I suggest to run scouting gradient using MeCN and water with 0.1% formic acid to see the pattern then you can see the effect of the solvents! How did you choose your mobile phase?

Try running a gradient

see

https://www.sielc.com/hplc-separation-of-biogenic-amines-on-primesep-100-column.html

Mr. Glynos,

Such quantifivation from the perspective of the Analytical Chemistry is carried our relible only my MS detection and quantification of the MS variables.

In this context consider this work:

1. Steroids, 164 (2020) 108750

Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II, Bojidarka Ivanova, Michael Spiteller

You are running basic amines in a Sodium acetate buffer pH4.5., did you check the pKa of each protonated amine? that will give you the idea in which form they will be in that solution, and them in what order they will elute.

It is clear from my point of view that to avoid one peak sharp from another broad, you need to use gradient phase. Best

Thank you all for your responses and recommendations. After much trial and error I have found that I was mistaken in the identity of the compounds eluting. Tryptamine was not eluting later than the more non-polar NMT and DMT, it was just that my methods were not sufficient to show noticeable peaks for NMT or DMT, because they were eluting so late.

So I modified the mobile phase to be very high in organic, and a higher buffer concentration--75% MeOH:25% 50mM NaOAc buffer.

The increase in mobile phase percent seemed to sharpen the peaks quite a bit, and the increase in buffer concentration brought the retention times down, such that all 3 compounds elute within ~6 minutes.

The only issue at this point is that NMT and DMT produce low, broad peaks, while tryptamine gives a tall, sharp peak. This problem is very bad for my sensitivity, because at a low concentration, NMT and DMT are not much larger than the noise. I will continue to tweak the methods to determine a method that might improve the sharpness of NMT and DMT.

Thanks again for all of your advice, and please comment if you have any other suggestions for peak sharpening.

p.s. Our lab does not have a mixer, so we are unable to run a gradient. We have multiple pumps but no way to program a gradient into the system.