huge hump in the chromatogram

[mimisandhu]

I am analysing drug in capsule(gelatin capsule) and getting a huge hump before the parent peak. The method works fine for the drug substance. the hump is coming where the two impurities are supposed to be. My mobile phase A is 5/95/0.1:ACN/H2O/TFA, mobile phase B is 95/5/0/1:ACN/H2O/TFA. The Column is C18 and the gradient is

Initial---------5.6%B
10.0 Min.-----50%B
15.0 Min.-----100%B

Please advice how can I get rid of the hump. Thanks in advance.

[Noser222]

Have you run a blank gradient? If not, TFA is notorious for causing baseline problems. Read a little more about the reasons in this post:

http://www.sepsci.com/chromforum/viewto ... hlight=tfa

[DR]

Water check:
Let sit pumping at initial conditions for ~20 min, then run back-to-back blank injections. If the hump is a lot smaller in the second blank, you need to clean up your water, then remake the A phase.

For details, search "Empore"

[mimisandhu]

When I run the drug substance only with same column, mobile phase, gradient etc. I don't see the hump at all. it is when I run the drug in capsule that I start to see the hump and it grows with each injection.

[Mark Tracy]

Is there dissolved gelatin in the drug sample? Run a gelatin sample and see if that is the source of your hump. If so, you may need additional cleanup prior to analysis.

[mimisandhu]

yes! I filtered the sample and also centrifuged it and by doing that I get a clear solution but It is causing that hump at 4-6min. where two of my impurities are eluting. If anyone had similar problem please let me know how to solve this problem. Thanks!

[JM]

What is in your capsule besides drug? if your standard run is Ok , it indicates that your sample containes some other excipients or your drug interecting with geletin . run one placebo and check.

JM

[Mark Tracy]

Dissolved gelatin will pass through an ordinary filter. They are kind of expensive, but centrifugal ultrafilters with a 3000 MW cutoff will remove dissolved proteins. Grace and Millipore are two sources. Another alternative is an SPE cleanup, but that is actually more work than the ultrafilter.