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If we had a validated assay for a compound in human plasma, and now wished to assay for the compound in whole blood, would we need to perform a full or a partial validation of the assay in whole blood?
Thanks.
Jennifer Norman
Jennifer Norman
************
QA Manager
Division of Clinical Pharmacology
University of Cape Town
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The following message was posted to: PharmPK
Hi Jennifer
Partial validations are used when there is a minor change in the method.
Changing matrix within species is normally considered a minor change hence
could be validated partially. Full validations are performed when the
method is generated for the first time or if a metabolite is added to an
existing validated assay. Though changes to existing validated assays could
extend to a full validation depending on the case.
Best wishes
Manish
Manish Issar, Ph.D
Project Manager
Applied Biopharmaceutics, LLC
2010 Cascade Drive
Corona, CA 92879
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Yes. Stability, interferences, recovery will be different as will your processing. If you are anticipating processing the same way--- whole blood to plasma, you can use the plasma portion of your validated assay.
For the most part assays should include the starting point of whole blood, since drugs may bind to cellular components and platelets as well as proteins. This is rarely done however. A concession is to look at drug recovery from and stability in whole blood using a validated plasma or serum method. Differences in recovery and stability may lead to more rigorous collection and harvesting requirements and may suggest the addition of other agents-detergents or anticoagulants to improve stability and recovery from whole blood. After all, that is what the drug is in in the subject and the last time I checked, most often it was at 37C for mammals.
--
Changing matrix within a species is not a minor change. The environment a drug "sees" in plasma is very different from the environment it sees in urine, CSF, vitreous humor whole blood.
Edward F. O'Connor
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The following message was posted to: PharmPK
Agree with Ed that change in matrix within species is not a minor change. Though that's what is written in the USFDA guidance on Bioanalytical Method Validation, we have seen that sensitivities of the assay greatly depends on the choice of matrix (and of course the analyte of interest) esp. in LBAs! According to guidance a partial validation range from as little as one intra-assay accuracy and precision determination to a nearly full validation; however, the latter is more apt.
Ed:
We have assessed the recoveries of the analyte in whole blood by spike studies. Whole blood was spiked with analyte and left at bench-top, 22-25*C for 15-20 mins prior to separating plasma. The recoveries were then assessed in separated plasma (this was done to mimic the study procedure). Though we achieved desired recoveries, majority of the times we got hemolyzed plasma. Is there a better way to perform these studies and avoid hemolysis?
Thanks
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Hemolysis occurs when the cell mechanisms are no longer able to oppose water in/ions out. The faster you can get cells out the more likely you will reduce hemolysis.
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