Cholesterol in Eggs

[Ann Lucas]

Hello,
I am starting a new project that I need a bit of help with. I have not been able to find any information on HPLC methods for determining Cholesterol and Fats in Eggs.
I am very new to the world of HPLC and any help with the methods would be greatly appreciated.
Thank you in advance,

Ann Lucas
NC A&T
Greensboro, NC

[Victor]

Ann- Gas Chromatography is a far better method for determining cholesterol than HPLC. I guess you might not have a sensitivity problem for eggs (there is only 1 isolated double bond in cholesterol if you have a UV detector on your HPLC) but the column efficiency in GC is much higher than in HPLC.

[Ann Lucas]

Victor,
Thank you for your reply. As I said, I am very new to this and I would like to have as much information as possible. Could you send me the method for GC? I do have a UV detector on my HPLC, but it is always better to have a plan B if needed. If you do not want to post that information here, let me know and I'll send you my email address directly. Still looking for HPLC method.
Ann Lucas

[Consumer Products Guy]

We also use GC for chlolesterol, with cholestane as internal standard. We updated the published procedure years ago (for example, to use capillary columns), but unfortunately the procedure is deemed proprietary.

[Victor]

Ann - I do not have a recipe for this method that you can follow blindly, but you should be able to find one in the literature if you want such a thing.

Basically, you can analyse cholesterol on a non-polar column (e.g. OV1) or a slightly polar column (e.g. SE-54, or anything with about 5% phenyl groups). It will also work on a polar column, but these are generally less temperature stable, so why bother?

You could use split injection probably for your samples. Maybe a temperature program up to 300 degrees centrigrade. People ususally make the trimethylsilyl derivatives of cholesterol to get rid of the -OH group although it will still work with the underivatised compound. You can make the TMS derivatives with the reagent BSA/TMCS. 5 alpha chosestane is an alkane which will do as an internal standard especially to get round any vagaries of split injection.

[Victor]

Sorry-that's 5 alpha cholestane. I'm talking about capillary colums too-choose one with film thickness 0.15-0.25um otherwise the temperature required to elute cholesterol may be too high. A standard FID will give you much better sensitivity than HPLC-UV, even though you might not need this with eggs......