Chromatography Forum

Hi every one,
I'm a Phd student in a french lab and one of my obectives is to measure cholesterol in different samples by GC-MS.
However i have some problemes with my internal standard (5alpha-cholestane) when i do the standard curve.
The area under the curve (AUC) of my internal standard increase in same way of the AUC of my molecule of interest (cholesterol) and in the first sample of my standard curve (whithout cholesterol but with internal standard) i can't detect the internal standard.

Have you ever seen something like that and if it the case how can I fix this problem ?

Thanks in advance

François

I think the most obvious possible cause would be human error during the preparation of your standards...

thanks for your reply
we have eliminate the human factor because i ve seen this every time i've reppeted this experiment.

If you do repeated injections of the first standard, the one with only the internal standard, does the 5 alpha-cholestane appear in the second or third injections? If so you may have a conditioning effect. If not see, LiVD's comment or think about why the internal standard is lost unless the target is present.
Is your internal standard at the same concentration in all standards? If its response tracks the target analyte it may not be your internal standard but a breakdown product of the target.

When I was doing cholesterol assays (milk, meat, etc.) about 2 decades ago, I didn't have any trouble at all. We also used cholestane as internal standard, and we made trimethylsilyl derivative of the cholesterol for better chromatography. Of course the cholestane itself does not derivatize, but chromatography was OK.

I'm not going to look up the details but remember the temperature settings were pretty high.

Thank you both for your reply.

after successive injections of the first standard (the tube with just internal standard) it don't appear in any injection.
to answer to your question, the internal standard is added at the same concentration in all of my samples and standards.

I don't fully understand what do you mean by "If its response tracks the target analyte it may not be your internal standard but a breakdown product of the target" (I have a biology/physiology backgroung and my chemical knowledge are not very large)

for the preparation of standards I evaporate under N2 just after I add internal standard and ressuspend the pellet in distillled H2O. Is that possible that come from here?

thanks again !

Be careful that the ions you use for quant and confirmation are unique to the compound you are looking for. Some duterated componds contain small ion fragments of the "parent" cpd.

Concerning the temperature this is how i do:

150°C-(50°C/min)-280°C-(0,5/min)-284-(50°C/min)-300°C
i never exceed 300°C
for information I use a HP-1MS (30,0m x 0,25mm x 0,25 µm) Agilent 19091S-933

Is that possible that some cholesterol is not eluted with this range of temperature, staying in the column and then be eluted in the following sample ?


thanks

To check to see if a compund is eluted in the next injection, make a blank injection (solvent only) right after an injection with the compund. If you see the peak - it has carried over from one injection to the next - either by haning on the column longer than your chromatographic run or by some other means.

Make an injection of the cholesterol you are using for the standard alone. Depending on the source, you may have cholestane or cholestene as an impurity in the standard. Cholestene would be formed by dehydration of cholesterol. Cholestene has a molecular ion two mass units lower than cholestane, but will have many shared ions - and may or may not elute close to cholestane (I don't remember the behaviour - and I was probably trying to avoid a coelution with compound on a DB-5 column.)

After modification of my programe it's fine now.
The 5 alpha cholestane leaves the collumn about 2 min before cholesterol and C13 chol (during the solvent delay) and the pick that i saw was not cholestane but a residue of chol or C13 chol. That why I can t see my internal standard when the other molecule was not here and the increasement of the amount of the internal standard in my sandard curve.


thanks all of you for your comments and your responsiveness !

Thank you for the update. And best of luck to you.

After modification of my programe it's fine now.
The 5 alpha cholestane leaves the collumn about 2 min before cholesterol and C13 chol (during the solvent delay) and the pick that i saw was not cholestane but a residue of chol or C13 chol. That why I can t see my internal standard when the other molecule was not here and the increasement of the amount of the internal standard in my sandard curve.


thanks all of you for your comments and your responsiveness !

Hi Francois,
I quoted your post because i got the same problem. I could not see the internal standard (both reference standard cholesterol and 5 alpha cholestane ), I run with the GC 6890 machine from Agilent.
I am really confused because I tried many differences GC conditions but only got hexane peak.
I would like to ask if you can share your GC condition which you got success. Thank you