Chromatography Forum

Hello
Thats my first post here. I have a few questions about LCMSMS analysis at basic conditions. I'm developing method for novel compounds new compounds that are synthesized in our company. At the "0" point of method development I check AUC in following conditions:
25 mM HCOOH in water (phase A) + ACN (phase B)
25 mM HCOOH in water (phase A) + MeOH (phase B)
10 mM NH4HCO3 pH 8.5 in water (phase A) + ACN (phase B)
10 mM NH4HCO3 pH 8.5 8.5 in water (phase A) + MeOH(phase B)
I'm using full gradient (from 5% to 95% B 2min)
Often I have to choose basic conditions with ACN, so I have few questions about these.
1) Now, I'm using Acquity BEH C18 column and still fighting with peak taling. I think about Eclipse Plus C18 column. According to the data sheet that column can work at pH range 2 to 9.
Is anyone working in these conditions with these column (or any analogue column)? I'm interested in column stability, reprodubility and lifetime in these conditions.
Generally, I wonder about the stability of the C18 column at a pH >7.
2) My compound degrades in basic conditions (0.1M NaOH pH 12.8 ) Can i observe these process during my analysis with NH4HCO3. I have not any data about compound in pH 8.5.
3) I want to use these conditions for plasma analysis.
Are there any contraindications or specific points on which I have to pay attention to?
4) At BEH AcQuity C18 data sheet I have read that Ammonium BiCarbonate is better (for column and MS detector), than Ammonium Carbonate, but why?
5) That question is not about basic conditions. I prepare calibration curve samples in plasma by spiking. What is the maximum amount of organic solvent (for ACN, DMSO, MeOH, EtOH and IPA) at plasma at which proteins will not be precipitated? I ask this because I'm still working with hard soluble (at water) compounds.
6) What is time for column equilibration? I have read that is 20 column volume. Now I'm using much less (about 8-10 column volume). I did not see any retention time drift. Is that correct column equilibation?
Sorry for terrible English

1) BEH columns will last a few hundred injections at alkaline pH, depending on the matrix and temperature. They will not fail catastrophically like silica columns (no voids), but selectivity will change.

2)It will likely degrade at pH 9 as well, just slower. Are you sure that the tailing you are seeing is not the degradation of the product, instead of chromatographic tailing?

3)Do not know

4)Ammonium carbonate will be always in equilibrium with ammonium carbamate, which is not volatile. Hence, you will have less reproducible buffers, pollution is the MS source, and higher pH (more aggressive) for the column.

5)It will be different for each solvent, but I'd say precipitation will start somewhere around 20%

6)No drift = good equilibration!

Just an idea. You are doing a matrix matched calibration, but you could also use an internal standard to further reduce matrix effects on quantification: a structural analoge of the target analyte (i assume you don't have isotope labelled versions of your novel compounds).

Out of curiosity, as i don't have experience with plasma, is there any sample prep or just dilute & shoot?

@carlo
1)Now I'm doing PPB for 4 compounds. I made about 1000 injections (500 in PBS buffer and another 500 in Plasma) and it still working,bBut I see some deterioration (little more peak tailing). Just for future questions - i'm using switching valve to avoid contaminations of MS source.
So the best columns for pH above 7 are these like Extend C18 or Poroshell PAH?
2) I'm using MRM so I'm sure (probably...)
5) I've read a lot of protocols for PPB. Usually I saw recommendation, that organic % in plasma not exceed 1%, so why? It's specific for PPB assay?
Thanks for another answers
@ Rndirk
I'm using internal standard in this assay of course

For plasma samples I'm using PPT (Protein Preciptation) and after that filter all samples, by 0.22 uM PTFE syringe filters. Im still waiting for 2D-LC, which allowing me to do SPE-OnLine.

5) I've read a lot of protocols for PPB. Usually I saw recommendation, that organic % in plasma not exceed 1%, so why? It's specific for PPB assay?

Because there is no organic solvent in real plasma. You want to keep it as low as possible, because it affects the binding between proteins and the drug, long before the proteins starts precipitating.

This means, if your drug is in an organic solvent, you'll want to use a high concentration to minimize the volume of solvent you add to the plasma.

Another one question about basic conditions.
Im usingBEH Acquity C18 2.1 x 30 mm x 1.7 um with ACN
and Ammonium Formate pH 8.2.
After ~250 injections I have to reverse and flush column with water, and then ACN.
In the past I used the same column but with a length of 50 mm and I had to reverse and flush column after 500 or 750 injections. Also I used Ammonium Bicarbonate pH 8.5 and ACN.
Why i have problem with shorter column?

Sorry for terrible language :_

I'm a bit puzzled why you are using basic conditions for the chromatography if your compound of interest tends to degrade in basic conditions? Is it impossible to get good chromatography under acid conditions? Or is your compound also acid labile?

I had 5 analytes with totally different Bjerrums plots. Also, usually I has better SNR at MS with basic conditions. I checked stability at 10 mM NH4HCO3 by degradation studies. All compounds are stability in this medium.
But thanks for reply