Bioequivalence and Bioavailability Forum

Dear Ohlbe,

Thank you for your answer and additional information, I reviewed the presentation of Tandem Labs and their problem looks similar to ours. Unfortunately I couldn’t see the publication of Tan, yet as I wait our information department to buy it. If it's not too much trouble to you I will be grateful if you can send it to me.

❝ Good ! Though personally I would avoid CPDA due to the high plasma dilution, and possible variations in this dilution if the tube is under-filled in case of a difficult blood draw.

We knew about that problem but unfortunately the only blank blood, we can get from the blood bank is with an anticoagulant CPDA1.

❝ Do I understand it correctly, that you do not mix your samples (either vortexing, shaking, manual inversion or whatever), at any time, after complete thawing and before extracting them ?

Yes, after thawing, we do not homogenize the samples, as it is necessary to centrifuge the samples (to prevent blockage of SPE cartridges) and this initial homogenizing seems pointless. We suppose that our problem is not coming from homogenization because as I mentioned before we simulated this with samples for freeze/thaw stability and long term stability. These samples were spiked with standard in the same Cryogenic vials as study samples and freeze and thawed 3 times without homogenization and there were no deviations in the results using the same procedure.
However, today we'll try to homogenize the samples without subsequent centrifugation to eliminate this potential reason for failure.

❝ Yes, I would also consider this to be very unlikely, though you can't totally exclude it. But that would not be my #1 hypothesis.

May be I should have to explain further why we believe that our problem may be caused by the presence of heparin or in a difference between the plasma from the blood bank and the plasma from the volunteers. In the sample preparation we used ion-pair SPE and it was noticed that by increasing the acid (from heparin in samples or any other difference in the matrix) loading the recovery decreases (probably by affecting the ionization of IPR). But it’s not our #1 hypothesis.

Best regards, Yasen

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Edit: Standard quotes restored. @Yasen: I activated the e-mail in your profile. [Helmut]