internal standard/analyte variations in cell medium

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

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Dear all,

I'm in the process of quantifying small organic molecules from cell medium. The cell medium is spiked with DMSO stocks of the respective compounds and a linear dilution row is made. After vortexing the internal standard dissolved in acetonitrile is added. Finally everything is centrifuged (14000rcf).The volume of the latter is twice the volume of the cell medium.
The problem I'm facing is that the response of the internal standard and also the area of the analyte is severely varying and my curve looks accordingly unfortunately.

I thought, one possible reason might be the recovery from the cell medium. However, would the internal standard be affected, too?

I'm doing right now multiple injections from the same vial to rule out any gradient formation within the vial.

I'm using ESI (+), MRM, with a water and Acetonitrile as mobile phases and 0.1 % acetic acid as additive.

Would you further recommend making one calibration for all compounds that need to be quantified or would you rather make a calibration curve for every compound? The first option is a lot faster but I suspect charge competition especially in the higher concentration range i.e. 20 µM


I'm thankful for every advice!

EDIT: Repeated injection from the same vial resulted in vastly differing peak areas.
first run:
analyte: 70000
ISTD: 19000

second run:
analyte: 310000
ISTD: 80309

The ratio in both cases is comparable. Both what could possibly cause this? Injection of the same samples merely in acetonitrile resulted in a very good reproducibility.


EDIT 2:
It is not due to ion suppression or enhancement since I observe the same problem with my DAD. So I assume there is somehow a gradient in my samples?!
If you inject a third time does it continue to increase?
Could there be multiple phases dispersed in your sample?
If you let is sit overnight what happens?
I have run into this recently using Acetic acid as mobile phase and deuterated Phenlyalanine as the internal standard. With the mobile phase fresh the area of Phenyalanine will start at 300,000 counts, and drop to 100,000 counts after about 30 runs at 15 minutes each. Rerun the sequence right after that one and the counts will start at 100,000 and drop to about 45,000. Wait overnight and reanalyze the same sequence and the counts begin about 40,000 and only fall to about 35,000 or maybe higher over the course of the run.

I am wondering if there is something about acetic acid that takes time to come to equilibrium and remain stable. The other internal standard of deuterated Uracil remains stable for all of the injections in the above testing.

Today I made up a fresh bottle of the acetic acid mobile phase but will not start the analysis until tomorrow to test this. Have you tried letting your acetic acid set overnight and see if that causes less variance?
The past is there to guide us into the future, not to dwell in.
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