Spectrophotometric determination of ascorbic acid and dehydroascorbic acid.
We present a method for measuring ascorbic acid in methanol/trichloroacetic acid extracts prepared from human plasma after enzymatic oxidation of ascorbic acid to dehydroascorbic acid by ascorbate oxidase. Samples 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 acid content. The detection limit was < 0.5 mumol/L. The calibration curve was linear (r > 0.995) over the range 0-1000 mumol/L. Analytical recovery of ascorbic acid added to plasma was 93-105%. The between-day variance was < 7%. Comparison of the spectrophotometric determination (y) with a chromatographic procedure (x) gave y = 1.02x - 0.653 (Sylx = 3.61) over the range of physiologically relevant concentrations. Total analysis time is < 10 min per sample and allows the simultaneous analysis of multiple samples.
Moeslinger
T
T
Institute for Medical Physiology, Vienna, Austria.
Brunner
M
M
Volf
I
I
Spieckermann
P G
PG
D001204:Ascorbate Oxidase / Q000378:metabolism; D001205:Ascorbic Acid / Q000097:blood*; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D002951:Citrates; D019343:Citric Acid; D003683:Dehydroascorbic Acid / Q000097:blood*; D006801:Humans; D007700:Kinetics; D000432:Methanol; D010084:Oxidation-Reduction; D010710:Phosphates; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods* / Q000706:statistics & numerical data; D013816:Thermodynamics; D014238:Trichloroacetic Acid
D002021:Buffers; D002951:Citrates; D010710:Phosphates; D019343:Citric Acid; D014238:Trichloroacetic Acid; D001204:Ascorbate Oxidase; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid; D000432:Methanol