Chromatography Forum

Hello Friends! I am doing some research on cholesterol analysis using the AOAC 994.10 method which calls for the use of a GC. I am using 5 alpha cholestane as an internal standard. Does anybody have any experience developing a standard curve that highlights the difference in response of cholestane vs. cholesterol in standard, known quantity solutions? Does the difference in response change depending on the amount of counts of both cholesterol and cholestane? When I vary the ratio of cholestane vs. cholesterol in known quantity solutions the ratio of counts of cholestane vs. cholesterol does not seem to reflect that same change.

Thanks in advance!

Steve

About 20 years ago R&D did a project which required cholesterol analysis. We too started with the AOAC procedure, didn't like it; unless things have changed, the cholestane internal standard was added near the end, thought that was strange.

A publication at about that time tracked cholestane recovery throughout the extraction process, authors thought adding it early worked pretty good. I liked this way of thinking better: add early in the process.

So we added cholestane early, did the extractions, then took up in dimethylformamide and made trimethylsilyl derivative of the cholesterol (cholestane doesn't have active site, does not get derivatized), then used capillary GC for assay. Derivatized cholesterol was more stable than GC "as is" for us.

I wanted to push for publication, but R&D management decided it wasn't worth the time and resources required. We did issue it as a company test procedure though.

Thanks for that bit of information!

When you obtained different fractions of analyte cholesterol, did you have have to change the response factor you used? In the work I have been doing it seems as if there is a changing response factor depending on the ratio of cholesterol to cholestane being injected into the GC.

Also, i have been doing my injections "as is" into the GC. When I attempted the TMS in DMF I got a split cholesterol peak as if only a portion of it derivatized. I doubled the amount of reagents I used and got a similar result. Any thoughts on this?


Thanks in advance
Steve

Thanks for that bit of information!

When you obtained different fractions of analyte cholesterol, did you have have to change the response factor you used? In the work I have been doing it seems as if there is a changing response factor depending on the ratio of cholesterol to cholestane being injected into the GC.

Also, i have been doing my injections "as is" into the GC. When I attempted the TMS in DMF I got a split cholesterol peak as if only a portion of it derivatized. I doubled the amount of reagents I used and got a similar result. Any thoughts on this?


Thanks in advance
Steve

Are you keeping the cholestane concentration constant while varying the cholesterol concentration?

If so, and the cholestane response remains fairly constant throughout the range of calibration standards then it would appear the cholesterol is behaving in a quadratic response to either the extraction or the GC. If you can take the response factors and model them on a quadratic fit then you should be ok as long as it always performs the same in the entire process.

So we added cholestane early, did the extractions, then took up in dimethylformamide and made trimethylsilyl derivative of the cholesterol

I checked, from back in early 1990s, and we took up in pyridine, not DMF. GC: single sharp peak for cholestane, and single sharp peak for silylated cholesterol. So here's some test information from then:

This method is applicable to the determination of total cholesterol in various meat products. This straightforward method (a modification and combination
of published methods) produces results compararable to or better than the
time-consuming, benzene-using official method (1984 AOAC, 14th edition).

Our method involves direct saponification of the sample after cholestane internal standard addition, petroleum ether extraction, petroleum ether evaporation, dissolution of the residue in solvent, and capillary GC analysis vs. a standard mixture containing cholestane and cholesterol. We found silyl derivatization unnecessary when using GC columns and liners in top-notch condition as consistent peaks of excellent shape were obtained; however, standards and samples can be derivatized if desired, using either ethyl acetate or pyridine as the solvent for both standards and extracted sample residue.

Even though cholesterol is not especially soluble in petroleum ether, complete extraction occurs because of the small amount of ethanol present in the petroleum ether when extracting. Recoveries of standards and meat samples spiked with additional cholesterol ranged from 97 to 103%.

Cholesterol (e.g. Aldrich #C7,520-9)

Cholesterol standard stock solution: weigh 0.25 g cholesterol (to the
nearest 0.0001 g) into a 50 ml volumetric flask; mix and dilute to volume with ethyl acetate. Store in a refrigerator and make fresh monthly.

5-alpha-Cholestane (e.g. Sigma #C-8003)

Cholestane internal standard stock solution: weigh 0.25 g cholestane
(to the nearest 0.0001 g) into a 50 ml volumetric flask; mix and dilute to volume with ethyl acetate or pyridine. Store in a refrigerator and make fresh monthly.

Standard mixture: pipet 5.0 ml cholesterol standard stock solution and 5.0 ml cholestane internal standard solution into a 50 ml volumetric flask; mix and dilute to volume with ethyl acetate or pyridine. Store in a refrigerator and make fresh monthly. (If derivatization is to be used, mix 500 µl dilute standard mixture with 500 µl BSTFA in a sample vial; allow to stand overnight if ethyl acetate solvent is used instead of pyridine.)

Weigh 5 g of well-mixed sample (to the nearest 0.01 g) into a 250 ml
Erlenmeyer flask with 24/40 standard taper joint. Add a magnetic stir bar.

Accurately add 500 µl cholestane internal standard solution.

Add 5 ml H2O.

Add 8 ml potassium hydroxide solution.

Add 40 ml 3A alcohol.

Attach a ground joint inner member to the Erlenmeyer flask to act
as an air condensor; heat at medium heat and stir for one hour,
adding additional 3A alcohol to make back to original volume if necessary.

Rinse air condensor with about 20 ml 3A alcohol.

8. Remove air condensor and add about 3 g NaCl and 60 ml H2O. Swirl.

9. Cool to room temperature in a water bath.

Add 80 ml petroleum ether. Stopper and stir for 3 minutes.

Transfer to a 500 ml mixing cylinder using 3A alcohol and petroleum ether from squeeze bottles. Add about 50 ml H2O, 40 ml 3A alcohol, and 80 ml petroleum ether to the cylinder.

Stopper the cylinder and shake for one-half minute. Allow the layers to separate.

Siphon off the petroleum ether layer into a 400 ml beaker through a funnel containing sodium sulfate granules held in Whatman #41 filter paper. Extract twice more, using about 100 ml petroleum ether for each extraction.

Blow off the petroleum ether using a steam bath and stream of air.

Add 5 ml ethyl acetate or pyridine to the residue in the beaker, swirling to dissolve the residue (use appropriate solvent to match that used for standard mix).

Transfer a 500 µl portion to a sample vial and cap. (If derivatizion is to be used, transfer a 500 µl portion to a sample vial, add 500 µl BSTFA, cap and mix; allow to stand overnight before injection if ethyl acetate is used instead of pyridine.)

GC: single sharp peak for cholestane, and single sharp peak for silylated cholesterol.

References
1. Adams, M.L., Sullivan, D.M., Smith, R.L., and Richter, E.F. (1986)
J. Assoc. Off. Anal. Chem. 69, 844-846.

2. Kovacs, M.I.P., Anderson, W.E., and Ackman, R.G. (1979) J. Food
Sci. 44, 1299-1301, 1305.

3. Bando, H., and Ishikawa, K. (1978) Tokushima Bunri Daigaku Kenkyu
Kiyo 19, 39-42.