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- Posts: 28
- Joined: Fri Mar 11, 2016 3:41 pm
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?!