SPECTROPHOTOMETRIC DETERMINATION OF ASCORBIC-ACID AND DEHYDROASCORBICACID

We present a method for measuring ascorbic acid in methanol/trichloroa cetic acid extracts prepared from human plasma after enzymatic oxidati on of ascorbic acid to dehydroascorbic acid by ascorbate oxidase. Samp les were assayed by spectrophotometrically monitoring the kinetics of the concentration-dependent absorbance changes of dehydroascorbic acid with phosphate-citrate-methanol buffers. Ascorbic acid was determined as the difference between dehydroascorbic acid and total ascorbic aci d content. The detection limit was <0.5 mu mol/L. The calibration curv e was linear (r >0.995) over the range 0-1000 mu mol/L. Analytical rec overy of ascorbic acid added to plasma was 93-105%. The between-day va riance was <7%. Comparison of the spectrophotometric determination (y) with a chromatographic procedure (x) save y = 1.02x - 0.653 (S-ylx = 3.61) over the range of physiologically relevant concentrations. Total analysis time is <10 min per sample and allows the simultaneous analy sis of multiple samples.