I’d like to grasp the finer points of method validation and know which guidelines to adhere to. For pesticides, I understand the Sante Guideline is applicable, but what about mycotoxins such as Aflatoxin, Ochratoxin, and Deoxynivalenol? Additionally, I’m seeking guidance on calculating the Limit of Detection (LOD) and Limit of Quantification (LOQ), especially since the slope affects these values with each linearity run. Also, my Blank, Solvent blank, and Reagent blank display small concentrations when processed by the machine software, even in the absence of a discernible peak. My CRM is in methanol and ethyl acetate . but i have prepared 1 ppm solution in acetonitrile and calibration points from 5 ppb to 200 ppb in MilliQ water Attached are my validation plan and the machine’s results. Suggestions for Refining the method validation and analysis of results if possible with example.
First off, I suggest you read ICH Q2 (International Council on Harmonization); I think this would be considered by most folk to be the defining standard for method validation. It has a usable definition of LOD and LOQ (which is calculated from the LOD). SANTE is required inside the EU
I personally use the USEPA method 40 CFR 136 Appendix B to calculate LOD; I find that this is the most restrictive of the methods but also the most reproducible. It gets you around the issues with changing slope. I then use the AOAC definition of LOQ (3.3 times the LOD); other places you will see it defined as the lowest calibration standard.
Your issue with small "hits" without apparent integratable peaks is an issue with your setting in your integration routine. You need to experiment with different settings until you find the correct combination that will not integrate noise. This is a very common issue.
I'm a bit uncertain as to why you prepared the calibration standards in water. The aflatoxins are quite soluble in HPLC-compatible solvents such as acetonitrile and methanol but not all that soluble in water. Is your sample matrix water with no preconcentration/cleanup steps?
For method validation on mycotoxin analysis:
- Consider guidelines from AOAC International, ISO, or FDA.
- Calculate LOD and LOQ based on signal-to-noise ratio.
- Investigate sources of contamination in blank samples.
- Ensure linearity of calibration curve and address matrix effects.
- Implement quality control measures for reliable results.
The guidelines for method validation for mycotoxins are similar to those for pesticides, and the European Union has published detailed guidance on this topic. The LOD and LOQ are typically calculated based on the slope of the calibration curve and the standard deviation of the blank measurements. Small concentrations observed in the blank, solvent blank, and reagent blank may be due to impurities in the solvents or reagents used, or to background contamination of the instrument. It is important to carefully select and purify solvents and reagents, and to run repeated blank measurements to assess the variability of the blank signal. In your validation plan, it is important to include details on the calibration standards used, the linearity of the calibration curve, the accuracy and precision of the method, and the LOD and LOQ. Additionally, it may be helpful to include a discussion of the matrix effects observed and any measures taken to mitigate these effects.
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