Chromatography Forum

Dear Friends

We are facing a very strange problem.
We are analyzinf plasma samples (100 ul) extracted with hexane. The mobile phase is acetonitrile - 0,05% formic acid (55:45) - 10% isopropanol. After the extraction, the residue is evaporated and ressuspended in 200 ul of mobile phase. The injection volume is 8 ul.
The column is an X-Bridge C18 (Waters). The run time is 3.2 min anf the flow is 0,6 ml/min (with split of 1:2)

All this study was done 6 months ago, with no problem at all. We are doing another study of the sampe analyte for another client.

At this study, with other plasma samples, also from volunteers, we have a kind of contamination, when, after 20 or 30 samples, the analyte area increases.

We change all the autosampler injector parts as needle, needle seat, rotor seal, peek tubes, and clean all the MS system.

We also introduced a wash step of the needle in the injector and a injection of a mobile phase in a different method (100% of organic for 4 min and 100% of mobile phase for 2 min) every 8 samples.

Nothing of my tests showed me the answer, in other words, the problem is still there.

Does anybody have this experience or could give me an idea ????????????

Thank you.

Doesn't have to be suppression, can be enhancement. See page 13 reference 7 on my webpage about enhancement due to phopholipids. If not eluted, the lipids will start to break through and can give enhancement or suppression depending the elution time of eluent..

http://users.chartertn.net/slittle/ see the matrix effects in LCMS section.

It depends on the analyte, but we also see very pronounced ion enhancements with certain analytes.
Matthew

I agree. Phospholipids are a likely cause of the problem. I would wash the column after the twenty or so samples with 100% organic to get rid of the non-polar contaminants. Alternatively, an improvement in the sample preparation step would solve the problem.

If you are going to wash, probably need to wash everytime a sample is injected or at least every 3-4 samples. After that, the phospholipids start to break through and affect analyte response.

However, does seem to reach steady state and doesn't change after a few injections.

See my paper at my website or in J. Chromatography

http://users.chartertn.net/slittle/