Webinar: Developing a Residue Analytical Method: The Critical Role of Extraction Efficiency
In this webinar, Derek Brown provides an overview of the key method development stages when developing a residue analytical method.
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Webinar Q&A: Developing a Residue Analytical Method: The Critical Role of Extraction Efficiency
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Derek Brown, Director, ResChem, a Smithers company, provided an overview of the key method development stages in the webinar, “Developing a Residue Analytical Method: The Critical Role of Extraction Efficiency.”
Read below for responses to the questions asked during the audience Q&A.
Q: In your experience, have you needed to address extraction efficiency for processed commodities? If so, is the same strategy used as for the "usual" non-processed commodities?
A: We haven’t conducted cross validation studies for processed commodities but have designed extraction methods for processed crops based on available metabolism data (following the same procedure as per “normal crop samples”). Our approach for processed commodities would effectively be the same as our approach for non-processed crops, i.e.:
- Design a suitable extraction method based on metabolism data.
- If there is no radiolabelled data for the processed crop but data is available for a crop from the same matrix group, it could be possible to bridge between the two matrix types, i.e., use the radiolabelled crop data to support the proposed extraction method for the processed crop (if they are from the same matrix group). As an example, if radiolabelled data is available for apple (high water content) this data could be used to support a proposed extraction method for apple puree.
A: This depends on the residue definition, the difficulty of the extraction procedure, and the nature of the sample. It may take anything from a day or two to a couple of months.
Q: How do you handle analytes that are NOT stable?
A: Develop method conditions to stabilize the analyte(s) as much as possible. Investigate the stability times of the analyte(s) and work within those parameters. Use isotopically labelled Internal Standards to compensate for any analyte losses during the extraction procedure.
Q: How do you develop procedures for very polar analytes?
A: It is likely that if we are dealing with very polar analytes, the extraction system will use a highly aqueous content which was generated from the extraction efficiency data from a metabolism study. It would be a good strategy to focus on the chromatographic conditions by looking at a suitable HPLC column such as polar embedded C18, ion exchange, HILIC etc. and if none of these give a good result, look at derivatization (this will increase the MW and make the resultant analyte less polar so that it may chromatograph on a RP system).
Q: With the rather recent EPA guidance on preference for extraction solvents with different dielectric constants rather than harsh extraction, I thought we were moving away from harsh techniques, but it appears that you still need to use harsh techniques for systemic residues, etc. How do you ensure that you do not break the molecule to something that was not a metabolite with the harsh techniques?
A: This can be evaluated by fortification of aliquots of control matrix with known amounts of the analyte of interest and then performing the ‘harsh’ extraction technique followed by quantitation (e.g. LC-MS/MS analysis). If the analyte of interest is present at the same level as fortified, then the ‘harsh’ technique is verified. If recovery values for the analyte of interest are low, we would analyze the extracted sample for relevant metabolites to determine if there has been conversion during the extraction step. If there is conversion to metabolites during the extraction procedure, an alternative extraction method would be investigated. Where a harsh extraction is required but breakdown of the analyte is observed a different approach would be to develop a common moiety method using a harsh extraction method where residues (and metabolites) are intentionally broken down to a common moiety. Residues would be determined as the common moiety. Common moiety methods are rarely needed but can be a good option to consider if all else fails.
Q: How much % recovery is accepted in the extraction efficiency step? Any range?
A: In the metabolism study, extracted radioactivity (TRR) should be > 70% and the sum of radioactive residues for all components of the residue definition should be >50% of the extracted radioactivity. For cross validation studies, residues calculated using the test method should differ by no more than 30% when compared to residues calculated using the extraction method from the metabolism study.
Q: Do we focus only on the % recovery in the extraction efficiency study or consider the metabolite(s) chromatographic profiling, which should match the sample analysis during the initial metabolism study?
A: In the extraction efficiency study, we need to consider the residue definition and the extraction efficiency for each analyte which is part of the residue definition. It should be noted that that only quantitative data for targeted known analytes is considered during the extraction efficiency study. Different extraction methods may be required for different analytes which form part of the residue definition, e.g. if metabolites are very polar relative to the active ingredient.
Q: How often do you do radiovalidation to show efficiency with incurred residues in water or soil matrices to support E-fate studies?
