Thank you so much for all the replies thus far! I appreciate all the help I can get really, but here's additional information about my LC-MS/MS method.
@AndyAlpert and @TylerSmith123 If I am missing any information please let me know! This is my first time posting on this forum haha.
@TylerSmith123 My apologies if I may be using confusing terminology. To be clear (hopefully!), I meant that my [M+H]+ ion is m/z 247, while my product ions (after fragmentation) are m/z 58, 160, 89, 77, 63, 115. I am not performing any derivitization during my sample prep. I share a similar hypothesis as you do, in that I was thinking whenever I spiked my drug in plasma, there might be some interference in the plasma that may be reacting with the analyte. But if this was so, I am unsure as to how I can overcome this issue.
Andy Alpert wrote:
You've provided no details about your chromatography, so I'm free to speculate about a scenario. The dimethylamine- group in acetylpsilocin's side chain is in fact ionizable. That means that it's going to have a counterion at any pH low enough for silica not to dissolve. Coming straight from plasma, it's reasonable to assume that the counterion for the amine is Cl-. Your precipitation step will get rid of much/most of the NaCl from plasma. Now, if you proceed to run the analysis on a reversed phase column with TFA in the mobile phase, then the amine group's counterion will be trifluoroacetate. That's more hydrophobic than chloride ion, and would account for the increase in retention time for the resulting ion pair. You can assess this by using a chloride salt in the mobile phase instead of TFA.
If this speculation is off-base, then kindly provide some details about your chromatographic conditions.
TylerSmith123 wrote:
Hi there,
I may be a little confused by some of the wording but when you say transition, this is just another way of saying the fragmentation patterns for your molecule? As in your base peak is the 247 while the fragments (from the MS) are to the right?
If that's the case, and all of the fragments are different post plasma derivatization, could it be the case that your derivatization or plasma is causing a chemical change of the molecule? This would result in some completely new fragments and potentially some identical ones.
I just looked up the structure and it has quite a few functional groups for such a small molecule, and it looks like it can also be protonated/deprotonated. What does your mobile phase look like? Additionally, what is the solvent used for injection of the extracts vs the standards? Could it be as simple as a proton? haha
Have you used your neat samples in a spiked plasma test? I suppose I'm not sure if these are coming from actual tissues or are just always spiked into the solution.
Anyway, get us some more information and hopefully we'll get to the bottom of this issue!