We have problems with analyzing cortisol by GCMS. After performing a 2 step derivatization (methoximation and silylation) with methoxyamine hydrochloride and MSTFA, we do not see peaks in the chromatogram. We used a DB5 MS column.
Does anyone have an idea what the problem is.
Thank you.
Yes, I do net see the derivatized ref standard.
I think the GC conditions are ok. I start with 70°C and go up to 300. I also tried up to 320. Also increasing the GC program time did not help. Derivatization seems also to work - at least for other substances like glucose.
Dear Benedikt
Did You try:
1) higher concentration of the standard
2) faster temperature ramp
3) use SIM mode?
I am not sure that any of these will solve the problem, but I would try, if You did not yet.
We used 0.5 mM (0.18 mg/ml) of cortisol, so really a lot compared to literature LOD & LOQ values, wich are in the ng/ml range.
I also tried to measure in SIM mode.
I ramp with 20°C/min so already fast. I also tried to ramp slower.
Why would a faster ramp help?
I have also the problem that the solvent comes in the first 10 minutes, but cortisol should come "very late". I checked the retention indices and so, it is no problem to skip the first 10 minutes for the solvent....
Thank you.
Dear Benedikt
Hm...
I just though that since it's a large compound, increasing ramp will protect You from finishing the run before elution.
What kind of solvent do You use? The same as for derivatization? Why it takes so much time - 10 min? Specifically under this fast ramp? What is the column length?
And You do not see any peaks at all?
I inject directly after derivatization, so the solvent in the sample is a mixture of pyridine and mstfa... I did that also for other substances and there it works. The GC solvent for the needle wash is hexane:iPrOH (80:20).
Column length is 30 m. I see a lot of the silylation reagent at the beginning - to avoid damage of the filament and detector I start now at about 10 minutes to acquire ms-data...
I see peaks, solvent related and also 2 fatty acids from sample prep/solvents used...
I also tried the same with glucose, there it works...
Dear Benedikt
It is a bit strange that You do not have selected peaks in SIM mode analyzing standard. That makes me think that either derivatization or column do not work for Your compound. But You said You are sure that both of theses are not the case.. Well, I have no idea.
What is RT of glucose under You conditions?
Dear Albert,
I now also tried a DB1 column and a much higher concentration of the standard. With no split and 3 µl injection I finally saw a little peak of the derivatized cortisol...
So there is a problem with the DB5 column and maybe with the derivatization efficiency...
We will work on that.
Thank you a lot for your help and suggestions!
(Glucose was coming at about 10 minutes...)
There is the possibility that something else in the sample matrix is consuming the derivatization agents.
Dear Benedikt
Sounds reasonable!
As for derivatization, try to replace mstfa with bstfa+tmcs.
Also, for SIM mode, try to chose more exact m/z. I understand that because You did not detect the compound yet, You can rely only on library. So just try to modify library suggested m/z to +/- 0.1 (if You did not try this yet). And the column, as You said.
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