Chromatography Forum

Hi all,

I want to determine vitamin E (tocoferol form, MW=430.71) assay using the succinate form (MW=530.79) as a standard.
The calculation I know requires the final result to be multiplied by a molecular weight correction factor that is MWtocoferol/MWsuccinate = 430.71/530.79 = 0.81

The person that I replaced (he left suddenly and not willing to be reached) had a different approach and actually multiplied by 1.2

How is the correct calculation?

I came from a pharmaceutical company and that was the way we would calculate the assay when we had to use a different standard. Do I miss something that is related strictly to vitamin E?

Thank you.

You also need to include the ratio of the molar absorbtivities at your detector wavelength and bandwidth. Tocopherol has a shorter wavelength maximum and higher absorbtivity than its ester forms. Unfortunately, unless you can find your predecessors old notebook, you may have to measure that ratio for yourself, or search the literature for it. Also, the international units for vitamin activity are expressed in terms of the racemic tocopherol acetate; if you use other forms you need a biopotency factor also.

Thank you for your answer. Could you please indicate a reference for your statement:

Also, the international units for vitamin activity are expressed in terms of the racemic tocopherol acetate

Thank you again.

Merck Index, 11th Ed, p1579-80.

What´s all this stuff, creeping up now, about a substance, different from the analyte, being used as a standard for the analyte???? One may use such a substance for internal standard, or if the analyte has to be converted to the acetate (precolumn). But building up the analysis (calibration included) with the acetate and then running the bare toco.? Am I misunderstanding something or are we reinventing the world?

It's common practice when a standard is too expensive, too unstable, or unavailable. None of those criteria apply to this situation, and I would not do it this way for Vitamin E, but it's not my lab.

I must have been too long in the ivory tower? Mark, how do you do a HPLC if the substance is too unstable?

Well, try doing carotenoids... I had to spectrophotometrically re-assay my standard stock solutions every day before making the working standard. The price of the standards was just barely tolerable. That is why the AOAC official method uses Sudan Orange as a surrogate standard for carotene: stable and cheap.

Well, I agree that it´s nice to have a standard which is stable, but am having trouble to see what good it does when one can not handle the unknowns due to instability of the analytes. If one can stabilize the unknown one can stabilize the standard. One gets the feeling that AOAC is delivering alibies?