Abstract

Abstract

This technical note describes the analysis of over 600 pesticides and mycotoxins in a single injection using a fast MRM acquisition rate method on the SCIEX 7500+ system. Good data quality was shown with an MRM acquisition rate as low as 3 ms (1 ms dwell time and 2 ms pause time) while maintaining instrument sensitivity, accuracy and precision. Most of the analytes (89%) had limits of quantitation (LOQ) of 1 ng/mL in the solvent-based calibration standards. Figure 1 shows extracted ion chromatograms (XICs) for 3 pesticides at 1 µg/kg in soybean extract, demonstrating good sensitivity in matrix at trace levels. The SCIEX 7500+ system features the new Mass Guard technology, which is designed to improve instrument robustness while maintaining optimal sensitivity, and the user-accessible DJet+ assembly, which increases flexibility for cleaning¹.

Figure 1. Overlaid XICs of 3 compounds at 1 µg/kg, acquired with a 1 ms dwell, 2 ms pause and 5 ms settling time in soybean extract. The images show overlaid quantifier and qualifier transitions for fenoprop, azoxystrobin and saflufenacil with ion ratio lines highlighting where the qualifier peak should sit to be within ±30% ion ratio tolerance.

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Key-features

Key benefits of the SCIEX 7500+ system for large panel contaminant analysis

• Fast MRM scan rate:  MRM time was reduced to 3 ms (dwell and pause time) without decreasing analytical performance, allowing more compounds to be analyzed within a single injection while still achieving ion ratio stability
• Continued high-end performance:  Of the pesticides analyzed, 89% showed an in-vial solvent LOQ of 1 ng/mL with an injection volume of 1 µL, resulting in lower matrix effects
• Positive and negative polarity switching:  Continued high-end performance was achieved with 5 ms settling time
• Enhanced robustness and usability:  Mass Guard technology extends instrument robustness while the DJet+ assembly allows increased flexibility for user cleaning when required

Introduction

Introduction

Detecting pesticides in food products is critical for protecting human health. The European Union (EU) regulates the presence of pesticides and mycotoxins in food of plant and animal origin according to the maximum levels specified in regulations 396/2005 and 2023/915.^2-3 These compounds are typically analyzed in food matrices using LC-MS/MS. However, analyzing large analyte panels can be challenging depending on instrument speed—insufficient speed makes it difficult to monitor multiple concurrent compounds while maintaining data quality.

In this technical note, the improved MRM acquisition rate of the SCIEX 7500+ system—as low as 3 ms per MRM—was used to analyze 560 pesticides and 43 mycotoxins, representing a total of 1,462 MRM transitions in a single injection. For brevity, only the pesticide results are discussed here. The system features enhanced robustness from the new Mass Guard technology and includes the DJet+ assembly, which allows for easier and more routine user cleaning, reducing the need for service interventions and increasing instrument uptime.

Methods

Methods

Standard preparation: Stock solutions of individual analytes were prepared at 1 mg/mL in organic solvent. Spiking solutions were prepared by volumetrically diluting stock solutions with methanol or acetonitrile.

Spiked sample preparation:  Representative samples from different groups were chosen, including food with a high oil content (avocado), food with a low oil content (soybean) and difficult or unique commodities (tea). The matrices analyzed were lemon, mango, avocado, tea and soybean. The samples were spiked at different levels (1, 3, 10 and 30 ppb) and extracted using the QuEChERS CEN method.⁴ For cleanup, a Normec Groen Agro Control internal procedure was followed.

Chromatography:  Separation was performed using an ExionLC AD system from SCIEX and a Phenomenex Kinetex™ C18 (2.1 x 100 mm, 2.6 µm, 100 A ) column (P/N: 00D-4462- AN). A 28-min gradient (including equilibration time) was used with 0.1% formic acid and 2mM ammonium acetate in water as mobile phase A and 0.1% formic acid and 2mM ammonium acetate in methanol as mobile phase B (Table 1). The column temperature was maintained at 50°C, the flow rate was 0.3 mL/min and the injection volume was 1 μL.

