Webinar Q&A – Navigating Sample Complexity in Residue Analysis

Webinar Q&A – Navigating Sample Complexity in Residue Analysis

In September, Smithers Study Director Sidney Bluemink presented the webinar “Navigating Sample Complexity in Residue Analysis”, an overview of challenges presented when working with a variety of complex sample matrices. Read below for responses to the questions asked during the audience Q&A.

Q : What kind of analytes are more common under hybrid Mass Spectrometry?
A: Accurate mass spectrometry such as time-of-flight and orbitrap systems, or hybrid techniques including differential ion mobility are useful techniques where there are isobaric interferences within the matrix at the analyte signal using common unit resolution mass spectrometry. They allow a much more refined window of analysis that increases detector selectivity and therefore signal to noise although sometimes at the sacrifice of sensitivity so it can be a balancing act. We find that these systems are incredibly useful when dealing with small and highly polar molecules, where clean up techniques and chromatography is more challenging.

Q: Please describe your SOP for making decisions (or determining) the LOD and LOQ.  Do you use a statistically based method in this decision?
A: Within pre-approval residue chemistry methods we follow SANTE/2020/12830 as our standard document. This has removed any statistical criteria for setting limits of detection or quantification, it instead looks at these from the assumption that these levels have been set. As such the LOD is equivalent to the lowest calibration standard and the expectation is this would have at least a 3x signal to noise ratio, and the LOQ is equivalent to lowest validated level with sufficient recovery, precision, and specificity (lowest fortification level) and this will usually be at least 3.3x the concentration of the lowest calibration point concentration in final extract form. Typically, the method is refined to be good enough to meet the limits we need to meet and so these parameters can be seen as being the lowest levels of method validation rather than true LOQ/LODs.
Therefore, in development we ensure our LOD level is >3x S/N and LOQ is >10x S/N, and reference 40 CFR Part 136 Appendix B where relevant especially if further robustness testing versus the levels chosen are required.

Q: Have you encountered deuterated ISTDS that are suppressed by matrix more readily than the target analytes during ESI-LCMS analysis? This would result in over-correction of the target analyte.
A: On very rare occasions we have seen some cases where the deuterated IS has not ionised in a consistent manner to the non-deuterated test item (e.g. Decalin). In such scenarios it is also possible that matrix suppression would differ. Likewise, on one occasion we observed what we considered to be deuterium exchange in the gas phase. When we set IS methods, we assess selectivity of the method for the IS, as well as confirming tracking of the IS versus test item for matrix effects. Avermectins have been a class of chemicals where we have seen divergence between the deuterated IS and the test item on occasion. Where we see variation, we would consider modifications to the method to improve the selectivity. An isotopically labelled IS can be extremely useful but problems can still occur. We therefore should not assume an IL-IS will prove to be a panacea for all analytical problems.
Q: Could you share the DEA bonded phase you're using if that's commercialized?
A: The DEA bonded phase was developed by Waters originally for SFC applications but has been tested in UPLC applications.
This document shows some of the work conducted in employing this column for polar pesticide separations.
Q: Once the method had been validated, is the method use by commercial laboratories?  Do you use the EPA ATP guidance?
A: Typically, the methodologies we develop are validated for the purposes of the pre-approval registration study only and are largely not used by commercial laboratories. Environmental Chemistry methods (ECM) are developed and validated to meet requirements of 40 CFR Part 158 and the ECM guideline (850.6100) and is intended to be used to develop data for submission to EPA under TSCA, FIFRA, and FFDCA (40 CFR Part 158). Methods developed for ECMs are typically single analyte methods for a targeted analyte of interest (including degradates or transformation products) so may not always be suitable for established multi-residue methods. Additionally, analysis of pollutants under 40 CFR Part 136 and the Alternate Test Procedure (ATP) program is governed by the Clean Water Act although this is not a space we have conducted much work in.

