chapter  3
6 Pages

Analytical enigmas in assaying for vitamers C

ByJohn E. Halver, Samuel P. Felton

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2 Vitamers C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.3 Extraction enigmas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.4 Assay techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Abstract L-ascorbic acid is labile and easily oxidized to dehydroascorbic acid. Early workers carefully extracted tissues using mild treatments in metaphosphoric acid to protect ascorbic acid. These extraction methods did not extract tissue bound ascorbate. More stringent techniques using stronger acids would extract bound ascorbate esters, but the acidic conditions would hydrolyze the ascorbyl esters yielding variable assay results. A modified technique using 5% TCA for extraction followed by centrifugation, filtration, and injection within one hour resulted in acceptable HPLC assays for several vitamers C. New columns without an ion-binding compound in the mobile phase eluted ascorbic-2-sulfate prior to ascorbic acid, and both compounds were confirmed with electro-ionizing mass spectroscopy.