HPLC analysis of bupropion

[Manar_fawzy]

It is my first time to post here. I hope I would find help. I have been investigating some xenobiotics for their inhibitory action on Liver CYP 450 enzymes. I am facing a problem in measuring hydroxybupropion as there is no peak detected by HPLC. I have used 0.4 mg/ml protein and final concentration of Bupropion 50 uM. The eluent I used was 75% 50 mM o-phosphoric acid buffer PH 3 : 25% acetonitrile. . Could you tell me how can I overcome such a problem and is it helpful if I use higher concentration of microsomes or Bupropion or both?
Thanks in Advance

[bbcmouli]

Dear Manar_fawzy,

Being high UV activity, Bupropion it should give peak at 50 uM.
Initially u should have to setup concentration range by injecting Individual solutions.
if u still not getting peak at 50 uM concentration, u may play with Injection volume, mobilephase & UV detection.
Ignore if u already did that.

Regards,
Mouli

[Manar_fawzy]

Hi
Thanks for your help. I would give more details. This is reversed phase chromatography. I used in all my trials 20uM injection volume. UV wave length is 214. Device is Shimadzu, Column is Waters symmetry C18 5um,3.9x150mm. What do you think I should try?
What do you recommend should I make the mobile phase more or less polar?

Kind Regards
Manar

[tom jupille]

Unless you are working from a published method, the easiest approach would be to start with a pure standard and run a gradient from, say 5%to 95% organic. Ideally, you will see a single peak in there somewhere. Look at the % organic at the time that peak elutes and back off about 10% from that as a reasonable starting point for developing an isocratic separation. Fine-tune as necessary from there.