I am about to run mice brain samples in the HPLC. I have optimised the standards however some methods are spiking their samples. Why do I need to do this?
Hi
If I correctly understand your question is about internal standard spiking. It's a common procedure that is very helpful in tissue analysis and methods validation. The same rules are used in plasma sample analysis in typical PK and LC analytics. Please find two useful documents below.
FDA
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
EMA
https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf
Best regards
Tomasz
Hi Jeannie,
Without knowing the details of your analysis, 'Spiking' can mean two things:
(1) Spiking/addition of an internal standard (IS): in that case you add a compound which is very alike your analyte of interest, but which is not naturally present in your sample. you just add a known amount of the IS to every sample, both calibrators and unknowns, and instead of basing the calibration on the absolute response of the analyte, the calibration uses the ratio of response between the analyte and the IS. If you add the IS before the sample preparation step, the IS will compensate for errors/variance/losses during the sample preparation step. If a IS is useful is dependent on your situation. In case the sample preparation is simple and the LC is working well an IS is not necessarily beneficial. John Dolan (LCGC) has a nice Q&A on this subject:
https://www.chromatographyonline.com/view/when-should-internal-standard-be-used-0
(2) Spiking/addition of a known amount of your analyte to your sample (Standard addition): this might be useful in case you want to rule out matrix effects caused by your specific mice brain samples. The main advantage of this method is that it can correct the matrix effect because exactly the same sample matrix is present both in calibration standards and the sample itself. A drawback of the method is, for example, that the amount of sample needed is much higher than in other methods because the sample is needed also for calibration standards. See for example:
http://zimmer.csufresno.edu/~davidz/Chem106/StdAddn/StdAddn.html
Hope it helps, Regards Hendrik
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