Pesticide testing laboratories are under ever-increasing pressure to analyze a greater number of pesticides with diverse physico-chemical properties in shorter turnaround times. Adding more pesticides to multi-analyte methods will require the analyst to compromise, as results for some pesticides may vary due to a variety of factors. This prompts the question: which analytical step is the weakest link in the pesticide residue workflow?As Dr. Katerina Mastovska (Associate Scientific Director, Nutritional Chemistry and Food Safety, Covance Laboratories) alluded to at the 1st International Symposium on Recent Developments in Pesticide Analysis (link to symposium) there are weaknesses in each step in the workflow. The presentation Never-Ending and New Challenges for Pesticide Routine Testing Laboratories is available on demand. A number of the pesticides can simply ‘go missing’ at each and every one of the steps in the process, leading to false or inaccurate results. Katerina summed up the situation poetically with a story titled ‘What Happened to Ten Little Pesticides,’ which begins:
Ten little pesticides were in the sample,
One didn’t get extracted and stayed behind,
And one degraded, poor little guy,
How many pesticides were in the extract?
Eight little pesticides were in the extract,
One was lost in the clean-up without a trace,
And one was diverted to the LC waste,
How many pesticides made it to the MS?
The fate of the remaining 6 pesticides can be found by viewing the presentation. In the case of targeted analysis it is common practice to spike samples or extracts with analytes of interest and determine the recovery. If the recovery is unacceptably low or the response unexpectedly poor, then it may be possible to take corrective action. This is all very well for targeted analysis, but the consequences for non-targeted or unknown analysis are more profound. Analysts beware!
Is solvent extraction a weak link?
The QuEChERS acetonitrile extraction method is probably the most popular extraction method used by pesticide residues chemists for both targeted and non-targeted analysis, because it has proven to be Quick Easy, Cheap Effective Robust and Safe. The principle of the method has been illustrated in the whiteboard video Introduction to QuEChERS Sample Preparation Technique. The base pH-sensitive compounds, such as captan, folpet, and dicofol, are degraded non-acidic matrices. Dr. Mastovska and colleagues modified the method by buffering the pH (link to article) by adding acetic acid and sodium acetate to improve the stability and recovery of these compounds. In doing so, the recovery of acid pH-sensitive compounds, such as pymetrozine, may decrease. The challenge of multi-analyte methods is always to reach an optimum compromise, since perfection is rarely attainable. In the case that pesticides appear to go missing because of matrix suppression of ionization, these can often reappear to some extent, after simple dilution of the extract, and hence matrix, prior to injection. Since mass spectrometry coupled to either liquid chromatography (link to LC-MS product page) or gas chromatography (link to GC-MS product page) can detect, identify and quantify hundreds of pesticides in a few minutes, detection surely cannot be considered the weakest link.
Is sample processing the weak link?
Perhaps 11 pesticide residues were in the sample received into the laboratory, but 1 residue was lost during comminution of the sample (typically 1-2 kg containing 5-10 units in the case of fruits and vegetables). Sample processing has been defined by Hill and Reynolds as the procedure (e.g., cutting, grinding, mixing) used to make the analytical sample (10-15 g for QuEChERS) acceptably homogeneous with respect to analyte distribution prior to removal of the analytical portion (link to article). It is surprising that analysts are prepared to expend substantial effort to improve the precision of the instrumental method by a few percent, but ignore the much larger possible errors associated with this unavoidable step needed to produce smaller analytical test portions for analysis. Fussell et al reported previously that the issues of insufficient homogeneity and/or losses of residues can be overcome to some extent by comminution at low temperatures in the presence of dry ice (link to article).
It now seems that sample processing is once again a hot topic. In a session titled “Going from Macro to Micro: The Future of Sample Processing in Residue Analytical Methods” at the 13th IUPAC International Congress of Pesticide Chemistry, the use of a two-stage cryo-processing procedure to allow the use of 100 mg samples and development of high throughput was discussed. Benefits include:
- Decreased usage of consumables (including solvents)
- Higher sample throughput
- Possible higher extraction efficiency due to smaller particle size
But is the homogeneity sufficient to represent the original laboratory sample? The same topic was also debated with equal enthusiasm in a session entitled ‘Advanced Sample Comminution Techniques’ at NACRW 2015 (link to workshop). A detailed summary of the discussion points is included in a recent publication titled Sampling and Sample Processing in Pesticide Residues Analysis (link to article).
There are many interdependent factors to be considered. A good starting point would be for laboratories to verify that the comminuted analytical sample is sufficiently homogenous to allow withdrawal of suitably-sized representative test portions.
What about Extraction Efficiency?
Decreasing the particle size of the comminuted test portion size will increase the surface area and may increase the extraction efficiency of incurred residues, but some difficulties may still occur in the solvent extraction of complex matrices like tea, spices and animal feedstuffs. In such cases, another option that can be considered is the use of Accelerated Solvent Extraction (link to ASE product page). The concept is simple: elevated pressure is used to keep the solvents in a liquid state as the temperature is increased above their boiling points. By using increased temperature and pressure, solubility of analytes is increased, viscosity of solvents is reduced, and hence analyte diffusion into the solvent is improved, thereby improving extraction efficiency and reducing extraction time. This process is described in the short whiteboard video Introduction to Accelerated Solvent Extraction Technique. Further information on the use of ASE was presented by Dr. Fabrizio Galbiati at the 1st International Symposium on Recent Developments in Pesticide Analysis.
As you can see, the answer to the question “which is the weakest link in the pesticide residues workflow?” is subject to opinion, but my vote goes to sample comminution. If you are interested in pesticide analysis and would like to make a comment or have a different opinion, then I’d like to hear your thoughts and experiences.
Additional Resources
If you’re interested in the analysis of pesticide residues in food, check out our Food Community , Pesticides Community, and virtual on-demand pesticide symposium, which feature the latest on-demand webinars, videos, application notes, and more. You can also visit these additional resources:
- Thermo Scientific Application Note: AN1138, (2015), Fast and Ultrafast LC-MS/MS Methods for Robust and Reliable Analysis of Pesticides in Food Using the... (downloadable PDF).
- Thermo Scientific Application Note: AN52291, (2012), Multi-Residue Pesticide Analysis in Herbal Prod...(downloadable PDF
- Selective pressurized liquid extraction as a sample-preparation for persistent organic pollutants an... (link to article)
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