Non-volatile Pesticides Residue Analysis by LC-MS/MS in Fruits and Vegetables

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) is one of the most powerful techniques for pesticide residue analysis for detecting and quantifying in ultra-low trace level in fruits and vegetables.

It is ideally suitable for highly polar, thermally liable and non-volatile compounds.

This technique provides excellent sensitivity/selectivity and discriminates analyte and matrix signal more efficiently than GC-MS (Alder et al) or LC-MS (Hernandez et al.)

LC-MS/MS is capable of simultaneous detection and quantification of multiple pesticide residues in a single run. This is especially important in fruits and vegetables, which may contain residues of different pesticides due to multiple treatments during cultivation.

PATRS OF LC-MS/MS

There are four basic components are, for the most part, standard in all mass spectrometers: a sample inlet, an ionization source, a mass analyzer and an ion detector. Some instruments combine the sample inlet and the ionization source, while others combine the mass analyzer and the detector.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) are techniques most often employed for multiresidue pesticide analysis in food due to their high sensitivity and selectivity, ability to screen many pesticides from various chemical classes in very complex matrixes in a single run.

The control of pesticide residues in food is required by law to assure human food safety and safeguard the consumers’ health. In Europe, maximum residue limits (MRL) of pesticides permitted in products of animal or vegetable origin that are intended for human consumption have been established by Regulation (EC) No. 396/2005 of the European Parliament and Council on pesticide residues.

Nowadays, a high-performance liquid chromatograph coupled with a QQQ tandem mass spectrometer, working in the multiple reaction monitoring (MRM) mode, is the most frequently used platform used in the analysis of pesticide residues in food. Figure below compares the types of mass spectrometers used in the analysis of pesticide residues in food in recent years.

Comparison of the usage frequency of the individual analyzers and tandem systems interfaced with liquid chromatography and used in the analysis of pesticide residues in food samples (notation explained in the text). According to the Scopus database, keywords: pesticide, food, multiresidue methods, LC-MS.

SAMPLE EXTRACTION FOR LC/MS/MS

Extraction is a fundamental process in pesticide residue analysis. Extraction may be defined as the procedure or step adopted to isolate the pesticides from the samples. This is accomplished by employing suitable solvents which remain in contact with the sample for a specific duration. Extraction and clean-up methods vary greatly with the matrix used. It largely depends on the moisture, fat and sugar contents of the commodity. The quantity of sample to be taken for extraction will depend upon the expected levels of residues as well as the sensitivity of technique or tool employed in the subsequent step of identification and quantification. An ideal extraction procedure should be capable of extracting 100% of pesticides only, leaving behind other materials. However, this is not the actual practice. An extraction method should satisfy the following requirements:
  • Rapid
  • Small solvent usage
  • Good recovery of the target pesticides: 70 to120%.
  • Robust: RSD (relative standard deviation) of the recoveries less than 20% at LOQ.

Advantages of LC-MS/MS for Non-Volatile Pesticide Residue Analysis

  1. Sensitivity and Accuracy: LC-MS/MS provides high sensitivity and accuracy, capable of detecting pesticide residues at levels well below regulatory limits (e.g., in the parts per billion or parts per trillion range).

  2. Multiresidue and Multiclass Capability: LC-MS/MS can detect multiple pesticide residues simultaneously, allowing the analysis of a wide range of chemicals in one single analysis, making it ideal for regulatory compliance where multiple pesticide residues are often present.

  3. Low Matrix Interference: The MS/MS capability minimizes matrix interference, which is often a challenge in complex food matrices like fruits and vegetables.

  4. Comprehensive Coverage: LC-MS/MS is suitable for detecting a wide variety of pesticides, including herbicides, fungicides, insecticides, growth regulators, and other agrochemicals, many of which are non-volatile and thermally labile.

  5. Non-Volatile Compounds: Unlike gas chromatography (GC), which is primarily used for volatile compounds, LC-MS/MS can efficiently analyze non-volatile pesticides that would otherwise be difficult to detect due to their lack of volatility and thermal instability.

  6. Versatility Across Pesticide Classes: LC-MS/MS can analyze a broad range of pesticide classes, including herbicides, insecticides, fungicides, and growth regulators, as well as other agrochemicals. This versatility is important in ensuring comprehensive monitoring of food products.

  7. Simultaneous Detection of Multiple Pesticides: LC-MS/MS is capable of detecting and quantifying multiple pesticide residues in a single run, making it ideal for multiresidue analysis. This capability is essential because fruits and vegetables are often treated with a combination of pesticides during cultivation.

  8. Matrix Effects Minimization: One of the challenges in pesticide residue analysis is the interference from complex food matrices (e.g., fruits, vegetables). LC-MS/MS, with its tandem mass spectrometry capabilities, can significantly reduce matrix effects, making it easier to obtain accurate results even in challenging sample matrices.

  9. Effective Clean-Up: Coupled with advanced sample preparation techniques like QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) or solid-phase extraction (SPE), LC-MS/MS can effectively clean up samples to minimize interference from co-extracted compounds, leading to more reliable analysis.

  10. Highly Accurate Quantification: The quantitative accuracy of LC-MS/MS is one of its major strengths. Using calibration curves and internal standards, it provides precise quantification of pesticide residues at trace levels, which is crucial for regulatory compliance.

  11. Low Detection and Quantification Limits: LC-MS/MS is capable of detecting pesticide residues even at extremely low levels, ensuring compliance with stringent regulatory limits set by agencies such as the FDA, EPA, and EFSA.

  12. Adaptability to Standards: LC-MS/MS methods are highly adaptable to meet regulatory standards for pesticide residue analysis. Regulatory bodies like the European Food Safety Authority (EFSA), the U.S. Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA) require methods that can detect pesticides at very low levels with high precision, which LC-MS/MS meets effectively.

  13. Reliability for Compliance Testing: Given its sensitivity, selectivity, and ability to detect a broad range of pesticide residues, LC-MS/MS is widely recognized and approved for compliance testing, making it the gold standard in many regulatory environments.

  14. Handling Complex Food Matrices: Fruits and vegetables contain complex organic and inorganic substances that can interfere with pesticide detection. LC-MS/MS, especially when combined with effective sample extraction and clean-up techniques, can handle these complex matrices and deliver accurate results, even in challenging cases such as high-fat or high-sugar content samples.

  15. Quantitative Analysis: LC-MS/MS provides accurate and reproducible quantification of pesticide residues, which is crucial for determining the compliance of food products with safety standards.

  16. Qualitative Analysis: LC-MS/MS also enables the qualitative identification of pesticide residues through the analysis of molecular ions and fragmentation patterns, adding an additional layer of confirmation for the presence of a pesticide.