Vit E stability-HPLC

[pokale2600]

Hello everyone,

I am having problem with Vit E stability study. i have a sample stored at room temperature at 20C (sample A) and one sample stored at 3C (sample B). every time i measure concentration of sample A it is increasing for some reason unlike sample B which is constant or slightly less. there isnt any change in retention time for any samples. Just wondering if there is any problem with HPLC method or something to do with stability of vit E.

chromatographic conditions are as follows;
method: isocratic
mobile phase: methanol
flow rate: 1.8 /ml
Agilent eclipse XDB-C18, 5μ Particle size, 4.6 x 150 (L x ID mm)
Temp: 25C
wavelength: 204nm

another thing is MP for Vit E is 3C. Though it is already in viscous liquid form but further melting might be a cause of my question me think??
Since there isnt any change in RT i think structure remain same at room temp.

Please let me know your thoughts on it. I will appreciate all sorts of suggestions.

Many tanks in advance

[Gerhard Kratz]

The stability of vitamin E depends on its form, dl-α-tocopheryl-acetate being the most stable. Vitamin E, occuring naturally in foods in the form of a-tocopherol, oxidizes slowly when exposed to air. However, vitamin E added in the form of α-tocopheryl acetate shows excellent retention in wheat flour. Losses of vitamin E occur only during prolonged heating such as in boiling and frying. So please let us know the matrix of your sample.

[Gerhard Kratz]

HPLC Analysis of Vitamin E by Conversion to a-Tocopheryl Acetate ...
von ST Mayne - 1988 - Zitiert durch: 3 - Ähnliche Artikel
copherol by conventional HPLC methods of analysis of vitamin E. A reversed-phase HPLC method was therefore devised to quantitate low levels of a-tocopherol ...
pubs.acs.org/doi/abs/10.1021/jf00081a019
Maybe that link is of interest for you. Canthaxanthine, a nonprovitamine A carotenoide, if it is in your sample, can coelute with your alpha-Tocopherol.

[craftti]

You haven't mentioned the concentration of your solutions. There is not typically any problem with solubility of vitamin E in methanol but this can be a problem with other fat-solubles. I noted that you are using 204nm for detection - most groups use 292nm to get away from non-specific absorbance of solvents and anything else at low UV. Fluorescence is a more sensitive and selective detection method. If you have one to run in-line, it would eliminate nearly all interferences from other components that might coelute.

There is no solvent evaporation at room temp - correct? Am I correct that you are keeping your MP at 3C? If it is just methanol, it may be better to maintain at room temp.

[HW Mueller]

Is pokale´s MP a melting point?
The only idea I have is that something evaporates out of the room temp. sample but not out of the 3° sample.
It seems that unless you have a biblical type miracle occuring an experimental error is behind increases of analyte.

[samsa]

hello wy you chose 204 nm, the best wave lenght is 292nm.

for the stability don't forget oxygen area if you use the same sample and

the light.

[pokale2600]

Thank you all for your response. Much appreciated.

@ Gerhard Kratz: I am using dl-α-tocopheryl-acetate (97%) for my stability.
So when i keep sample at room temperature it is 10 gm of above sample. then i analyse sample at various time points by dissolving and further diluting the samples.

@ Craftti: As mentioned above i keep 10 gm of vit E outside at room temp. i take 10 mg of sample dissolve it in 20 ml methanol. then i do 10 times dilution with methanol before i analyse samples in HPLC. So there is no solvent evaporation.

@ HW mueller: Yes! MP is a melting point in my case

@samsa: i have run UV scan for standard Vit E sample on HPLC i get good seperation peak at 204nm, However literature says that Vit E is detected at 292nm. I tried scaning same sample on UV-VIS spectrophotometer and i get the same result. Though in both scans (UV/HPLC) there is a tiny peak at 292nm as well.
There are some research papers which used 210nm wavelength for Vit E analysis. following is one of those,
http://pubs.acs.org/doi/abs/10.1021/jf00081a019

Can you please explain bit more about oxygen area and light??

Again thanks every one. Please let me know if you have some more suggestions

Best regards,
AMIT