I need to validate a method that determines compound X in human plasma. However, this compound is endogenous to plasma and is present in a wide range of concentrations between "blank" plasma lots.
How do I approach the validation of this method to satisfy the FDA's GLP bioanalytical method validation guidance?
In other words, how do I prepare calibration curves and QC? Usually I would use a "blank matrix". But since I cannot obtain a blank matrix what do I do?
Would it be acceptable to have calibration curves in solvent and use a bulk plasma matrix (with a low analyte concentration) for QCs. I could add a QC0 to the method and perform blank subtraction to calculate QC recoveries?
Anyone have experience with this or can suggest a better approach?
Dear Fabiola,
in general you can use different approaches. First of all you could show and validate that external calibration is applicable. Some researchers also use surrogate matrices like e.g. saline buffers + BSA to create an artificial matrix or you can also do charcoal stripping of your sample to remove your analyte. However, all these approaches will not let you determine the true matrix effect. I prefer the surrogate calibrant/analyte approach. Here you prepare calibrants and QCs by spiking isotopic labelled analogues of your analaytes into the real matrix and using these signals for quantification of your target analytes.
Here´s also some literature on the topic
Article Quantitation of Endogenous Analytes in Biofluid without a Tr...
Article Quantification of steroid hormones in plasma using a surroga...
You can subtract the values already available in the blank sample, however, you can also mimic the blank whether it is urine or blood... Etc. But the former choice is better I mean to consider the same blank then subtract , check also
Article Novel liquid chromatographic determination of cystatin C in ...
http://www.eurjchem.com/index.php/eurjchem/article/view/1658/pdf_1658