In June, Smithers Study Director
Andrea McGuinness presented a
SANTE method validations webinar which included a review of
SANTE/2020/12830 rev. 1, a discussion of strategies and lessons learned from its use, and an overview of what to expect from rev2.
Our audience posed several questions during the interactive Q&A session. Some questions were answered live and are included in the
webinar recording. Due to time limitations, responses to questions that could not be included during the live event are listed below.
Q: What are the acceptance criteria for residual plot data?
A: SANTE2020/12830 does not have specific criteria, only citing that residuals should be randomly distributed upon visual inspection. Our interpretation is that this should look more like a scatter plot rather than a trendline.
Q: Would the modification of linearity range be something that would be performed during method the development phase, prior to the "official" validation?
A: Yes, all aspects of linearity are evaluated prior to validation so that we have confidence in quantitation of the fortified recovery samples.
Q: Can we use Dichloromethane in liquid-liquid extraction for water analysis?
A: No, due to dichloromethane’s toxicity classification under Regulation (EC) No. 1272/2008, it cannot be used in any aspect of analytical method validation. The only exception to this is in a bridging study where the performance of a replacement solvent is being demonstrated.
Q: What are the acceptability criteria for final extract stability?
A: Final extracts are considered sufficiently stable if the recoveries of the fortified samples are within the acceptable range of 70-120%. We also consider the %RSD relevant in assessing stability, as a higher RSD can be indicative of instability or adsorption in some cases. Additionally, evaluating how close to the original value percent recoveries are is also important. If the original results were all ~115% and re-analyzed samples are now ~75%, this could also allude to potential instability.
Q: Can hexane or dichloromethane be used in normal phase HPLC methods?
A: Hexane may be used in analytical method validation. Due to dichloromethane’s toxicity classification under Regulation (EC) No. 1272/2008, dichloromethane may not be used.
Q: How do you handle validation of crop group matrices. Do you only do representative crops?
A: If crops fall within the same matrix group, they do not require a separate full validation. A reduced validation set is still recommended, in addition to procedural recoveries demonstrating the method is performing adequately. If not, a full validation would be required. Additional information is provided in OECD ENV/JM/MONO(2007)17.
Q: Can 1-chlorobutane be used for SANTE validations? It is a chlorinated solvent but not classified as Category 1A or 1B carcinogenic, mutagenic or toxic for reproduction.
A: If a solvent is not listed as Category 1A or 1B carcinogenic, mutagenic or toxic for reproduction, then it can be used in analytical methods.
Q: Can we exclude L1 and the highest linearity concentration in a PA batch?
A: Assuming L1 is the lowest calibration standard, this should not be excluded from a PA (pre-application) batch as the low standard is considered the LOD. The highest linearity concentration could be excluded assuming there are still an adequate number of linearity points, however we would recommend running the same method as the validation.
Q: Is the hazardous reagents the only change from SANTE/2020/12830 Rev.1?
A: Yes, the re-classification of hazardous reagents is the only content change to SANTE/2020/12830.
Q: What does the term “extraction efficiency” refer to? Is this in the guideline?
A: True extraction-efficiency, by definition, is an assessment of method performance using samples with incurred residues, typically conducted with radiolabeled material. Refer to the guidance document SANTE 2017/10632 for more detail. In this context of analytical method validations to support non-radiolabeled ecotoxicology studies (for example) this is also a term used to describe the acceptable percent recovery and repeatability of a method, such as during liquid-liquid extraction or extraction of soil.
Q: What concentration is used for stability determination of standard solutions?
A: There is no specific criteria outlined in the guideline. As a conservative approach, we recommend performing the stability determination (5 replicate injections) for both the lowest and highest calibration standard or working solution being used, which remains consistent with the referenced document SANTE/11312/2021 (chapter F).
Q: If recoveries of dose concentration verification samples during a stability study are outside of acceptance criteria due to the solubility of the analyte, what are some options to address this?
A: Solubility of the test material is a separate issue from stability
. Ensuring that a material is soluble during processing is critical, so evaluating the extraction procedure would be a good first step. Additionally, ensuring the samples are homogenous via adequate mixing could help, or extracting the entire sample to remove the homogeneity variable. If final extracts or stock solutions used during stability assessment are exhibiting solubility issues, further method development may be required.
