New to GC-MS need help with some Sterol Stuff

[BrianGumble]

Greetings,

I am new student, new to using the GC-MS and have been having some problems quantifying and identifying some sterol standards. I have no tested all the standards but 2 of them so far: Cholesterol and 5a-cholestan. I made up these standards individually and both are in hexane (1ug/mL). I blew down the solvent under a stream of nitrogen and added 100uL BSTFA+TMCS for derivitization at 60C for 20 minutes then ran them on the GC-MS using a DB-5 60m column. Without the derivitization step the standards can been seen, but with the derivitization I cannot seem to find the derivitized product. It is expected that they would come off at a sooner retention time, but they remain almost identical to the retention time of the standards that have not been derivitized. We are running at 50C at 20C per min to 300C held for 20-30 mins. I was wondering if I am doing anything wrong?? Thanks in advance.

[Alexandre]

Keep der. condition anhydrous. What you do after derivatiztion? Straight injection, or you wash with something?
Looks like your der. reagent does not work. Open fresh ampoule.

[Don_Hilton]

What did the spectra look like? With the fast ramp the derivatized cholesterol may come close to the cholesterol. With 5a-cholestane - if we are not talking about something like the 3-ol, there is not a hydroxyl group present; no derivative will form.

[BrianGumble]

Thanks for the reply. I tried adding the BSTFA + 1% TMCS straight into a 1ug/mL solution of cholesterol and 5a-cholestan in hexane, then straight running it. I've tried blowing down the samples under N2 gas and adding the derivitizating agent then running it and I still do not get the peaks that I am looking for. The BSTFA+TCMS was replaced with brand new solution from sigma aldrich and it still does not produce the peaks we want. I am going to try blowing down the BSTFA and reconstituting in hexane before running it, but I do not think it will work.

GC-MS is 50C for 3 mins ramped to 320C at 15C/min and held for 10 minutes. Total runtime is 31 minutes.

We are using epicholestanol (5a-cholestan) not sure about the 3-ol. and the spectra when ran in SCAN produces a lot of peaks and when I signal out for specific ions, they do not line up at the same retention time (same goes with cholesterol). When ran WITHOUT derivitizing, then it does produce the peaks we want when identifying the ions in SCAN mode, when in SIM Mode it produces one nice sharp peak.

[Peter Apps]

[quote="

We are using epicholestanol (5a-cholestan) not sure about the 3-ol. and the spectra when ran in SCAN produces a lot of peaks and when I signal out for specific ions, they do not line up at the same retention time (same goes with cholesterol). When ran WITHOUT derivitizing, then it does produce the peaks we want when identifying the ions in SCAN mode, when in SIM Mode it produces one nice sharp peak.[/quote]

This sounds very much as if you have closely co-eluting peaks. If you extract the ions that are typical of the derivative and not the original compound, do you see a peak ? If you do, Try slowing down the temperature ramp to get the peaks separated.

Peter

[Alexandre]

Salut Brian,

You need to cook your derivatisation. Check condition on the net or books. I would do it from 60C or up to 80 (90?) for 30-60 min.
Be careful, reagents are hazardous; keep cooking inside a fume hood. Verify with your superviseur the safety procedure.

To blow out the garbage after derivatisation is good idea, but don’t overdo it as your products are volatile, just evaporate until you see a little drop left or use keeper, e.g. 10-25 uL of undecane.

[Don_Hilton]

I would not blow down the derivitization mix. Common practice is to add BSTFA/TMCS and cap the vial to keep air out. Heat at 60 degrees for an hour or so and run. If the concentration is sufficient to see the cholesterol, it is sufficient to see the TMS derivative.

you will have many of the same ions for the underivatized compunds as for the derivatized compunds. Thus, the importat question: do you see the ions for the molecuar ion of the derivatized cholesterol or epicholestanol? I see these kinds of compunds in work that I do and the derivatized peak does not elute far from the underivitized peak under the conditons that I use.

[BrianGumble]

Thanks for all the responses. I lowered the ramp time, and reconstituted the mix in hexane after derivitization, it still produced the same peaks I think. I lowered the ramp time so it is getting eluted at a slower rate. I still think it is being eluted very closely to the underived form (for cholesterol). For 5a-Cholestan (epicholestanol), I ran in SIM Mode for ions 217 and 129, and I do not think any of the peaks line up. Ion 129 is coming off of the derivitizing agent and 217 is a qualifier ion for 5a-cholestan (derivitized) I found from a journal article. I do not know where the derivitization is working for 5a-Cholestan.

My supervisor told me that derivitized forms of sterols should be coming out noticeable faster? However, when I am running it, it comes out almost identically to the underivitized form? What are your suggestions? Thanks again.

PS. I lowered the ramp from 15C/min to 10C/min and cholesterol (derivitized) comes out at almost 35mins whereas it was 28 mins before.

[BrianGumble]

I would not blow down the derivitization mix. Common practice is to add BSTFA/TMCS and cap the vial to keep air out. Heat at 60 degrees for an hour or so and run. If the concentration is sufficient to see the cholesterol, it is sufficient to see the TMS derivative.

you will have many of the same ions for the underivatized compunds as for the derivatized compunds. Thus, the importat question: do you see the ions for the molecuar ion of the derivatized cholesterol or epicholestanol? I see these kinds of compunds in work that I do and the derivatized peak does not elute far from the underivitized peak under the conditons that I use.

I saw some journal articles where they do blow down the derivitization agent and reconstitute (I was trying to see if it made any difference in peak resolution).

For ions, when I type in the ions for derivitized cholesterol a peak shows and all the ions match up, however retention times are almost identical to the underivitized form. For 5A-Cholestan, I am not sure if the derivitization is working or whatnot, when I look at the derivitized 5a-cholestan, the ion peaks do not align.