Is it Time to Embrace High-Resolution, Accurate-Mass Mass Spectrometry for Pesticide Analysis?

Using targeted methods, laboratories test on average between two and five hundred pesticides from a predefined list. If a pesticide present in the sample is not included in the predefined list, it will not be detected. These false negative results can raise concerns that the misuse of pesticides, or use of counterfeit pesticides, may go undetected. The development and implementation of ever more sensitive multi-residue methods enable more effective monitoring of the hundreds of possible pesticides, in hundreds of different types of foods.

Hundreds of thousands of maximum residue levels (MRLs) or tolerances have been set worldwide, creating an interest in the use of high-resolution accurate-mass (HRAM) spectrometers. These instruments can be operated in full-scan mode (no predefined list required) to screen for a higher number of pesticides in a single multi-residue analysis. Ideally, the method should not only detect, identify and quantify the targeted compounds, but should also provide additional information on a larger number of other pesticides not on the predetermined list. Evaluations of the main operational parameters and modes of HRAM instruments for pesticide analysis in fruits and vegetables have been outlined in two publications by the European Reference Laboratory (EURL).^1,2 (See section on high-resolution accurate mass pesticide detection below.)

Multi-residue screening

Triple-quadrupole (QqQ) mass spectrometers coupled to gas and liquid chromatographs are limited by the need to optimize acquisition parameters for each compound (only those from a target list will be detected) and their inability to perform retrospective data analysis.² HRAM technology,³ on the other hand, works by radially trapping ions around a central spindle electrode, providing high mass resolving power (>100,000), high mass accuracy, mass/ charge up to 6000 and dynamic range greater than 10^3.4

Thermo Scientific Orbitrap analyzers (Thermo Fisher Scientific, Waltham, Mass.) are available as single-stage mass spectrometers and benchtop hybrid instruments. The single-stage instruments provide high resolving power, but cannot isolate precursor ions prior to fragmentation, and thus can generate a number of false detects when relying on retention time data and one diagnostic ion. To overcome this issue, benchtop hybrid instruments incorporate a quadrupole mass filter. This enables isolation of the parent or precursor ion that is subsequently fragmented into product ions or fragment ions, providing improved selectivity and effective analyte identification. Combined with excellent linearity (6 orders of linear dynamic range, with no detector saturation) and high mass accuracy, these are the desired characteristics for the simultaneous analysis of targeted compounds and screening of a much larger number of unexpected residues, allowing laboratories to increase the scope of pesticide residue analysis.

High-resolution accurate mass pesticide detection

A benchtop hybrid system operating in full-scan mode with a simultaneous single MS/MS scan was used to analyze 139 pesticide residues in QuEChERS extracts of tomato, orange, pepper and green tea.1 Full-scan data were obtained at a resolution of 70,000 (at m/z 200), while MS/MS data in various working modes were acquired at a resolution of 17,500 (at m/z 200). Operation in full scan at a resolution of 70,000 generated a sufficient number of data points for quantification and selectivity to overcome most matrix interferences. With the exception of a few pesticides, excellent reproducibility and good peak shapes were attained, even in the crowded areas of the chromatograms and for difficult matrices.

To verify method efficacy, 100 fruit, vegetable, herb and marmalade samples were analyzed using the Thermo LC/Q-Orbitrap MS/MS, with the LC-QqQ-MS/MS used as a reference. The number of pesticides detected at a concentration above 10 μg/ kg was 183. For 93% of the samples, the difference between concentration determined by the QqQ and LC/Q-Orbitrap MS was less than 20%. High-resolution accurate mass full-scan mass spectrometry thus provides better selectivity and permits data analysis that is not possible with QqQ.

Researchers used the same instruments to analyze 170 pesticides at concentrations of 10, 50, 100 and 500 μg/kg with resolutions of 17,500, 35,000 and 70,000, respectively (Figure 1).² At a resolution of 17,500 with a 5-ppm mass tolerance, 91% of the pesticides at a concentration of 10 μg/kg were detected in tomato and 83% in tea. These rates increased to 98% and 88%, respectively, at a resolution of 70,000. At 17,500 there is a risk of false negative results due to deviations in mass accuracy (Figure 2). The peak shape improves at higher resolutions because of an increased number of data points due to better mass accuracy, especially for complex matrices, or in cases of low ion abundance (Figure 3).

Figure 1 – Influence of resolution (full scan) on detection rates of pesticides at different concentations in green tea matrix. (Figures reproduced with permission from Prof. Amadeo Rodríguez Fernández-Alba, University of Almeria, Spain, and head of the EURL for pesticides in fruits and vegetables).

 Figure 2 – Influence of resolution on selectivity and detection. Spectrum at RT = 3.95 min. Exact mass of pymetrozine 218.1036.

 Figure 3 – Influence of matrix on the number of data points acquired at different resolutions. Chlorpyrifos (XIC m/z 349.9336 ± 5 ppm) at 100 μg/kg.

Resolving power is one of the most critical parameters for avoiding false negative results. The low number of false negatives that were observed was attributed to a lack of analyte sensitivity exacerbated by ion suppression effects in the experimental conditions applied.

Linearity of detector response was excellent, even at a resolution of 70,000. This is in contrast to other high-resolution MS technologies such as time-of-flight, in which poor linearity of response often occurs due to detector saturation.

Conclusion

A benchtop hybrid quadrupole-Orbitrap mass spectrometer working in full-scan mode at 70,000 resolution with a mass tolerance of 5 ppm is an effective tool for screening hundreds of pesticides in a single analysis, even at concentrations as low as 10 μg/kg. HRAM instruments operating in full-scan mode with simultaneous MS/MS detection offer high levels of sensitivity and selectivity, a wide linear dynamic range, reproducible analyses and fragment ion information sufficient for compliance with European Union (EU) pesticide identification guidelines.⁵

Continuous software improvements are alleviating the data burden associated with rapid, automated multi-screening methods. HRAM-based methods accurately screen large numbers of pesticides, save time and money and more readily identify restricted or banned pesticides in food or animal feed.⁶

References

1. del Mar Gómez-Ramos, M.; Rajski, L. et al. Liquid chromatography Orbitrap mass spectrometry with simultaneous full scan and tandem MS/MS for highly selective pesticide residue analysis. Anal. Bioanal. Chem. 2015, 407(21), 6317–26.
2. Rajski, L.; del Mar Gómez-Ramos, M. et al. Large pesticide multiresidue screening method by liquid chromatography- Orbitrap mass spectrometry in full scan mode applied to fruit and vegetables. J. Chromatogr. A  2014, 1360, 119–27, 2014.
3. Makarov, A. Electrostatic axially harmonic orbital trapping: a high-performance technique of mass analysis. Anal. Chem. Mar 2000, 72(6), 1156–62.
4. Hu, Q.; Noll, R.J. et al. The Orbitrap: a new mass spectrometer. J. Mass Spectrom. Apr 2005, 40(4), 430–3.
5. SANTE/11945/2015. Method Validation and Quality Control Procedures for Pesticides Residues Analysis in Food and Feed; http://ec.europa.eu/food/plant/docs/plant_pesticides_mrl_guidelines_wrkdoc_11945_ en.pdf (accessed Dec 14, 2015).
6. 1st International Symposium on Recent Developments in Pesticide Analysis; http://info1.thermoscientific.com/pesticide-analysis (accessed Aug 17, 2015).

Richard Fussell is global applications manager, Thermo Fisher Scientific, Hemel Hempstead, HP2 7GE, U.K.; tel.: +44 1442 233555; e-mail: [email protected]; www.thermoscientific.com