[ablake]

Hi All!
I confess, I am no analytical chemist, only a PhD student who has absolutely no clue what I am doing. So please, bear with me.
Here is the rundown of my problem with a little GCMS background to cover all my bases:
I recently acquired this quantitative GCMS assay from two graduating students. We ran a batch of standards together in May before they left, and the extracted ion chromatograms (EIC) came out weird for some compounds (they picked ions to monitor for each compound in the development of the assay; EIC should be very consistent  because ion ratios/fragmentation patterns are inherent to compounds). I have since ran 6 or 7 batches of standards that all have the same problem, and have replaced almost every part on the instrument that could relate to the issue. BUT, the confusing part is that a) it only happens to certain compounds (i.e. in sterol standards, 5 out of 15 have EICs that consistently don't match what we would expect); b) the effect does not dilute down (in dilutional series, the ratio of ion2 to ion1 can vary from 60-400% and there is no pattern to this variation); and c) the EICs of calibration verification vials (pure compounds at the concentration of the middle standards, aliquoted [from the same stock solutions we run standards from] and stored in freezer to be run with every batch of samples) come out great and as expected every time! We prep and run the calibration verification at the same time as our standards as well.

My thoughts so far:
a) If something was wrong with the instrument, I don't see how it could selectively choose ions from certain compounds to have inconsistent ratios. Ions from across the entire scan range are affected
b) If certain compounds were degrading, shouldn't the effect dilute down?
c) The only difference in the calibration verification vials, is that they are not tightly sealed and the chloroform evaporates off. Both calibration verifications and stocks are stored in glass vials in -20C; stocks have teflon-lined screw cap, and CVs have plastic pop tops. This makes me think that some compounds are not favorable to being stored in chloroform solution. Does anyone have experience or advice for this?

More possibly relevant information would be how we prep the standards/CVs:
We dry them under Nitrogen at 65C, add Sylon HTP (HMDS+TMCS+Pyridine, 3:1:9), incubate, dry, then reconstitute in hexane and inject onto the instrument. I have tried new Sylon and hexane to no avail.

Anyways, I am open to any ideas, and I would very much appreciate any questions/comments/advice/etc!

Kind regards,
Amanda

[hypervalent_iodine]

How certain are you that the standards that aren't running as you'd expect, are what you think they are? Have you checked their structures in any other way? Chloroform does contain small amounts of HCl in it. If your compounds are very acid sensitive, there is a possibility that they are degrading over time. Do you notice a difference when you run samples that have been freshly prepared in chloroform vs. solutions that have been stored for a while?

[ablake]

Chloroform does contain small amounts of HCl in it. If your compounds are very acid sensitive, there is a possibility that they are degrading over time.

This explains a lot! We add HCl to samples during sample prep, but not to standards because it degrades some compounds when they aren't in sample matrix.

Technical support for the instrument company has been adamant that it is an instrument issue, so I am just now getting around to running the compounds individually in full scan to check purity. In hindsight, I should have done this as soon as the issue arose. Now I will try freshly prepared standards as well.
Thank you so much for the excellent advice!

[MOTOBALL]

If memory serves, dichloromethane does not undergo the degradation that affects chloroform.

Can you prepare samples/standards in CH2Cl2 ?

Regards.

[MOTOBALL]

If prepn. in dichloromethane (or other solvent) resolves the issue, keep all of your old data for

(1) important part of thesis
(2) possible publication

Regards