Chromatography Forum

Can any one tell me the best way for precipitation of proteins in plamsa before analysis by HPLC-DAD. (Determination of creatinine in plasma ).

Thank you.

Blood plasma one part add with acetonitrile or methanol equal part or more for example,
plasma 0.2 ml mix with acetonitrile 0.8 ml and centrifuge to separate the supernatant for next step.

What you have left in the supernatant depends on the ratio of plasma to acetonitrile:
1:1 Anything smaller than 25 KDa
1:2 Anything smaller than 6 KDa
etc.
See my poster from ABRF 2003 per the following link: http://www.polylc.com/downloads/ABRF_20 ... poster.pdf
There are also two papers in print from two groups that followed up on this study in a more methodical manner.

Thank you,

But can i inject directly the supernatant in HPLC , or i have to evaporate and dissolve the residue in mobile phase.

I don't know the specifics of creatinine analysis, but other ways to crash out proteins would be acids like 20% TCA, perchloric acid or TFA. The advantage here is that you can use less (100uL plasma and 20uL 20% TCA for instance) and then dilute the supernatent with mobile phase and inject.

After addition of organic solvent, you can inject directly provided you're using a column in the HILIC mode and there isn't a big mismatch in the % organic solvent in the sample vs. the mobile phase. The drawback is that the sample is more dilute under these conditions, per Paul's comment. One solution would be just to inject more of it. The other is to collect the supernatant, evaporate the solvent, and then redissolve the residue in whatever you want.