Hello Everyone,
We are developing an amino acid HPLC method where we will use internal standard Norvaline. Can anyone share their ecperience of working with internal standrads? How do we go about it? Do we add the internal standrad at a much higher concentraion in the beginning? Kindly share your experiences/tips.
Thanks,
Hi Gurnoor Judge,
FDA, Evaluation of Internal Standard Responses During Chromatographic Bioanalysis: Questions and Answers Guidance for Industry (2019)
https://www.fda.gov/media/130451/download
FDA, Bioanalytical Method Validation Guidance for Industry (2018)
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
EMA, Guideline on bioanalytical method validation (2012)
https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf
UNODC Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized Materials and Biological Specimens (2009)
https://www.unodc.org/documents/scientific/validation_E.pdf
and check the topic:
https://www.researchgate.net/post/How_to_use_internal_standard_on_HPLC
You should spike all your samples by the same vol and concentration of IS. In such case all your samples for extraction procedure should have the same volume. Then when you have IS in your samples you can start with extraction, evaporation etc and finaly make analysis. The same with calibration curve. If you have detection like UV, FL EC concentration of IS should be at level which get peak more less close to your mean peak of analyte. For example if you have calibration curve between 1-100 then your IS should be close to highest of 50 for analyte. If you working with MS/MS its not so important.
Best regards
Tomasz
Hi Gurnoor,
take a look here:
https://www.researchgate.net/post/What_internal_standard_can_be_used_for_the_analysis_of_amino_acids_using_Pre-Column_HPLC2
Regards
Markus
Gurnoor,
IS has to be something which is not present in your sample matrix nor can be a degradation product of anything present in the sample. So, the best way to achieve this is to have a deuteriorated version of any endogenous molecule, for which it should be an deuteriotated amino acid. And no, the concentration does not need to be high. You have to check the sensitivity of your IS before you can add a channel for this in your method. We use 1 10 uM C13-D-Ala for our amino acid analysis by LC-MS/MS. I am attaching our paper here in case you might find it useful:
Article LC-MS/MS-Based Separation and Quantification of Marfey's Rea...
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