Chromatography Forum

Good morning everbody.
I'm dealing with an HPLC/MS-MS method to evaluate the presence of my analytes in human plasma.
With my C18 column I'm experiencing a serious problem of Retention time shifting, sometimes gradual and sometimes more severe (from one inj to another aometimes there's a big difference in Retention time) Once the retention time chances there's no turning back (it's not a matter of mobile phase, bubble and so on)
To extract my analytes from the matrix I'm doing a liquid liquid extraction (LLE) with Et2O.
The chrom. method is in isocratic conditions with 45% organic.
I had validated the method using pool of donors plasma and although the column life was not long I could make the analysis. Now with palsma of real samples colums won't last an analysis. Everything is right with curve and QC (made with pool of donors plasma) until the real samples begin...than shifting of RT and back pressure increase.
I'm thinking the problem might be the dirty matrix, phospholipids stucking in the column.
Has anybody experienced the same problem? What can I do from a chrmathograpy point of view or to better clean up the sample?
Thanks in advance,
Mass

I would recommend to use Solid Phase Extraction to clean-up your sample. Your column life will increase.
SPE cartridges are available on the market from many manufacturers. Recently I got some brochures and catalogues on my table from UCT, United Chemicals Technology. On www.unitedchem.com you can look for the right application. I'm sure UCT and all the other manufacturers will send you some selected cartridges to test in your lab.
Good luck.

have you ever check pressure of the pump during the run?if the Rt is shifting one of the possible reason could be the fluctuation in the pump?