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Mass spectrometry:  The SCIEX 7500+ system was used for data acquisition. The optimized source and gas parameters are listed in Table 2. Optimized compound-specific MRM parameters were used for the 560 pesticides and 43 mycotoxins, representing a total of 1,462 MRM transitions in a single injection.

Data processing:  Data collection and analysis were performed in SCIEX OS software 3.4.0.

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Fast-MRM

Fast MRM acquisition rate while maintaining sensitivity

When analyzing over 600 pesticides and mycotoxins in a single injection, instrument speed is critical for data quality— the instrument must provide low cycle times and sufficient data points across the chromatographic peak. The SCIEX 7500+ system is capable of <1 ms dwell times in combination with pause times of <2 ms and a settling time of 5 ms. The practical implications of these fast rates are that each MRM transition only requires a 3 ms MRM acquisition rate and, when combined with the Scheduled MRM algorithm, can result in the analysis of very large compound panels in a single injection.

To evaluate the MRM acquisition speed of the SCIEX 7500+ system, 4 different acquisition rates were compared:

• Treatment 1: 2 ms dwell, 3 ms pause and 15 ms settling
• Treatment 2: 2 ms dwell, 3 ms pause and 5 ms settling
• Treatment 3: 1 ms dwell, 2 ms pause and 5 ms settling
• Treatment 4: 1 ms dwell, 1 ms pause and 5 ms settling

In general, across the entire range of pesticide analytes, the sensitivity of the SCIEX 7500+ system was unaffected until the fastest acquisition rate (treatment 4) was used. Figure 2 shows a comparison of the different rates using butoxycarboxim as an example. Specifically, a minimal reduction (14%) in area count was observed under the 1 ms dwell, 1 ms pause and 5 ms settling time conditions (treatment 4). For this reason, the 1 ms dwell, 2 ms pause and 5 ms settling time conditions (treatment 3) were used for subsequent experiments. However, the data generated with treatment 4 could still be acceptable if the benefits of the faster acquisition rate offset the minor reduction in sensitivity.

Figure 2. Sensitivity performance as a function of total acquisition rate for butoxycarboxim.  The line graph (top) shows the peak area percent, normalized to the highest peak area, for butoxycarboxim at the 4 different acquisition rates: 2 ms dwell, 3 ms pause and 15 ms settling time (treatment 1); 2 ms dwell, 3 ms pause and 5 ms settling time (treatment 2); 1 ms dwell, 2 ms pause and 5 ms settling time (treatment 3) and 1 ms dwell, 1 ms pause and 5 ms settling time (treatment 4). The y-axis of the line graph is normalized to the area count in treatment 1. The graph shows a minimal area count decrease at the fastest acquisition rate. This trend is also shown in the XICs (bottom).

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In addition to instrument sensitivity, the MRM acquisition rate for treatment 3 (1 ms dwell, 2 ms pause and 5 ms settling time) showed good data quality with respect to precision and accuracy. Figure 3 shows the precision (%CV) and accuracy across the solvent-based calibration curve (n=3) for 3 example compounds: butoxycarboxim, methamidophos and saflufenacil. The results show that the mean %CV was <10% for all calibration standard levels in the 3 compounds, and the mean accuracy was 85%–111%.

Figure 3. Accuracy and precision of the solvent-based calibration standards (n=3) for butoxycarboxim, methamidophos and saflufenacil using the acquisition conditions of 1 ms dwell, 2 ms pause and 5 ms settling time (treatment 3). The data showed good accuracy and precision across the calibration standard levels for all 3 compounds. The mean %CV was <10% and the mean accuracy was 85%–111%. The concentration units are ng/mL

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Finally, the MRM acquisition rate of 3 ms (dwell and pause time) allowed for >10 data points across the chromatographic peak, which is critical for accurate quantitation. Figure 4 shows XICs for 3 compounds—methamidophos, dimethoate and triazophos—that were selected based on their retention times covering the entire gradient. The data show adequate data points despite the high degree of analyte concurrency.

