Response factor decreasing in time

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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Hello everybody. I’m using an UPLC-QDa for quantify two related substances in fermentation broths. These impurities are not justified and there’s no reference standard, but they are chemically likely to the main compound, so i assume that their response facors are the same of the main compound (same fragmentation pattern).
I use ammonium formate 25mM in water as mobile Phase A and acetonitrile with 0.05% acetic acid as mobile phase B; the elution is in gradient mode.
When i run analtsis with fresh mobile phase, on the first day for example, i have a response factor about 1 x 10e-5 for the standard and areas of around 70000 for the imputities. On the second day, i obtain a RF of about 0,7 x 10e-5 for the standard and areas of around 90000 for the impurities. On the third day i obtain a RF of about 0.5 x 10e-5 and areas of around 100000 for the impurities. And so on. When i renew the mobile phase, RF and areas return at 1 x 10-5 and 70000. The RF stabilizes at about 0,35 x 10-5 until the mobile phase finishes. I exclude the standard degradation because i prepare new standard solution everyday, in triplicate, and i always observe the phenomenon. Even the carryover is excluded by several tests.
What’s going on according to you?
Sorry for my poor english, i hope you can help me
Ammonium Formate is volatile and it could be a loss of this to atmosphere that causes some drop.

I have also seen a similar problem with Acetic acid in the mobile phase, it seems to take a long time to reach an equilibrium, but once it does the response of the analytes become stable.

These two together may be why it is unstable when it is fresh. Have you tried making the mobile phases in advance, and using them only after a few days? If the mobile phases sit for a few days maybe they will reach an equilibrium and be stable to use until they are empty.
The past is there to guide us into the future, not to dwell in.
What adduct are you monitoring?
I run a method with ammonium acetate 10mM and my analyte ions are the nh4+ adducts. Over time my ammonia evaporates as nh3 and signal drops, i was suprised how quick it was since we have proper bottle caps that should slow down evaporation. I guess the process would be slower at low ph where most ammonia is protonated. I did not see similar trend for the h+ adduct of my analytes so pretty sure its nh3 evaporating in my case. When I weigh in the ammoniumacetate salt you really see how quickly it evaporates on the scale.

To avoid this i split my mobilephase batch into smaller bottles 500ml with little head space and run a new bottle each day but retain same rt as the day before since its from same batch prep. It is nice to have exatly the same rt througout a study set in particular when mrm windows are small. I try to keep organic at 10% also in A to prevent microbial growth. If you need 100% aq you may need to prepare fresh daily.
per_oxid wrote:
What adduct are you monitoring?
I run a method with ammonium acetate 10mM and my analyte ions are the nh4+ adducts. Over time my ammonia evaporates as nh3 and signal drops, i was suprised how quick it was since we have proper bottle caps that should slow down evaporation. I guess the process would be slower at low ph where most ammonia is protonated. I did not see similar trend for the h+ adduct of my analytes so pretty sure its nh3 evaporating in my case. When I weigh in the ammoniumacetate salt you really see how quickly it evaporates on the scale.

To avoid this i split my mobilephase batch into smaller bottles 500ml with little head space and run a new bottle each day but retain same rt as the day before since its from same batch prep. It is nice to have exatly the same rt througout a study set in particular when mrm windows are small. I try to keep organic at 10% also in A to prevent microbial growth. If you need 100% aq you may need to prepare fresh daily.


I do not see adducts, and my bottles are always closed with appropriate caps. Instead, using smaller bottles seems to be a good idea. Do you think that stirring for while minutes the phases could give better result?
Franofi wrote:
per_oxid wrote:
What adduct are you monitoring?
I run a method with ammonium acetate 10mM and my analyte ions are the nh4+ adducts. Over time my ammonia evaporates as nh3 and signal drops, i was suprised how quick it was since we have proper bottle caps that should slow down evaporation. I guess the process would be slower at low ph where most ammonia is protonated. I did not see similar trend for the h+ adduct of my analytes so pretty sure its nh3 evaporating in my case. When I weigh in the ammoniumacetate salt you really see how quickly it evaporates on the scale.

To avoid this i split my mobilephase batch into smaller bottles 500ml with little head space and run a new bottle each day but retain same rt as the day before since its from same batch prep. It is nice to have exatly the same rt througout a study set in particular when mrm windows are small. I try to keep organic at 10% also in A to prevent microbial growth. If you need 100% aq you may need to prepare fresh daily.


I do not see adducts, and my bottles are always closed with appropriate caps. Instead, using smaller bottles seems to be a good idea. Do you think that stirring for while minutes the phases could give better result?


I guess my situation is not the same as yours since i need the ammonia to get my compound ionized.
I usually sonicate my bottles 10 min to get everything solvated and mixed as this is so easy to do and no risk of adding contaminants. I guess splitting your mobile phase into two smaller bottles could be a test to see if the effect is the same when you install a bottle that has been sealed with little head space vs installed with the LC-lines.
I always mix phases for some minutes and than sonicate them in order to get proper degasing and mixing; it seems like the lc-ms system needs some time to equilibrate with fresher phases, but i can’t explain Why, even if the cause could be the evaporation of both formate and acetic acid, the areas increase in that way. My molecule must be in buffer solution because they are really poorly soluble in water. I really don’t know
Ionisation efficiencies depend enormously on solvent. I note you say you don't have adducts? I assume you mean that it runs as a hydrogen adduct, or ammonium, and isn't forming anything else?
What commonly happens with aqueous solvents that stand a few days is that sodium leaches out of glass bottles, and if your analyte has a particular affinity for sodium, it starts to form a sodium adduct. These don't fragment well, so if you've been following either an ammonium/hydrogen adduct, or a fragment derived from one of them, your signal shrinks.
The awkward thing in your case is that whatever the reason this is happening, the fact that your main analyte and the impurities behave differently (they vary in opposite directions, and yet you've demonstrated that it's not degradation of the standard), you can't justify the claim that the impurities have similar ionisation efficiencies to the main analyte; they clearly don't. I'm not sure what you can do about this, in the absence of reference standards or a more independent way to detect them.
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