Chromatography Forum

I have been working with this thing for the past 6 months. In all, I am pretty happy with the universality. Unfortunately it is too universal to be used to detect low levels of analyte in complex matrices. Does anyone have any advice for methods that I could use to clean up, say plasma, to eliminate this background. I have tried protein precipitation and SPE, but so far much of the background remains. I need good recovery with no optimization, so liquid liquid extraction is pretty much out. Maybe ultracentrifugation would help?

Thanks,

Nick

No ultracentrifugation won't work. You will have to give more information about the compounds that you want to detect (i.e. positive or negative ionizable groups, hydrophobicity etc.). But my guess would be that you won't be able to eliminate the CAD background in such a complex mixtures such as plasma... unless your analytes have significant different properties than the matrix compounds...

There are several SPE techniques around that are optimized for analyte classes. They use mixed-mode sorbents. No background can be entirely clean (based on such a general principle), but the methods can be finetuned for a given analyte and generate a rather clean background. What SPE method have you tried?

I have tried the Waters Oasis SPE and Phenomenex Strata-X SPE. With both I see some drop of the baseline, but it is not significant enough to be much help. I am afraid to use a selective SPE because I do not know what I will be analyzing. I also worry that the large matrix peaks in my plasma are from endogenous small molecules which can never be eliminated. My clean up method must be universal for high throughput reasons. From what it sounds like, this may be a unrealistic goal.

Thanks,

Nick

Have you considered to use mixed-mode sample preparation (ion-exchange + RP; Oasis MCX and Oasis MAX). With the optimized techniques for these SPE packings, you reduce the background further. With a generic technique, you will still elute all positively or all negatively charged analytes, but you cut your background significantly (- neutrals, zwitterions, and oppositely charged analytes). If this is suitable for the problems that you are trying to solve, this may be worth a shot.