Separative determination of ascorbic acid analogs contained in mushrooms by high-performance liquid chromatography

Analogs (6-deoxyascorbic acid, erythroascorbic acid, and associated glycosides) of L-ascorbic acid (AA) contained in mushrooms were allowed to react with hydrazine to form osazones, and the conditions for separative determination by HPLC using a Zorbax SIL column were examined. Separation was started using solvent system 1 (ethylacetate/n-hexane/acetone/acetic acid, 50:50:1:1, v/v) as the mobile phase, and switching after 15 min to solvent system 2 (ethylacetate/acetone/acetic acid, 100:1:1, v/v). Detection was performed by absorbance at 500 nm. Because these analogs showed different formation rates for osazone, calibration curves were prepared for each substance. The recovery rate in the load test was 93-105%. By this method, AA and the analogs contained in eight species of edible mushrooms have been determined. The results revealed that: (1) the main constituents of all mushrooms are AA analogs rather than AA itself; (2) only one species contained AA in a very small amount (2 mumol/kg); (3) the types of AA analogs present differed according to the species of mushrooms, and (4) the total amount of AA analogs was between ca. 100-500 mumol/kg (2-9 mg per 100 g, converted to AA). In addition, a new AA analog was found in Pleurotus ostreatus and identified as 5-O-(alpha-D-xylopyranosyl)-D-erythroascorbic acid in structural analyses by NMR and other methods.