I work with derivatized neurotransmitter molecules. I was using C-18 SPE cartridges previously for desalting them before LC-MS, but now I would like to adopt in-house made stagetips.
These are a version of ziptips without the horrendous costs associated with them.
I was wondering if anyone has used ziptips or staegetips for desalting their non-peptide/protein samples and what their experience was.
Thanks for your help!
There is always a sample loss associated with SPE devices, commercial or custom-made. I recommend doing a recovery study on a standard for your neurotransmitters of interest before applying custom stage tips to valuable samples.
Depending on sample carrier and derivatization reagents you need to remove, a 4:1 isopropanol: acetonitrile mixture may work. A general procedure for such desalting would include adding the 4:1 IPA : ACN mixture to the sample (prepared in 50% acidified MeOh in our case), centrifuging, and taking 80-90% of the liquid volume (to make sure not to disturb the pellet at the bottom of the tube). Then dry to concentrate with the speed-vac and reconstitute in desired solvent for injection. This procedure effectively removes inorganic salts, we did not test with anything else. Best wishes in your work!
Elena V Romanova Thanks for your reply!
I will definitely quantify recovery before using them on my experimental samples, there's no question about that! Especially for my low abundant compounds.
My samples are AQC derivatized amino acids and biogenic amines.
Previously I had used Phenomenex C-18 cartridges and used 100% Methanol (wetting), 0.1% aqueous formic acid (equilibration) and 4:1 ACN:MeOH containing 0.1% formic acid for eluting. The rest of it (lyophilizing and reconstituting) was the same as you mentioned.
I wanted to bring this method over to stagetips, and wanted to see if someone had tried this before. Especially for the amount of substance that can be adsorbed etc, which would then affect the stack size of the disks, the elution volume and so on.
I will have to test it all out anyway, but any lead would give me a good place to start.
@ Divya Ramesh, I was suggesting liquid-liquid extraction protocol without any SPE materials. SPE did not work for our purpose when applied to amino acids and biogenic amines. Kind regards.
LLE is a good way to desalt. I previously gained experience method based on EZ: Faast. OPA, Chloroformates, and other derivatizing agents can also be used with the method. Isooctane and chloroform included extraction solvents that can solubilize the derivated amines and can keep the salt interferences out of the aliquot. Alternatively, you may have a look at the protocols for AA analysis kits from Phenomenex, cation exchange resin is implemented as stage tip application just before derivatization. This may also contribute the cleaning but simple derivatization and further LLE using hydrophobic solvents would be much helpful as Elena V Romanova indicated...
Good Luck
You may want to chat with Dr. Mark Carroll with the ARS-USDA based at Carl Hayden Bee Research Center in Tucson, AZ. He has been the chemist who has helped me resolve amino acids and biogenic amines with GC-MS. We also use the EZ-FAAST method.
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