Strange SPE protocol

[Gaetan Glauser]

Hi,

I have came across a strange protocol using C18 SPE cartridges (Bond Elut). The authors analysed steroids such as cortisol, testosterone etc. in plasma or serum by LC-MS/MS. After cartridge conditioning, they loaded serum or plasma samples diluted with water, then washed the cartridge with water and hexane, and finally eluted steroids with ethylacetate. To me, this is quite suspicious: I would expect steroids to be eluted with hexane. But they claimed that this process enabled them to obtain a clear reconstitution solution (MeOH:water 50:50), that would be cloudy otherwise (this I can understand), and that recovery in the ethylacetate phase was 87-101%. Does someone has a rational explanation for this?

If you are interested in the paper, here is the reference: Plos One 2012, Vol 7, Issue 2, e32496.

Thanks for your comments,
Regards

[Don_Hilton]

It is worth a try. I am working on a method where we put the sample on an SPE cartridge in water, wash with a solvent that I would have expected to remove the analyte (and it does not remove it, but does remove other stuff from the matrix) and then we remove the analyte with a third solvent - that third solvent is supposed to be between water and the first solvent in ability to strip an analyte from the cartridge.

I was puzzling over this and then remembered that the solvent triangle for LC has three corners, not three solvents in a line...

[Gaetan Glauser]

I see what you mean, this looks rational. Thanks!

[passy]

I'm not surprised that the hexane doesn't remove the steroids but am somewhat surprised by the hexane following a water wash. The "wetting" of the SPE sorbent by the hexane after the water wash must be problematic. I suppose this would be overcome if you put through a sufficient volume.

Tell us how it goes.

[Gaetan Glauser]

Sorry for the delay. I finally had time to test that procedure on pure standards and it actually worked well. Water and hexane fractions were free of steroids which were concentrated in the EtOAc final elution fraction.