A: Radiovalidation with incurred residues for soil or water are not routinely conducted by Smithers but we routinely validate methods used in laboratory soil and water simulation studies (non-radiolabelled). We routinely perform radiovalidation for metabolism (plant or animal) studies and confined rotational crop studies and the same principles could be applied to soil and water to support environmental fate studies.
Q: What about extraction efficiency for animal matrices when no radiolabelled samples are ready, and no repetition of livestock residue is allowed?
A: Extraction efficiency in food of animal origin must be demonstrated for commodities showing residues >0.01 mg/kg in metabolism/feeding studies at the 1N rate. As no repetition of livestock studies is allowed, the extraction method used in the metabolism study would have to be used for the residue analytical method. Alternatively, a cross-validation study could be conducted if there are samples available with incurred non-radiolabelled residues e.g. from non- radiolabelled feeding studies. Both these options assume there is metabolism data available.
Q: What incurred residue levels are optimal in samples for use in a cross-validation study?
A: Residues would be expected to be >0.01 mg/kg for relevant analytes. As a general rule, the higher the residue level the more reliable the data will be from the cross-validation study. Ideally, we would want residues ca. x10 LOQ or higher. Optimal incurred residue levels may be increased by application of the formulation of interest at higher than 1N rate during the associated field trials.
Q: To perform cross validation, extraction procedures coming from metabolism studies must be available; what if there is no inforamtion?
A: If no information on the extraction solvents used in the metabolism study is available, it is recommended to approximate extraction efficiency by performing an indirect cross validation with samples containing incurred residues and testing different solvents e.g. QuEChERS (acetonitrile/water 1/1 v/v), DFG S19 (acetone/water, 2/1 v/v), Chemelut (methanol/water, 2/1 v/v) and EN 12393:2013 SweEt (ethyl acetate). See section 6, page 17 of SANTE/2017/10632 Rev. 5. If the analyte(s) are very polar it would be sensible to consider water as an additional solvent. It would also be worth considering analyte solubility i.e. if the analyte is very soluble in one particular solvent it would be sensible to include this solvent. There could be useful information in the public domain e.g. renewal assessment report which could be worth considering.
Q: How do you usually obtain samples with incurred residues, as it requires a field trial? Is the trial made especially for the validation study?
A: If sample matrices containing incurred residues at appropriate levels are already available then additional field trials to show cross validation are not required. If samples are not available, field trials should be conducted with application of the pesticide of interest contained in a suitable formulation to generate required incurred residues. Matrices containing incurred residues are then extracted using the methods of interest and compared to the extraction method developed in the metabolism study which showed acceptable extraction efficiency.
Q: How do you see the need for evaluating extraction efficiency for rather liquid matrices such as milk? Are such samples really extracted or are residues simply dissolved in the applied "extraction" solvent? E.g., for honey the guideline states that no extraction efficiency is required.
A: For liquid matrices there is a strong argument for these to be considered in the same way as honey. The guideline states “Honey is considered as a relatively simple matrix without possible incorporation or residues bound to matrix components. No separate studies to address the extraction efficiency in honey are needed.” However, any available metabolism data for high water content crops should be considered when selecting the “extraction” solvent for liquid matrices e.g. if there is suitable metabolism data to support acetonitrile/water (50/50, v/v) as an extraction solvent for high water content commodities it would be sensible to use this solvent for extraction / dilution of liquid matrices. If the proven extraction method for high water content crops is relatively simple and not time consuming it would be worth considering the use of the same procedure for liquid matrices so that any potential questions related to extraction efficiency can be navigated.
Q: Regulators are pushing for multiresidue (QuEChERS) method for all actives. If the typical ACN/water QuEChERS extraction procedure does not sufficiently extract residues from sample matrices, what have your developers tried to optimize extraction for QuEChERS methods?
A: It largely depends on the nature of the residues, but the regular QuEChERS procedure can be adapted to optimize extraction efficiency. Parameters to consider include optimizing the pH of the extraction solvent, increase the amount of time of the extraction, use a high speed dispersing device (IKA Ultra Turrax, GenoGrinder), addition of water to hydrate dry crops prior to extraction, incubate samples at a higher temperature etc. ACN/water will not be a suitable extraction solvent for all analytes, in which case a procedure other than QuEChERS would have to be considered.
Q: What is the approach if the recovery of the extractable TRR in the metabolism study was low?
