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Advice from the group would be most appreciated.
I have finally managed to successfully validate a robust and
sensitive LC/fluorescence method for a test article, in its free base
form.
As is always the case, a succinate salt version has since been
manufactured and we are now due to perform several animal pk studies
with this compound.
Historically, standards of the free base have been prepared in
methanol and this is how the method has been validated. I have since
compared stock solutions of freebase methanolic solutions with
succinate salt aqueous solutions and have demonstrated an approx 20%
increase in response (peak area) with the salt solutions.
Obviously this will translate into a similar error when measuring
plasma from animals dosed with the succinate version, being
artifically higher.
Question is....
How do I amend my validation to have a viable method for measuring
the succinate salt, do I need to completely revalidate with correct
std/qc solutions or can I perform a quick cross validation as the
analyte is essentially the same?
Regards
Simon
*********************************************
Simon Clark M.Sc.
DMPK Specialist
Analytical Pharmacology,
Antisoma Research Laboratories,
St Georges Hospital Medical School,
Cranmer Terrace,
London SW17 0QS.
Direct +44 (0)20 8772 7071
simon.clark.-a-.antisoma.com
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The following message was posted to: PharmPK
When you prepare stock solution using succinate salt,
did you consider the salt factor. I mean if you have
the same amount of weight of free base and succinate
salt, for the salt, you need to add less solvent (how
much less depends on salt factor).
If you molecular weight is X, then for the salt, the
MW becomes X+118.09. So the salt factor Y is
(X+118.09)/X.
When you add the solvent to salt, if for free base you
need add Z mL, then for the salt, you only add Z/Y mL.
Hope this can help.
Xiaodong
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The following message was posted to: PharmPK
Hi Simon,
Doesn't it strike you as odd that you see a 20% increase in peak area
using LC/fluorescence?
Regards,
Frederik Pruijn
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.-at-.auckland.ac.nz
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The following message was posted to: PharmPK
Hi Simon,
As you have validated method for free base, now you should prepare
the stock with the salt form after weight and salt correction.
So your method remains the same. The comparison (20%) of salt and
base form should be in same solvent and with salt correction.
If you are changing the solvent, then the response may change based
on solubility. You can run P&A and stability.
Further, for animal studies you can go for dose calculation as free
base. 10 mg/kg of free base, in that case you will be estimating
correct concentrations corresponding to dose may be 10+x of salt form.
Hope this helps
Gurpreet
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Hi Xiaodong
yes I have factored in the Mr for the salt/free base ratio.
I have since found that its has been my injection matrix that has
determined my difference in response, ie varied percentage of MeoH/
aqueous, thus when diluted in same phase, I now have 96% response in
freebase vs succinate salt.
Therefore, I presume my original validated method is viable for
analysing samples from animals (dosed with the salt). And I intend to
construct plasma calibration stds with succinate compound as per free
base method.
I still believe that I am required to perform a cross validation check.
What would this involve, single inter run (usual response parameters
linearity, sensitivity, repro etc)
or just a stock solution comparison
BTW Fredrick, solubility has been considered here, but have now
resolved the 20% issue. My main focus now is, is it ok to start using
the succinate salt instead of the freebase in the validated method?
Thanks again
Simon
*********************************************
Simon Clark M.Sc.
DMPK Specialist
Analytical Pharmacology,
Antisoma Research Laboratories,
St Georges Hospital Medical School,
Cranmer Terrace,
London SW17 0QS.
Direct +44 (0)20 8772 7071
simon.clark.-a-.antisoma.com
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The following message was posted to: PharmPK
Hi Simon,
I don't feel I am qualified to answer your question since you and I are
operating in different environments with a different 'set of rules'. My
view (.....) is that you can simply compare stock solutions as long as
nothing else changes (e.g., varied precentage of MeOH/aqueous). Should
be o.k. to use succinate if the comparison doesn't throw up any
'surprises'.
On a different note, if dosing calculations etc. are done on a
mole/molar basis, simply using the Formula Weight (FW), it is easier to
avoid mistakes between different salt forms of the same compound IMHO.
Good luck with it.
Frederik
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.aaa.auckland.ac.nz
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The following message was posted to: PharmPK
Hi,
You can make two stock solutions, one using the free
base and the other using the salt. Then you can use UV
detecor to check peak height or peak area of these two
solutions. This experiment can eliminate matrix
effect.
If the responses are same using UV detector, then you
can draw the difference was caused by matrix effect.
Then you can prepare two calibration curves in plasma
matrix, one curve using the free base and the other
using the salt.
In addition, how much std working solution do you use
to spike into plasma?
If you think the solvent you use caused this
discrepancy, I guess you may need to perform at least
an intra-day validation and also stock stability
evaluation.
However, I have not come cross with this kind problem.
One thing I would suggest is to put switching valve
there and send salts 9solvent front to the waste.
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