Assessment of Provitamin A Determination by Open Column Chromatography/Visible Absorption Spectrophotometry

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Abstract
Determination of provitamin A content by open column chromatography/visible absorption spectrophotometry is assessed using food samples of varying carotenoid composition. A general method consisting of extraction with acetone, transfer to petroleum ether, saponification (optional), concentration, separation on activated MgO:Hyflo Supercel column developed with 1 to 15% acetone in petroleum ether, and quantitation of individual provitamins spectrophotometrically demonstrates repeatability comparable with that of high-performance liquid chromatographic (HPLC) methods. Overnight saponification (10% methanolic KOH, ambient temperature) does not degrade the provitamins and is unnecessary for kale, tomato, and squash; however, it is required for good separation of papaya carotenoids due to the presence of carotenol esters. The current Association of Official Analytical Chemists (AOAC) method is found to be inappropriate because (1) the volume of extracting solvent is not adjusted to the type of sample; (2) the less active α- and γ-carotene, α- and β-cryptoxanthin, and 5,6-monoepoxy-β-cryptoxanthin (50% active) are quantified as β-carotene (100% active); (3) inactive carotenoids such as ζ-carotene and zeinoxanthin are also quantified as β-carotene.