Q: For aquatic studies of pesticides where the test solutions are not homogenous, what are some strategies used to mitigate this issue?
A: Undissolved material can negatively impact many test organisms and study designs. Testing to the limit of solubility, preparation of a water accommodated fraction (WAF) via filtration or other preparation method, adding a co-solvent or adjuvant, or using a specialized test design can all be viable options.
Q: How do you identify an outlier in linearity of the calibration range?
A: SANTE/2020/12830 does not outline any criteria for determining an outlier for calibration standards specifically. As a result, our standard practice is to use the accuracy value to determine if the calibration standard should be included in the calibration line. Generally speaking, 80-120% is considered acceptable and anything outside of this range would be excluded (providing the remaining standards satisfied the minimum criteria set forth in the guideline). Smithers has our internal criteria for calibration curves defined in an SOP.
Q: Do existing method validations, fully validated under SANCO guidance few years ago, need to be repeated according to the new SANTE guideline? What about methods that were validated recently under rev 1 of the SANTE guidance?
A: If all studies utilizing the previously validated SANCO method are complete, then revalidation would not be required. Our experience is that new studies should be supported with a SANTE validated method in most cases. As a result, we recommend “upgrading” SANCO methods to SANTE compliance to ensure acceptance. Methods validated according to SANTE/2020/12830 revision 1 do not need to be re-validated following the release of revision 2.
Q: When conducting “bridging” work for an ILV study, do all method validation requirements need to be met when using the hazardous reagent and the new replacement? Or are there only certain validation aspects that need to be conducted and bridged.
A: Conservatively, all validation requirements should be met for the method with the replacement solvent. There may be cases where some parameters are not required, but this would be evaluated on a case-by-case basis.
Q: Can the highest linearity standard be excluded if it appears to be an outlier?
A: The linear range of a method should range from 30% of the LOQ to 120% of the highest concentration in solution. It would not be recommended to exclude the highest calibration standard during a method validation if you are no longer able to quantitate 120% of the highest concentration in solution within the calibration range. If this is the case, we recommend a repeat validation or re-analysis with a newly prepared calibration curve. If however your curve range still meets guideline criteria while rejecting the highest standard, it would be recommended to simply define your validated range as the shorted range and exclude that concentration going forward.
Q: Are both transitions (for LC-MS/MS analysis) monitored during concurrent or in-study validation?
A: Yes, both transitions (where required) would be monitored both for standalone and concurrent SANTE method validations. Additionally, the confirmatory transition may also be monitored for subsequent supported studies, as that data can be a useful tool for interpretation of out of specification results.
Q: What are some strategies one could use to salvage a method that is being validated concurrently and is not performing as expected (i.e., it is non-compliant)?
A: It would largely depend on the nature of the failure. If recoveries are high or low, we’d evaluate stock solution preparation and matrix effects to determine if it was a processing error. If we’ve lost sensitivity on the instrument, maintenance might be performed and investigative samples run. There are some parameters on instrumentation that are considered “non-critical” to the method validation and are widely accepted as needing to be optimized per instrument. Re-tuning these parameters to ensure optimal performance would be something that can be done mid-study to avoid holding samples for analysis at a later time.
Q: What is the percentage between nominal concentration and measured concentration that is acceptable for calibration standards?
A: This information is not directly outlined in SANTE2020/12830. It is our standard practice to consider each calibration standard to be acceptable if the measured concentration is within 80-120% of nominal concentration, provided all other linearity parameters are also met as described in the guideline.
Q: Why there is difference in validation recovery ranges for the LOQ and High recovery samples (70-120%) vs. dose concentration samples (80-120%)?
A: Acceptable ranges of mean recoveries are specified as 70-120% with a precision ≤20% RSD. For food/feed of plant and animal origin, the range of mean recoveries as well as the precision will vary based on the concentration levels being validated (high concentrations which are “easier” to extract and analyze have a more conservative recovery range, while lower or more “difficult” levels to extract and analyze have wider recovery ranges). Certain experiments, such as in support of ecotoxicology studies, may have specific dose verification recovery ranges identified in those particular guidelines. If you have a stricter recovery range required to support testing, it would be our recommendation to validate that specific range to ensure your method can routinely achieve it.
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