Chromatography Forum

Hello,

I am working with this basic peptide in plasma. I am having problem with the sample prep. Our groups usually use common regular protein precipitation in our previous projects. This particular peptide I am working on disappear after I precipitate the protein out of plasma. If I crash a blank plasma, take out the supernatant then spike the compound to the supernatant, I can see the compound. If I spike and mix the compound just after I crashed the protein without separating the precipitate, I can not see the compound. So far I tried mix of solvent and acids (TFA, FA, TCA, Phosphoric) to try to disrupt the binding, but none worked.
have anybody experienced this before ?
I also tried liquid liquid extraction. I basified the compound with NH4OH, but found nothing in organic layer.
The only thing so far that worked to clean up the plasma sample seems to be using solid phase extraction, which I am not really familiar with. Anybody have suggestion which SPE brand to go ? I am looking at C18 96 well plate and vacuum manifolds to go along with the sample collection.

Thanks folks

SPE is reality in sample preparation, you can read about visiting several publications. I do not use spreading brands because of all them can offer us good products. Many researchers use to start from using the same brand of their analytical columns, which can be a normal way. So, I would recommend you ask your local vendor of analytical LC columns, but requesting a formal training with his/her SPE Specialist, that may explain you about SPE. Moreover, you should study deeply in sites and or catalogues.

Hello,

I am working with this basic peptide in plasma. I am having problem with the sample prep. Our groups usually use common regular protein precipitation in our previous projects. This particular peptide I am working on disappear after I precipitate the protein out of plasma. If I crash a blank plasma, take out the supernatant then spike the compound to the supernatant, I can see the compound. If I spike and mix the compound just after I crashed the protein without separating the precipitate, I can not see the compound. So far I tried mix of solvent and acids (TFA, FA, TCA, Phosphoric) to try to disrupt the binding, but none worked.
have anybody experienced this before ?
I also tried liquid liquid extraction. I basified the compound with NH4OH, but found nothing in organic layer.
The only thing so far that worked to clean up the plasma sample seems to be using solid phase extraction, which I am not really familiar with. Anybody have suggestion which SPE brand to go ? I am looking at C18 96 well plate and vacuum manifolds to go along with the sample collection.

Thanks folks

Phenomenex and Waters are two of the biggest names out there when it comes to SPE, so you should be fine with either of them, espeically when it comes to something like a C18 96-well plate. If you're looking for a polymeric sorbent (higher loading capacity, dry-resistant), check out Strata-X or Oasis HLB as alternatives to C18, but if C18 works for you, then go with it!

As a SPE technical specialist, I can tell you that SPE won't help you to separate a covalently bonded compound from a protein. After hydrolysis and separation, then we can help you with extraction/purification.

Don

Hello,

Within the same subject, I work with proteins and phenolics and proteins bind to phenolics.

Due to the difficulty in working with this kind of mixtures, I was thinking to do SPE firstly in order to "clean" all the protein from the mixture. I am only interested in studing the phenolics, not the proteins.

Did anyone performed an assay of this type? I am thinking about base hydrolysis followed by SPE and HPLC.

I have Oasis HLB available to test, but not sure about the best option.

Thanks
Ric

@rchagas

I'm not sure about alkaline hydrolysis - will you manage to destroy your matrix proteins without destroying the analytes? Phenolics are prone to oxidation in alkaline medium, so if you go with hydrolysis, you should definitely add some antioxidants (e.g. BHT)!

Just a thought: maybe to try liquid-liquid or solid-phase extraction (either way, using a hydrophobic phase) of phenolics from sample, with proteins kept in as hydrophilic as possible (by adjusting the pH) and discouraging them from forming hydrogen bonds with your analyte (e.g. by using urea)?