Figure 4. Data points across the chromatographic peak for 3 example compounds using fast MRM acquisition rate conditions.  The XICs demonstrate sufficient data points for good quantitation, even when using acquisition parameters of 1 ms dwell, 2 ms pause and 5 ms settling time (treatment 3).

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Trace-level

Trace-level sensitivity

The fast MRM acquisition rate (1 ms dwell, 2 ms pause and 5 ms settling time) also showed low ng/mL (ppb) LOQ concentrations for the pesticides in the solvent-based standards. Figure 5 shows the LOQ breakdown in the calibration standards across the 560 pesticides. The majority (89%) of the pesticides achieved an LOQ of 1 ng/mL, which was the lowest concentration analyzed, with 97.9% and 99.5% achieving an LOQ of 5 ng/mL and 10 ng/mL, respectively. These low LOQs were achieved while injecting only 1 µL, highlighting the excellent sensitivity of the SCIEX 7500+ system. The low injection volume is important for food extracts to reduce matrix effects, and the lower matrix load can help prolong optimal instrument performance, reducing instrument downtime and service intervention.

Figure 5. LOQs determined for 560 pesticide compounds analyzed within the mixed standard solution using the fast acquisition rate conditions of 1 ms dwell, 2 ms pause and 5 ms settling time.  The chart above shows that most compounds (497) achieved an LOQ of 1 ng/mL with 51 and 9 providing LOQs of 5 ng/mL and 10 ng/mL, respectively.

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Figure 6 shows the overlaid XICs for 8 compounds (4 in positive ion mode and 4 in negative ion mode) at the lowest concentration analyzed (1 ng/mL), highlighting the sensitivity of the system along with the consistent ion ratios observed.

Figure 6. Overlaid XICs of 8 different compounds (4 in positive ion mode and 4 in negative ion mode) at the lowest concentration analyzed (1 ng/mL) using the fast MRM acquisition conditions. The 3 XICs above show overlaid quantifier and qualifier transitions and ion ratio lines with a tolerance of 70%–130%.

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SCIEX-7500+

SCIEX 7500+ system performance in spiked samples

The performance of the fast MRM acquisition rate method (1 ms dwell, 2 ms pause and 5 ms settling time) was evaluated in food matrix spikes (soybean, avocado, mango, lemon and tea) at 1, 3, 10 and 30 µg/kg. Figure 7 shows an example of carboxim (positive ion mode) and triclopyr (negative ion mode) at 1 µg/kg for all matrices. This figure highlights the sensitivity of the SCIEX 7500+ system and the use of 2 MRM transitions (qualifier and quantifier) with the qualifier ion ratio tolerance lines of 70%–130%.

A retention time shift between the matrices was observed, with triclopyr showing the most impact. Specifically, a portion of the triclopyr peak in the tea extract was outside of the retention time window. Retention shifts can occur when analyzing complex and diverse matrices, and the tea matrix data illustrate a potential risk when performing acquisitions using the Scheduled MRM algorithm with short retention time windows. However, the fast acquisition rate of the SCIEX 7500+ system can minimize this risk with the ability to widen the retention time window while maintaining relatively short cycle times and sufficient data points across the chromatographic peak. Ultimately, this reduces the need for retention time maintenance and frustration from sample reinjections to correct retention times.

The analysis utilized an external calibration curve (in solvent) for the quantitation of all pesticide compounds in the 5 matrices. Acceptable recoveries were shown for many of the target analytes in the 3 ppb spike (Figure 8). This was possible due to the sensitivity of the SCIEX 7500+ system, which allowed for a 1 μL injection volume and reduced matrix effects while maintaining LOQs down to 1 µg/kg

Figure 7. Overlaid XICs of carboxim and triclopyr at 1 µg/kg for all 5 matrices tested.  The images show the sensitivity of the SCIEX 7500+ system along with overlaid quantifier and qualifier transitions with ion ratio lines depicting 70%–130% tolerances. The XICs illustrate the retention time shifting, which occasionally occurs when analyzing diverse and complex matrices. The potential risk of shifting outside of the retention time window can be mitigated by the fast acquisition rate of the SCIEX 7500+ system, which allows for increased retention time windows while maintaining sufficient data points across the chromatographic peak.