A: There is information in SANTE/2017/10632 Rev. 5 (page 12 – 13). If <70% of the TRR is extracted in the metabolism study, extraction efficiency may still be okay if the remaining non-extracted radioactivity is:
- represented by radiolabels incorporated into biomolecules or integrated into the crop matrix
- cannot be released from the crop matrix without chemical conversion (e.g. using strong acid or base)
- attributed to known metabolites which are not part of the residue definition
Q: Do we need demonstrate matrix effect in extraction efficiency assays?
A: When comparing methods it is usual to prepare ‘matrix-matched’ standards for residue quantitation. During the method validation according to SANTE/2020/12830, rev.2, matrix effects are evaluated.
Q: What is procedure for volatile analytes? Is there any specific guideline for nullifying the loss during procedures?
A: For volatile or unstable analytes, the use of isotopically labelled Internal Standards to compensate for any analyte losses during the extraction procedure may be valuable.
Q: What can we do if we don't have the metabolism study for the commodity group that we have to analyze?
A: A field trial could be set up to generate samples with incurred residues. A cross validation study could be conducted where the extracted amount could then be compared using at least three different extraction solvents. If metabolism data is available for a sample from a different commodity group, it would be sensible to include the extraction method from this study in the cross-validation study. This procedure results in a relative extractability, demonstrating which solvent is the most suitable for the extraction of incurred residues. The uncertainty with this option is the lack of reference values from samples with radiolabelled incurred residues. Bridging between high water content and acidic matrices is acceptable for slightly acidic matrices.
Q: The guideline states that we have to test different solvents, do you have any suggestions on which solvents we could to test?
A: It’s dependant on the nature of the analyte(s) of interest but a good starting point would be using the solvents from monitoring methods e.g. QuEChERS (acetonitrile/water 1/1 v/v), DFG S19 (acetone/water, 2/1 v/v), Chemelut (methanol/water, 2/1 v/v) and EN 12393:2013 SweEt (ethyl acetate). If the analyte(s) are very polar it would be sensible to consider water as an additional solvent. It would also be worth considering analyte solubility i.e. if the analyte is very soluble in one particular solvent it would be sensible to include this solvent. There could be useful information in the public domain e.g. renewal assessment report which could be worth considering.
Q: Is this true? The extraction efficiency test should be performed with the radiolabelled samples containing the residues incurred from the metabolism studies?
A: Yes. In an ideal situation, extraction efficiency for new methods would be integrated into the experimental design of metabolism studies using samples with incurred radiolabelled residues. For cross validation studies, samples with non-radiolabelled incurred residues can be used.
Q: How to improve extraction efficiency?
A: Extraction solvent composition can be optimised (considering solubility data), pH of the extraction solvent can be optimised, mode of extraction (shaking, maceration), length of the extraction procedure, and temperature can all influence extraction efficiency. For stable analytes more “aggressive” conditions can be employed without loss of analyte. Where stability of the analyte is a problem, this needs to be considered when using more aggressive extraction conditions.
Q: We do not understand the bullet in the final summary slide that "extract efficiency CANNOT be estimated....", please explain more...
A: Procedural recoveries are performed by addition of the analytes of interest to a sub-sample of the matrix. They are therefore NOT incurred residues and therefore cannot be used to determine extraction efficiency. Procedural recoveries are used as checks on method volumetrics and also the effects on analyte stability in various situations (pH, solvents etc.), analyte(s) loss during extract purification steps or solvent evaporation.
Q: How do you prove hydrolysis step without available commercial conjugated standard?
A: If hydrolysis efficiency is proven in the metabolism study then the same procedure can be used for the residue method without the need for further verification.
Q: What are the factors that affect extraction efficiency?
A: Extraction solvent composition (considering solubility data), pH of the extraction solvent, mode of extraction (shaking, maceration), length of the extraction procedure and temperature can all influence extraction efficiency.
Q: If different extraction metabolism studies for same crop, then extraction efficiency by cross validation versus all of them?
A: For cross validation, the most appropriate extraction method (e.g. the one where the most amount of the residue is extracted) could be used as the reference method for the cross validation study. As long as the metabolism method selected is acceptable, it should not be necessary to include all metabolism extraction methods.
Note: In general, it is difficult to give an answer that will work for every scenario and each case needs to be considered based on the information available. We would always advise our clients to discuss specific requirements with the relevant authorities and, if possible, have an agreed approach in place ahead of any experimental work being conducted.