysis achieves the expected mass accuracy and baseline separation. However, at 15K and 30K resolution, chloroaniline failed to separate from interfering ions, reducing mass accuracy. Especially at 15K resolution, mass accuracy is severely affected, with a mass difference of up to 18.4 ppm. According to the screening criteria used in this study, even a further relaxation of the tolerance for quality differences to 10 ppm would still result in a false negative result for chlorpheniramine. These test results indicate that a mass resolution of more than 60,000 is necessary, and the specific value of this minimum resolution depends on the complexity of the sample and the concentration of the target compound and interferent.

Figure 6. Resolving power affects the mass accuracy of analytes in the matrix. Mass spectrum of an ion of 10 ng/mL chloroaniline in the leek extract collected at 15K, 30K, 60K and 120K mass resolution. The 15K and 30K resolutions are not sufficient to separate the chloroaniline ion and matrix interference peaks, resulting in poor quality accuracy. At 15K resolution, the pesticide will not be detected according to the screening criteria of this study, resulting in false negative results.

Figure 7. View the extracted ion chromatogram and calibration curve for butyl morpholine in leek in the TraceFinder software. The calibration sample was injected in three parallel replicates with good linearity.

Quantitative analysis of pesticides

In routine analysis, the pesticides detected in the samples should be quantitatively analyzed. We evaluated the linearity of the pesticide test with matrix-matched samples at a concentration range of 0.5–50 ng/mL, and each calibration standard was analyzed in triplicate. In all cases, the coefficient of determination (R2) was > 0.99, the mean was R2 = 0.997, and the regression residual was < 25%.

Figure 7 shows the quantitative calibration curve of the compound using butyl morpholine as an example. The complete quantification process for the detection of compounds is beyond the scope of this study, but is described in detail in the Thermo Scientific application document 10449 ³ .

in conclusion

The results of this evaluation show that the Thermo Scientific Q Exactive GC combined quadrupole-Orbitrap mass spectrometer, along with TraceFinder software, is an extremely effective tool for routine pesticide screening for food and feed. The resolution, quality accuracy and sensitivity of the Orbitrap mass spectrometer are excellent.

* Screening with high-resolution full-sweep mass spectrometry is an effective way to expand the range of analysis. With this technology, more compounds can be analyzed in a single experiment without prior optimization of acquisition parameters.

* Fast GC analysis and acquisition rates increase laboratory productivity and sample throughput. Superior mass accuracy combined with extremely high sensitivity makes reliable routine pesticide screening possible.

* Data acquisition at a mass resolution of 60,000 FWHM (m/z 200) eliminates interference from homogeneous compounds and increases the confidence in the results of screening pesticides in complex matrices. Sub-ppm levels of mass accuracy are consistently provided for all compounds to ensure reliable compound identification.

references

1.SANCO/12571/2013. Method validation and quality control procedures for pesticides residue analysis in food and feed.

2. Kellmann, M., Muenster, H., Zomer, P. & Mol, H. (2009) Full Scan MS in Comprehensive Qualitative and Quantitative Residue Analysis in Food and Feed Matrices: How Much Resolving Power is Required? J Am Soc Mass Spectrom, 20, 1464-1476.

3. Thermo Scientific Application Note 10449: Fast screening, identification, and quantification of pesticide residues in baby food using GC Orbitrap MS technology. Runcorn, UK

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