Chromatography Forum

hi guys ,
I am using USP analytical method for rifampicin isoniazide capsule , and there is something a bit confusing
for assay test it is gradient method solvent A ( phosphate buffer pH 6.8 : acetonitrile 96 : 4 respectively )
solvent B ( buffer : acetonitrile 45 : 55 )
run starts isocratically 100 % A for 5 minutes then gradually increase in solvent B till reach 100 % within 1 minutes then from minute 6 to 12 isocratically 100 % B then it didn't specified any re equilibration time , this because rifampicin is highly non polar, but dissolution is isocratically method with only 5 % methanol ,
assay method on column c18 25 cm but dissolution method on c18 30 cm
on performing dissolution test I didn't get rifampicin peak for at least 1 hour ( time I left for first run ) I expected something like that before working but i thought it would be a some trick and tolerence is specified to be NLT 75 % for rifampicin and 80 % for isoniazide ,
did I miss something ?!!!

Hi Hossam,

Nope, you don't seem to have missed anything. As to the re-equilibration, you could add a step into the gradient program to run for 5-10 column volumes of Eluent A--this usually works well enough to get back to starting conditions. (You may be doing this already).

As to the Dissolution LC method...those are the correct conditions as per the USP monograph. My best advice is to try the exact LC column mentioned in the PF and see what happens. Any well-covered, modern C18 column will likely give you fits with the method as written. As I recall, the PF-recommended phase is Waters' Microbondapak.

Best Wishes!

Hi Matt
Thanks a lot for your precious reply
I was in real confuse because it is my first time to face such a problem , I thought I missed something , I will try bondapack packing thanks for your advise
but I feel that the mobile phase is week for rifampicin eluting

Hi Hossam,

I agree, the eluent for the dissolution is quite weak, with luck the microBondapak will not hold on to these analytes very strongly, either. Best Wishes!