Q: Why have some of the emerging technologies you mentioned not been widely adopted?
A: For the most part, it comes down to getting the return on investment. Instruments are costly to purchase and maintain and in residue chemistry most analytical problems can be resolved using LC and GC and their iterations. Residue chemistry labs usually work on tight budgets, so investment needs to be clearly justified. Initial uptake of new technologies can be slow where cost of changing processes is high, but once that uptake is past a certain threshold, it tends to accelerate. In my presentation the range of techniques and adaptions covered range from very low cost to considering hefty investments, but sometimes the large investment in instrumentation is needed to support the lower cost sample extraction procedures that come as a result of the increased instrument performance. Eventually I see laboratories making those investments when the analytical requirements merit it. Currently papers covering the adaptions to QuEChERS methodologies are a fraction of those detailing the first QuEChERS iteration, that can be because of the relative specificity of some of those adaptions, but also because it takes time for the wider scientific community to build that literacy.  For example, it took over 15 years for pre-filled SPE cartridges (developed in 1977) to be widely adopted across the scientific disciplines.

Q: How can automation be employed for the types of analysis you spoke about?
A: When using QuEChERS and its iterations in a standardised approach, there are already robots available that can employ these workflows with minimal human intervention. There are modules available that can dilute, shake, centrifuge, transport, heat and cool your sample among others. They can perform automated liquid/liquid extraction and solid phase extraction. These robots can be directly connected to the instrument and most of the human work goes into programming the workflows. Automation has been around for a while but in risk assessment labs, methods can vary a great deal which can make automation more difficult and the time to programme many different methods particularly time consuming. In contrast automation makes a lot of sense in labs which use very routine workflows and large sample sizes. However, the advance of application software’s makes the transition to automation easier and easier as user interfaces become more intuitive. Sometimes very basic automation can be used to remove routine, simple tasks where errors result from lack of concentration rather than technical difficulty and allow chemists to focus on more specific/critical parts of the sample workflow.

Q: Do you see methodologies involving TOF-MS or orbitrap technologies for regulatory residue studies like MOR’s and residue declines?
A: There seems to be a much slower uptake in accurate mass techniques for quantitative analysis in regulatory residue studies. That can be down to the training element, cost, and perceived robustness of higher-end instrumentation, where the majority of challenges can still be successfully overcome with traditional QqQ instruments. There can also be the need for global methods that can be transferred across a lot of laboratories: the more specific the technology the harder that will be. Increasingly though these mass specs can solve analytical problems that triple quadrupole technology cannot. This is due to their inherent selectivity coupled with sensitivity improvements that can now match that of QqQs in many cases. For this reason, there is an increasing consideration of these instruments for specific analytical challenges as the relative price point lowers and they are able to meet the performance required of them. It should also be considered that using accurate mass spectrometry often removes the need to quantify a confirmatory ion of a given analyte, which can reduce the amount of data to process.  For multi-residue methods there is now a lot of literature for their use with labs taking advantage of their high scan rate alongside mass resolution and excellent signal to noise. The high scan rate is important to distinguish multiple and sometimes co-eluting analytes in a single analytical run.

Q: Is the QuEChERS approach limited by the solvent that can be used versus extractability requirements as detailed in your presentation. Most QuEChERS methods use acetonitrile, this seems a key step.
A: The workflow I showed was an example and there are ways to adapt the initial extraction if that is required. It is possible to use other solvents such as methanol, isopropyl alcohol, and ethyl acetate, for example. QuEChERS also allows pH adjustment by using buffered salts or simply by adding acids/bases to the solvents and sample better comminution is also a useful way to improve extraction efficiency. Conventional QuEChERS generally works best when there is 3:1 organic to aqueous ratio at the salting-out step. With the initial solvent we can dilute, evaporate, and add water to achieve that ratio before the salting out step.

Q: Are there challenging matrices that are simply around how you process the sample before extraction and analysis?
A: Yes, some commodities can be tough to process or it can be challenging to get a homogeneous mixture. Some are just difficult to handle, and it might depend on who you talk to. Fibrous plant materials, nuts and sugary commodities can present particular difficulties. Matrices can also become challenging simply by not having the correct processing equipment for that commodity. In my personal experience, sieving soil that is very clay-like is enough to bring a person to the brink of despair! For tissues and plant material, switching from dry ice to liquid nitrogen often helps with processing tough materials. The colder temperature makes the material more brittle and easier to break up. Even for tissues others pose little challenge when processed with dry ice can show improved processing quality when processed with liquid nitrogen. For clay-like soil though, I’m sorry to say I’ve not seen any solutions to make sieving it any easier.

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