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Dear sir,
I am developing a method for simultaneous estimation Sildenafil (2-1000ng/mL) and its metabolite DesMethyl Sildenafil (2 - 150 ng/mL) in human K2EDTA plasma, I have used Sildenafil D3 as internal standard, The problem I faced is, there was a drop in IS response to even less than 50% when the concentration of Des Methyl Sildenafil increased, and so the accuracy keep on failing for Des Methyl Sildenafil, while the sildenafil P&A comfortably passes. Is there any method to stop the response variation in internal standard, particularly deutriated standards.
Mobile phase used was 10mM Amm.Acetate pH 5 and Methanol (20:80) using a ACE C18 column 4.6 *50mm, with a flow rate of 0.600mL/min
Please give me some guidance regarding this issue.
With warm regards
S.Mythies Kumar,
India.
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The following message was posted to: PharmPK
Dear S.Mythies Kumar
First check the internal standard stability in solution & matrix also
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Hi,
You should see if there is IS supression with increase in metabolite concn. If so, you can probably reduce the inj vol and check;else, try with a longer gradient program. Hope it helps.
-AB Rao
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The following message was posted to: PharmPK
Hi,
Does your Sildenafil co-elute with the metabolite DesMethyl Sildenafil in chromatography?
XD
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The following message was posted to: PharmPK
My first approach would be to separate the elution time of the des methyl peak from the Sildenafil peak as much as you possibly can.
Longer column
lower flow rate
etc..
Good luck!
Rachel
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