Da. Schell et Am. Bode, MEASUREMENT OF ASCORBIC-ACID AND DEHYDROASCORBIC ACID IN MAMMALIAN TISSUE UTILIZING HPLC AND ELECTROCHEMICAL DETECTION, BMC. Biomedical chromatography, 7(5), 1993, pp. 267-272
Reliable measurement of the reduced and oxidized forms of ascorbic aci
d (AA) is challenging because they are highly reactive and unstable co
mpounds. Detection of small amounts of AA and dehydroascorbic acid (DH
AA) is essential for determining the biochemical function of the vitam
in. While a variety of techniques exist for measurement of AA with det
ection limits in the millimolar range, a need exists for highly reliab
le assessment of picomolar levels of AA and DHAA in tissues. The prese
nt study presents a method for measuring AA and DHAA that combines hig
h performance liquid chromatography with the advantages and increased
detection limits and selectivity available with coulometric electroche
mical detection. The difference between AA and ''total AA'' in tissue
samples gives an assessment of DHAA concentration. Verification of the
reliability of the assay is by the successful linear recovery of exog
enously added AA and DHAA in tissue homogenate. Optimal conditions for
reducing DHAA in tissue samples include a pH of 7.2, reaction time of
10 min, reaction temperature equal to room temperature, and a 10 mm c
oncentration of the reducing agent, beta-mercaptoethanol. AA and DHAA
are measured in several mammalian tissues using the method presented.