I am trying to increase the final yield of acetylcholine (ACh) in my biofluid samples. I need to get the yield high enough for high confidence annotation of ACh (currently all I can really say is that I've got something that matches the ACh expected mz).

My first samples were prepared using C18 solid phase extraction using a water liquid phase and methanol to elute. Based on my understanding of C18 SPE, I assume I've lost a lot of ACh at this step, because I assume the positively-charged ACh would have been washed away during the wash step.

Now that I have a polar analyte to target, I need to prepare more samples with a higher ACh concentration. I should be able to increase the concentration by using an SPE protocol more amiable for ACh extraction, but I can't find a protocol like this in the literature. How does one design a targeted SPE protocol? Can I assume any HILIC procedure that targets charged molecules will be appropriate here, or is there more to consider?

My problem is made more complicated because I need the final, concentrated version of my extract to be compatible with my downstream bioassay (i.e. no carry over from acid, no strong solvent that can't be removed)

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    $\begingroup$ Maybe you can clarify what you mean by this: "Now that I have a polar analyte to target, I need to prepare more samples with a higher ACh concentration." Are you quantifying ACh in your samples? Maybe you should be using a cation exchange column? $\endgroup$ – Buck Thorn Aug 15 '19 at 10:00
  • $\begingroup$ Thanks for pointing that out. What I need to do is identify this mass trace as acetylcholine unambiguously, and for that I will use either FTICR-MS/MS or LC-MS. We've tried MS/MS and found that the concentration wasn't high enough, which is why I am trying to target it directly. Ultimately, I would like to quantify the amount of acetylcholine in my samples. $\endgroup$ – HarD Aug 18 '19 at 13:19
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    $\begingroup$ I found the following supelco catalog informative : supelco.com.tw/C-2%20%20150-163%20New.pdf I can't claim to be an expert but you may want to try something like the DSC-WCX columns. $\endgroup$ – Buck Thorn Aug 18 '19 at 15:34

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