Bioequivalence and Bioavailability Forum

Dear all,

That's a nice question (at least for me! It makes a change from statistics ).

Have a look at the paper of the 2006 Crystal City conference:

There was recognition of a distinct difference between
EDTA and heparin-containing plasma and that a bioanalytical
method validated for one could not be used for the other
without some revalidation of the method but no consensus
was reached for the need for cross-validation when using
the same anticoagulant with a different counter-ion. As
an example, attendees could not agree on the degree of
cross-validation necessary for a method validated using
sodium-heparinized plasma when it was applied to a
lithium-heparinized sample.

Heparin is something quite special, as lithium can play funny tricks in LC/MS/MS (lithiated adducts. You can have sodium adducts too, but you will get these even without sodium heparin as you have sodium in plasma anyway). Switching between sodium and lithium heparin can lead to some surprises.

But what about EDTA ? The counter-ion is the same: K. And you have potassium in your samples anyway, so the small difference in K concentration between K₂ and K₃ EDTA may not make any difference. During the meeting the FDA guys (who wanted a revalidation) mentioned a difference in pH which could lead to differences in stability, but they failed to give any specific example. I haven't seen one in the litterature, but I didn't do an extensive search.

Actually what worries me more in the switch between K₂ and K₃ EDTA is rather the sampling tube itself. If you are using BD Vacutainer, you will have noted that K₃EDTA comes with glass tubes, while K₂EDTA comes with plastic tubes. Have a look at this paper for more details on matrix effects due to polymers and plasticisers:

Mei H, Hsieh Y, Nardo C, Xu X, Wang S, Ng K, Korfmacher WA
Investigation of matrix effects in bioanalytical high-performance liquid chromatography / tandem mass spectrometric assays: application to drug discovery.
Rapid Commun Mass Spectrom. 2003;17(1):97-103

❝ You surely can, but chances are high that it will not be accepted.

There are chances for US submission. What about EU ? For the time being the assessment of the bioanalytical part by EU regulators is... well... not consistently as thorough as it could be. Sometimes the counter-ion is not mentioned in the analytical report ("heparin", with no precision), but no question is raised. Sometimes the anticoagulant used in the validation and to prepare the standards and QCs in the study is different from the study samples (typically, the lab gets blank CPDA plasma from a blood bank, and analyses heparin or EDTA plasma), and no question is raised...

Due to possible analytical problems, and to avoid spoiling my samples, I would do some partial validation when switching between K₂ and K₃ EDTA. But rather for my own peace of mind than for the regulators'.

Regards
Ohlbe