Chromatography Forum

Hello Everyone,

I am trying to develop LC-MS/MS method for steriodal drug, I optimized compound+ source dependent parameters for transition m/z 495.1/101.0. When I ran Flow injection analysis (FIA) @Mobile phawse 0.1% formic acid and 10mM Ammonium formate (50:50) without column (only union) I observed good intensity. But when I ran the same thing with C18 column I couldnt detect the peak intensity. Please let me know suggestions.

Thanks,
Sri

This may be obvious, but did you run the method long enough for the active to come out? Do you have another C18 column you can use that you're sure is good, or even another type of column just to check. If you don't have a column, or even if you do, I would recommend you run a caffeine solution through the column to a basic UV detector to make sure active is getting though (a simple 50:50 organic:water MP works and the peak comes out in a couple min at ~254nm).

If all that checks out, the column may just need a little work. I would start by back-flushing into a beaker (not to the detector!!) for maybe a 1/2 hour or so. If that doesn't work, try running your MP through the column for an hour (or even overnight at a low flow). I've seen compounds not show up if the column isn't properly condition overnight like this.

Hello,

I tried all the ways you mentioned. The drug was lipophilic, so can we run normal phase with LCMS? Can you please suggest me regarding the mobile phase selection??

Thanks,
Sri

Hello,

I tried all the ways you mentioned. The drug was lipophilic, so can we run normal phase with LCMS? Can you please suggest me regarding the mobile phase selection??

Thanks,
Sri

It might be a bit early to switch to normal phase. Have you tried a post column derivatization yet? I found this article by Agilent that may help you a bit, seems to explain things nicely for a steriod analysis: http://www.lcms-connect.com/uploads/tx_ ... eroids.pdf

If you really want to switch to normal phase, I would try a 50:50 mix of pretty much any of the following: hexane, dichloromethane, chloroform, ethyl ether, or isopropanol. Also, be careful of the system you're using, some don't do that well in normal phase. I would double check with the manufacturer before making the switch to be sure.

Good luck!