Uncovering the hidden potential
Researching pesticides, a lab stumbled on something interesting about supercritical fluid chromatography (SFC)
European Union Reference Laboratory for Pesticide Residues in Fruit & Vegetables, University of Almeria.
At the University of Almeria in Spain, Víctor Cutillas and a team of analytical chemists were using the conventional standard method to analyze pesticide residues in fruits and vegetables. In the interests of time, the team was also applying a secondary, “back-up” method. In the process of doing their work, however, the team became aware of how well the secondary method was working. Was it perhaps performing even better than the primary method? Víctor Cutillas decided that he needed to know the answer to that question. So he embarked on a comparative investigation of the two systems.
Víctor Cutillas analyzes pesticide residue at the EU Reference Laboratory for Pesticide Residues in Fruits & Vegetables (EURL-FV) at the University of Almeria in Spain. Pesticide residue analysis is critical for ensuring food safety and regulatory compliance, and the EURL-FV plays a crucial role in keeping consumers safe by improving the quality, accuracy and comparability of the food-safety test results.
The most commonly used method in pesticide residue analysis is reverse-phase liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). This is due to its robustness and broad applicability for semipolar and some thermolabile pesticides. However, supercritical fluid chromatography (SFC) – widely used in areas such as the pharmaceutical sector – is also applied occasionally in pesticide residue analysis because of its advantages, e.g. in ionization.
One day, Víctor and the EURL-FV team were very busy with their analyses, using both LC-MS/MS and SFC-MS/MS methods. As they ran more and more samples, they began to notice that thermolabile compounds, which usually suffer in the ion source, showed remarkable stability using SFC. In addition, certain chemical groups displayed far better sensitivity using SFC than they had ever seen using LC. And, while they expected some reduction in matrix effects using SFC, the results often went beyond their expectations – sometimes dramatically.
Comparative performance of LC and SFC for multiresidue pesticide analysis in food
Wondering whether the potential benefits of SFC for their work had been underestimated, Cutillas decided to conduct a formal study of the two methods. The study was to directly compare the performance of LC and SFC on 215 pesticides in fruits and vegetables using the same mass spectrometer (a Nexera UC coupled to an LCMS-8060, both from Shimadzu). The focus was on sensitivity, matrix effects and ionization efficiency across different food samples, including tomato, leek, onion and orange.
Residues in Fruit & Vegetables, University of Almeria.
Sensitivity and ion source temperature
The study assessed the optimum electrospray ionization (ESI) interface temperatures for both techniques, which is crucial for achieving the highest sensitivity in pesticide detection. This information is summarized in Figure 1. LC exhibited slightly higher sensitivity at 350 °C, with 95 % of pesticides identified in a pure solvent and 92 % in the tomato matrix at this temperature. In comparison, SFC showed consistent results across a broader temperature range, with 99 % of pesticides determined in a pure solvent and 95 % in the tomato matrix even at lower temperatures, such as 200 °C and 125 °C.
In complex matrices such as leek and onion, the differences became more pronounced. For leeks, LC identified 80 % of pesticides at 350 °C but only 69 % at 125 °C. SFC, however, maintained a higher identification rate of 83–85 % across all temperatures. Similarly, in onions LC determined 89 % of pesticides at 350 °C but dropped to 67 % at 125°C, whereas SFC identified 86–89 % consistently. This indicated that SFC can be more versatile in handling various ion source temperatures without compromising sensitivity, offering significant benefits for laboratories dealing with a variety of food samples.
Chemical group sensitivity
Different chemical groups responded uniquely to each chromatography technique. For instance, among the 70 compounds showing higher sensitivity in LC, 27 % were organophosphates. Carbamates, urea derivatives and triazines also showed high sensitivity in LC, with 10, 7 and 6 pesticides, respectively, exhibiting more than 50 % higher area values than SFC.
Conversely, among the 77 compounds showing higher sensitivity in SFC, 22 % were triazoles, such as bromuconazole and cyproconazole. Other groups, such as benzoylureas and neonicotinoids, also performed better in SFC. The nonpolar nature of the supercritical CO2 used in SFC, coupled with its unique elution mechanism, likely contributed to this enhanced sensitivity. The average logPow values were similar for both techniques, indicating that polarity alone does not determine the best technique for each pesticide. This nuanced understanding helps laboratories select the most appropriate technique based on the chemical nature of the analytes they encounter.
Matrix effects and ion suppression
Matrix effects can significantly impact the accuracy and reliability of pesticide residue analysis. The study revealed that SFC had a lower degree of ion suppression than LC, particularly in complex matrices such as leek, onion and orange. For instance, in the leek only 5 % of pesticides in LC had low or non-existent matrix effects (0–20 %), whereas 28 % of pesticides in SFC fell into this category. In the onion, the results were similar, with 11 % of LC-analyzed pesticides showing low matrix effects compared to 29 % for SFC.
In the orange matrix, the difference was even more striking. Only 7 % of pesticides analyzed by LC had low matrix effects, compared to 53 % for SFC. These findings are crucial, as they demonstrate that SFC is more effective at reducing ion suppression caused by coextracted matrix components. This can be attributed to SFC’s nebulization and sampling efficiency, which minimizes coelution with matrix components. Furthermore, the different elution profiles of SFC and LC play a vital role, with SFC’s unique mechanism reducing the overlap between analytes and interfering substances, thereby enhancing sensitivity and identification accuracy (Figure 2).
Cutillas’ comparative study revealed that, while both LC and SFC have their merits, SFC offers distinct advantages in analyzing complex food matrices with high coextracts. Its ability to maintain sensitivity at lower ion-source temperatures makes it particularly suitable for thermolabile compounds. Routine laboratories can benefit from integrating SFC into their workflows for enhanced pesticide residue analysis. The choice between LC and SFC should consider the specific needs of the laboratory, including the types of pesticides to be analyzed and the complexity of the food matrices involved. With its consistent high-sensitivity performance across a range of temperatures and lower matrix effects, SFC provides a robust and versatile alternative for modern pesticide residue analysis.
Cutillas also notes that SFC is a more sustainable method of analysis as it produces less waste. The CO2 it uses is recyclable, whereas LC relies on higher amount of organic solvents. In addition, by using equipment such as the Nexera UC, labs can run both methods on the same platform without additional hardware.
Knowledge is a complex construct, and sometimes what we think we “know” is only a mask for our ignorance. In this case, what was thought to be a backup turned into a breakthrough. The true knowledge had been sitting there all along, waiting for a curious scientist to notice it. And now, thanks to Víctor Cutillas and the team at the EURL-FV in Spain, a more efficient, more sensitive and more sustainable way to ensure food safety has been revealed. The result is progress: for labs, for science and for humanity.
Equipment Víctor used in this comparative study
- A unified chromatography system: Nexera UC from Shimadzu
- A CO2 pump
- A back-pressure regulator
- A triple quadrupole mass spectrometer: LCMS-8060 from Shimadzu
- An electrospray ionization (ESI) source
Cutillas V., Ferrer C. & R., Fernández-Alba A. (2021). Liquid chromatography versus supercritical fluid chromatography coupled to mass spectrometry: a comparative study of performance for multiresidue analysis of pesticides. Anal Bioanal Chem. 413: 5849–5857. https://doi.org/10.1007/s00216-021-03565-4.
