Problem met in carotenoid extraction

[angel00123]

Hello, guys~

I am now working on carotenoid extraction from rice mature seeds. To estimate the extraction efficiency, I added beta-carotene as an internal standard before organic extraction. After extraction, HPLC was performed, and the peak area is compared with that producted by the same amount of standard alone without extraction. And the result shows only 30-40% of beta-carotene is recovered.

Please help me~~~~~ Thanks alot.

[angel00123]

As carotneoids are easy to be oxidized and is light-sensitive. I performed the extraction in nearly darkness (Because it is not quite possible to turn off the light in the lab, there are people doing lab work nearly 24 hours everyday........... I am hidding in a DIY dark chamber when adding the solvent, use a aluminium foil covering the tubes when took them to sonicate and centrifuge), and centrifugation have been performed in 4℃. Samples were kept on ice whenever possible. I tried hard and changed the method of extraction once, but the efficiency is still about the same.

Is this because the problem of using beta-carotene standard? Or the problem of dissolving the standard in n-hexane for storage, and diluted in ethyl acetate before HPLC? Or is it because my standard have been stored in -20℃ for two weeks already?

Can anyone give any sugestions? Thanks alot.

[angel00123]

By the way, I used acetone and tert-butylmethylethane for extraction. Residual organic solvent is evaporated by either nitrogen evaporation or vaccum drying. Then, a certain amount of ethyl acetate is used to resuspend the extracted product.

HPLC column used is C30 reversed YMC carotenoid column. Solvent used is a gradient solvent system composed of methanol, tert-butylmethylethane and water.