No constructive remarks can be obtained without some basic info. Please provide us with the basic HPLC method details. Esp the column dimensions (L x ID x particle size). The column dimensions allow us to calculate the most basic HPLC parameters needed to understand the method used. Your issue may be sample carryover contamination, column overloading, inappropriate detector settings, poor quality method design (i.e. low Kprimes), excess deriv reagent, impure material....

BTW, the technique is called "HPLC", "UPLC" is a Waters brand name/trademark. As scientists, terminology is important to us.

You describe part of your method as using "5% to 100% acetonitrile". Please explain. Bottle 'A' = ?, Bottle 'B' = ? pH ? Additives ?

Gradient Time program and steps please (0 time to end).

You state that "Flow rate 0.5 mL/min". OK

BEH C-18 column. Again, we need dimensions please.

You are running a gradient in ACN (vs something aqueous), but are dissolving your sample in DMF, DMSO, or ACN. *Ideally, samples should be dissolved in the mobile phase.

What is your injection volume? Is it 100 ul?

Are you aware that most suppliers offer this derivatization reagent for HPLC applications with a purity of only 70%? What is the purity of yours and by what method was it tested for purity? During the derivatization steps unreacted material is often left over which can contaminate the system.

- The column details are: ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 mm X 50 mm

- Regarding sample carryover, we have been injecting nothing but these controls, and using a needle and valve wash of 10% acetonitrile/90% water.

- We are injecting 10 µL on the column

- Bottle A is HPLC-Grade water

- Bottle B is LC-MS-grade acetonitrile

- Gradient is 95%A/5%B to 0%A/100%B over 6 minutes, held there for 5 minutes, then back to starting conditions prior to the next sample.

- There are no additives at the moment because we were not sure of their interference or reactivity with the 1-pyrenyldiazomethane (PDAM)

- We are not controlling the pH at all (a mistake?). I thought we would control the pH once we determine that we can get some sort of confirmation that the negative control (for carboxylic acid) yielded a relatively clean chromatogram.

- We originally tried ethyl acetate to dissolve PDAM, but got the same issue (I thought maybe there was residual acetic acid in the ethyl acetate from production, reacting with the PDAM)

-PDAM is not soluble in the starting conditions (95% water), and we need a high concentration (2 mM) for our derivatization reaction since we are going to eventually be derivatizing in cell lysate with several other carboxylic acids naturally present and able to react with the PDAM. I suppose I was starting very aqueous because I have no knowledge of the derivatized PDAM hydrophilicity/retention on the column. We aim to derivatize oxalate, but we cannot even get a clean negative control.

-Regarding PDAM purity, we are very concerned about this issue, and due to the expense $377 for 25 mg we are considering in-house synthesis and purification. I found this thesis that has a promising, or at least facile, synthesis for PDAM. https://www.semanticscholar.org/paper/Synthesis-of-1-Pyrenyldiazomethane-for-of-Fatty-Kalvaitis/0bcdcda50c878eca4b60e04f8682c804c0efbec0

-We are avoiding other derivatizing reagents because we need to have some compatibility with water, since our samples will be aqueous, to which we will add a water-miscible solvent (10% water, 90% solvent with PDAM). I cannot find a good substitute that has been used in aqueous environments.

Thank you for your time and expert opinion!

Thank you for the info. Too many things which are not following good HPLC fundamentals is one problem. No verification that anything works (and I like to assume nothing is doing what it is suppose to...). Way too much to address on a free web forum like this (I make my living as a professional scientific consultant and can only help so many people each day for free).

Constructive Suggestion: In order to simplify, it appears at this stage you are just trying to run the "pure" deriv agent on the column to confirm that it does not indeed react with anything (when no carboxylic acid is present in the solution). So, start with fresh solutions, a brand new column, clean the HPLC system (wash down the flow path, insure injector is clean, perform any needed maintenance, run performance tests). Next modify your method to end at 95% ACN (not 100%). Make sure your FLD's gain is not set too high(!). Monitor the baseline. NO INJECTION. Run a slow, gradient from 5% to 95%, then hold for several minutes and make sure NOTHING elutes. Go back to initial conditions. When the baseline is stable, repeat the run, but this time inject 10 ul of your initial mobile phase. You should see the same clean baseline plus the injection pulse. If you have a UV/VIS detector inline with the FLD (and you should), make sure that it is on and monitoring the baseline ~ 230 nm, 4nm or so too (extra info regarding impurities and contaminates will be useful). That is where I would start. You must first verify that (1) the instrument is working properly, (2) that what you think is happening IS in fact happening and (3) what should be happening IS in fact happening. Until these basic things have been confirmed, no need to run samples or standards. If everything works well, you may also want to switch over to MeOH instead of ACN, but nothing wrong with sticking with ACN for now (I always like the idea of changing as few things as possible when troubleshooting). Good luck.

Rule #1 when troubleshooting:

Keep It Simple, Check Everything Twice, Assume Nothing.