Highly Retained NonPolar Compound LCMSMS METHOD DEVELOPMENT

[shemesh199]

More recently we are working on a larger small molecule compound called Irganox 1010 with a molecular weight near 1178Da on a 6410QQQ, which is a larger non polar molecule (image attached). From review of the literature most people perform APCI + for better response, we have an ESI source, and I have also seen ESI + Ion trap performed to monitor the molecular ion. The mobile phase usually 20mM aqueous ammonium acetate and IPA as the organic ( I guess more non polar than ACN). The columns I have seen used are C18 so I prepared a 1,000 ng/mL standard and attempted a full scan injection without column to make sure we could detect the precursor ion, and we did which might indicate this compound ionizes to some extent. Then I plugged on our Agilent Zorbax C18 (2.1x100mm, 3.5micron) column with a guard column and attempted a very long ~30min scouting gradient, based on prior knowledge that this compound elutes nearly at 100% organic after quite a long time due to the large affinity towards stationary phase. I started the gradient at 85% organic and ramped to 100% over time. To make the story short, after the injection around 0.3mL/min, after a few minutes the system went overpressure, the lines popped off and a leak occurred, then it took me nearly 2 hours to rinse and restore the column to appropriate running conditions ( to lower the pressure). I believe this compound plugged my column because it was so highly retained. Since then I have been hesitant to reinject. Do you guys have any experience with this sort of situation, and how would you go about method development for this sort of compound? I have attached a figure of the compound .

Thanks so much

[bisnettrj2]

What is your injection solvent, and how much did you inject? Also, did you run 100% organic in your no-column run to see if it will ionize under your proposed elution conditions (100% IPA?)?

[shemesh199]

Injection solvent is 15:85 20mM aqueous ammonium acetate: Isopropanol, injection was 15uL of a 1,000ng/mL reconstituted in this starting mobile phase.

[bisnettrj2]

Hmmm, seems ok. You could try a shorter-chain column, like a C8 or a C4. Also, I edited my previous post to ask about whether you tried your no-column run under 100% IPA to see if the compound will actually ionize under those conditions.

[shemesh199]

The run with no column was using the 15:85. Do you think I should try a smaller injection volume and lower flow rate? Im under impression that the ammonium acetate is required to generate some form of ionization for this complex analyte.

[bisnettrj2]

As to why the column went over-pressure, that to me says something about the injection solvent or volume - injecting anything in isopropanol is difficult, so maybe try a smaller injection of a more concentrated standard and see if the same thing happens.

[bisnettrj2]

Your ionization occurs at the elution conditions of the compound, not at the conditions under which you infuse or inject. From what I know, most analytes don't ionize well in 100% organic, so you might need to add a post-column infusion of acid or ammonium acetate to get your compound to ionize.

[shemesh199]

i will probably do a 1uL injection at a lower flow rate while keeping the 15:85 ratio.

[bisnettrj2]

If you don't get the pressure spike at 1uL, then try to increase your concentration 10-fold and repeat the 1uL injection. If the concentration doesn't spike at the 10X concentration, go up another 10-fold and keep watching the pressure. If you go up 100-fold in concentration without a pressure spike, that says the pressure spike was due to the injection volume and not the analyte concentration. If you do these tests and no pressure spike is shown, go back to the original concentration and shoot 1, 5, 10, and 15uL and see if there is a pressure increase on injection, and see if it increases with injection volume. If there is, I would try a different injection solvent, like water:methanol.

[Camisotro]

Isopropanol-water mixtures have a significantly higher viscosity, hence back-pressure, than methanol-water. Try running a low flow of 85% IPA through your column until the pressure stabilizes, and then you can figure out how much you can increase that flow while maintaining acceptable pressure. Try again at 100% IPA. Don't just blindly run a gradient without testing out the pressure.

Also, if this compound is really really highly retained... why not just take out the column, and use only the guard column? You may well be able to get adequate retention.

[Alp]

I agree with Camisotro. IPA will give you higher pressure.

I'd also check if your 20mM AmAc is soluble in IPA or is it precipitating out.

Alp

[Alp]

I am also wondering if this compound would give a better signal in negative mode? It looks mildly acidic.