Hello,i'm new in this forum.

I've been working for many days in a method Development for isolate ephedrine from blood plasma for biodisponibility assays. We've tried with SPE technics using C18 cartridges,conditioning with acetonitrile.No rinsing was made.Eluting solvent we use buffer phospate:acetonitrile(90:10).We mantain the pH of 6,0 in the system.The method has not succesfull.

Aprecciates collegues:
would someone to give us some guidelines to continue our investigation .Thanks a lot.

how about a quick ACN protein precipitation? It works for many compounds in my lab.

Many drugs are in the blood bond to the proteins.Can the quick precipitation with ACN to precipitate the analyte in question too ?

It looks like your elution solution is too weak. Try to use more ACN or lower pH in your elution solution.

The idea behind using ACN, MeOH, etc., for extraction is to increase the solubility of the analyte in the liquid phase while precipitating the protein, thus removing it. The ensuing denaturation of the protein may contribute to the analyte solubility by weakening the bonding to the protein.
If you use a protein precipitant like trichloroacetic acid, which has no effect on the analyte solubility, you indeed have a very good chance to coprecipitate your analyte. (Demonstrated in J Chrom B, 678, 137 (1996)).
If your analyte bonding to the protein is considerably stronger than to a SPE surface you can also loose your analyte. If you use a protein precipitant copiously in SPE, the protein will get hung up in the cartridge.....

ephedrin is not the stuff that is strongly bound to plasma proteins. I doubt that this is the problem. You may have a problem with avoiding the proper conditioning of the SPE cartridge by loading the sample directly without conditioning with a controlled aqueous environment. It is also possible that the ephedrine sticks to your C18 SPE, if it is full of silanols. You may want to change the pH to acidic to get it off the C18, or you could consider using a packing that does not have this problem, like Oasis.

Better polystyrene cartridges -- they are sign. more hydrophobic than Oasis (Envi+, Isolute). They can completely adsorb such analytes as your even at high conc. of organic solvents (20-30%)even al a very low pH.
So you can firstly prec. proteins and then SPE on polystyrene.
But remember -- this mat. may be "full of chlotometh."!!! So? or example, you can use 3chloroac. acid, followed by SPE on polysyrene
Constantine

Constantine:
Oasis is a water wettable divinylbenzene-based packing. It is the one with the highest retention properties of well functioning sample preparation packings without the difficulty of dewetting, as a pure polystyrene packing will do.
You do not need to do first protein precipitation. Protein precipitation is done in a high concetration of acetonitriel. If you now want to adsorb it on a reversed-phase packing, you are in trouble. You either need to evaporate to dryness, or dilute with a ton of water until the sample adsorbs again. In addition, a decent packing for SPE does not bleed strange things and is not "full of chlotometh."

Uwe:
Excuse for I meant HYPERCROSSLINKED polystyrene -- it`s about two times more hyrdophobic than Oasis and does not suffer from swallowing.
As for prec. -- so, I see, ater it sample may diluted to 30% for ex. of AcN, and then concentrated. Believe -- hypercrosslinked polyst. will do it well.
Problems may occur with desorption -- perfet way is to desorb it with ch2cl2 followed by evaporation, but as I`ve mentioned, some of these materials "are full of chloRomethyl groups" It`s bad, so I am interested in this material to be completely chemically inert.