Bioequivalence and Bioavailability Forum

Dear Ohlbe,

Thank you for your fast answer. I had tried to be as short as possible, but as I see I will have to explain in details our problem to clarify the picture.
We validated bioanalytical method using plasma from a blood bank with a CPDA1 anticoagulant. The same anticoagulant was used in the S-Monovette for the study samples. In the stage of development and pre-study validation we didn’t encountered any problems with all parameters of the method (very good precision, accuracy, selectivity, linearity, recovery et.ec.). During study validation all quality control samples (more than 150) had very good precision and accuracy, but for our great surprise we have a big difference in the results for incurred samples. We didn’t have any problem with IS (the area of QC samples and study samples had CV~2%). The big part of the second results (ISR) had highest concentration to the primary results, like there is a difference in concentration (or pH – because we use ion-pair solid phase extraction in sample preparation) between different levels in Cryogenic Vials of the volunteers plasma samples.
The samples of volunteers are thawed at room temperature (without homogenization) and centrifuge, we simulated all condition of different stages of samples preparation and storage conditions (have proof even stability for the transportation in dry ice) and didn’t get any deviation from the results of the QC’s samples.
It looks very strange to have such difference in concentration levels, as Metformin is negligibly bound to plasma proteins and we didn’t meet such problem with QC’samples, or with the samples from freeze/thaw stability which are also not homogenized after thawing and are centrifuge before sample preparation.
The only difference between samples for quality control and samples of volunteers for which we suppose is the existence of another anticoagulant (possibly heparin) in samples of volunteers. We assume that in the clinical center it was used to prevent blood clots in cannula and in this way it came to the samples.

It looks very unlikely to have such problem from small quantities of heparin, but it is the only difference which we found from the QC samples.

We will tried to homogenized study samples with vortex and without further centrifugation before taking the aliquot, to tried to eliminate this strange problem if the reason is unhomogenized study samples.
We would appreciate if anyone shared his opinion if it is possible the reason for this problem to be unhomogenized samples or presence of heparin in the samples or if anyone have any other ideas about the reasons for this results.

PS: In the method we used UV detection.

Best regards, Yasen

---

Edit: Full quote removed. Please delete anything from the text of the original poster which is not necessary in understanding your answer; see also this post! [Helmut]