Hi all,

What's the problem of injecting plasma directly into a short analytical C18 column, like 2.1 x 50mm, using LC-MS/MS?

I was told it is not good, especailly due to the large amount of proteins in the plasma, but why so?

thanks a bunch!

Even if you filter the plasma, the proteins can denature and precipitate out, clogging your column. There are what I would call "online extraction" columns that are meant for this application, Cohesive being one vendor. These columns have larger particles (30 to 50 micron) and larger frits, and can handle about 100 microliter injections of filtered plasma.
These columns will eventually die by clogging as well, but all accounts I have read say 100 to 500 injections can be made before this happens. The large particle size gives reduced chromatographic efficiency, and such columns are used either with a step or steep gradient (after injection and washing) along with a mass spectrometer, or by coupling with an analytical column with some switching valves (e.g. Cohesive sells an LC / extraction system especially designed for this application).

On the other hand, at this year's ASMS conference, there were some posters where plasma was being directly injected onto analytical columns, so it apparently can be done. If you try it, let us know how it works out. Good luck!

We have been using the direct injection of a plasma sample onto Oasis columns for years now. The particle size is 25 micron, and the column dimensions are 2 cm x 2 mm. A high flow rate during the sample loading step minimizes the residence time in the column. Thus proteins do not interact with the packing and are flushed out. Afterwards, you can run a standard gradient at low flow to maximize resolution and bring all your analyte into the MS system for maximum sensitivity.

MG and Uwe,

Thanks for the help. I tried to inject plasma onto a small analytical column, 2.1 x 20mm, 5um particle size, 1.5ml/min flow rate. After just 2 injections, the peak got really broad, indicating a column problem. The pressure was fine. I am thinking maybe the problem is the small particle size silica I used (5um)? Both Oasis and Cohesive use large particles (25-50um) for direct plasma application. Any idea why small particle size (like 5um) does not work?

Thanks!

The only thing I can think of right now is, perhaps there are components in the plasma that are being strongly retained, and not being washed off by your mobile phase. Maybe try washing your column with a strong solvent at the end of each run. If you are already doing this, then the only other idea I have is to try a backflushing step, but you'll need switching valves.

To Anonymous: I have done the same thing as you, with the same results. With a small particle size, you need to use a packing with an external surface that prevents the adsorption of protein. With larger particles and higher flow rates, you do not need this.

Recently we developed a column for direct plasma injection (Primesep D - D stands for Direct) It is a reverse phase column with 5 um particles for analysis of hydrophobic and acidic compounds. Plasma does not retain on the column and comes out of the column in the void. The results are not posted yet on our website (www.primesep.com) but will be posted shortly. If you have a specific question regarding column for direct plasma injections please contact us at mail@allsep.com.

De ser posible le solicito hacerme lleagr informacion relacionada con la inyeccion directa de plasma en una columna ¨ analñitica¨.

Atte.

Aquí está la conexión para la inyección directa del plasma:
http://www.allsep.com/Technology_DirectPlasmaAnalysis.php

Sorry my Spanish is a litlle rusty.