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Streamlined-data

Streamlined data processing using SCIEX OS software

Processing large data sets can often be laborious and time-consuming. SCIEX OS software enables custom calculations, data flagging and filtering to streamline the data review process and ultimately save time. An example is presented in Figure 8, where a “Counter” column was built to calculate the number of matrices that passed the 80%–120% accuracy criteria in the 3 µg/kg spikes for each target pesticide. Then, the “Counter” column can be filtered to only show analytes with passing accuracy criteria for all food matrices.

Alternatively, when the “Counter” column is set to “4,” the results table shows pesticides in which a matrix fails the accuracy criteria. In this example, a custom filter was applied that shades matrices with low accuracies (<80%) in blue and those with high accuracies (>120%) in red. These customized, user-built features in SCIEX OS software help reduce the data review time by quickly visualizing all matrices in a single results table and rapidly highlighting compounds that passed or failed the performance criteria. Custom calculations can be tailored to any data set with numerous functionalities available.⁵

Figure 8. Easy implementation of custom calculations in SCIEX OS software to aid data review.  This table illustrates how custom calculations and flagging rules can be implemented in SCIEX OS software to visualize which compounds fall within 80%–120% accuracy. Here, the “Counter” column is used to filter the data to show how many compounds have passed the accuracy criteria, with 5 indicating all criteria are met and 0 indicating none were met. The last 3 compounds listed show that flagging rules were applied due to accuracy values falling outside of 80%–120%. For quick and easy visualization, custom flagging rules have been used to shade cells with values <80% in blue and cells with values >120% in red.

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Instrument-performance

Instrument performance during unscheduled analysis

In addition to using the Scheduled MRM algorithm for analysis, an unscheduled method was assessed to determine the number of unscheduled MRM transitions possible when considering the enhanced speed of the system. Over 830 MRM transitions were analyzed using a 1 s cycle time, equating to a 0.5 ms dwell time and 0.7 ms pause time. All data were collected in positive ion mode only. Figure 9 shows all transitions overlaid along with some chosen examples to focus on individual compounds (100 ng/mL). This demonstrates that sub-ms dwell and pause times are possible while still achieving acceptable performance.

Figure 9. Over 830 MRM transitions, collected using a 1 s cycle time, overlaid along with 3 highlighted examples. The top image shows all MRM transitions overlaid, and the bottom images show 3 chosen compounds—methamidophos, dimethoate and triazophos—from the total number of transitions analyzed.

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Conclusion

Conclusion

• The fast MRM acquisition rate of the SCIEX 7500+ system enables more compounds to be analyzed within a single injection, with dwell and pause times below 1 ms, while maintaining data quality
• The majority (89%) of compounds provided an in-vial solvent LOQ value of 1 ng/mL, maintaining the high levels of sensitivity expected from the SCIEX 7500+ system
• Matrix samples provide spiked LOQ values down to 1 µg/kg with minimal matrix effects observed
• Custom calculations and layouts in SCIEX OS software can be used to streamline data review and processing

References

References

1. Setting a new standard for instrument resilience. SCIEX webpage. Triple Quad 7500 Plus System (sciex.com)
2. European Union. Regulation (EC) No 396/2005 of the European Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin. http://data.europa.eu/eli/reg/2005/396/2024-02-26
3. European Union. Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain contaminants in food.
   http://data.europa.eu/eli/reg/2023/915/2023-08-10
4. QuEChERS. About the method.
   https://www.quechers.eu/method
5. Custom calculation and filtering packages for SCIEX OS software based on EU guidelines. SCIEX technical note, MKT-30732-A. https://sciex.com/technotes/technology/custom-calculation-and-filteringpackages-for-sciex-os-software

Acknowledgements

Acknowledgements

We would like to thank Said El Ouadi (SCIEX) for helping to make this collaboration possible.