Chromatography Forum

Hi everyone,

I'm currently developing an assay for paracetamol in plasma however I'm having major problems with the precipitation of the plasma proteins. My assay (mobile phase KH2PO4/THF(0.1%)/isopropanol(1.5%) pH3.7, c18 150 x 4.6mm 5um column, 1mL/min flow rate) elutes paracetamol at ~ 9.6min however there is aplasma protein peak eluting at 10.6 min. This peak is present when I use 30% perchloric acid (using which I am obtaining the best results - though still unacceptable), acetonitrile (1:1), methanol (1:1), 20%TCA (1:1) and acidified methanol. If anyone has any ideas about alternative methods of precipitation of the plasma proteins to eliminate the peak or any ideas for manipulating the assay to resolve the two peaks and stop them interfering I would be most grateful!!

Thanks in advance for your help.

Cheers,

Merrin

How do you know this is a protein peak? Anyway, your precipitation methods seem a bit weak, also there are SPE and ultrafiltration methods, millions of them.

You can try direct injection with Cadenza HS-C18.
It's a novel design - you can run gradient elution and the proteins
will not precipitate inside the column.

Here's a chromatogram showing the large proteins being excluded from the column:
http://www.imtakt.com/TecInfo/TI290E.pdf

In the chromatogram below - you'll notice the bovine serum elutes off quickly, while the small molecules are retained:
http://www.imtakt.com/TecInfo/TI295E.pdf