Fluorometric determination of total vitamin C in whole blood by high-performance liquid chromatography with...

A reliable and semi-automated high-performance liquid chromatographic (HPLC) method is described for the determination of total vitamin C in whole blood. After deproteinization of whole blood and enzymatic oxidation of l-ascorbic acid to dehydro-l-ascorbic acid, the latter is condensed with o-phenylenediamine to its quinoxaline derivative. This derivative is separated on a reversed-phase HPLC column and detected fluorometrically. Total vitamin C in whole blood can be determined in concentrations as low as 0.2 mumol/l. Special attention was paid to the stability of vitamin C in whole blood and of its quinoxaline derivative in the extract. Results of our investigations showed that total vitamin C in whole blood is stable for eight days at -20 degrees C, provided ethyleneglycol-bis-(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid and glutathione are immediately added to the blood sample. The quinoxaline derivative of vitamin C in the blood extract is stable for at least 24 h if stored in the dark at 4 degrees C. Routine vitamin C determinations can be carried out in a series of 100 samples within 48 h. The within-assay and between-assay coefficients of variation were 3.7% and 4.6%, respectively. The between-assay analytical recovery of l-ascorbic acid added to whole blood samples was 97.0 +/- 7.0% (mean +/- S.D.). Reference values of vitamin C in whole blood of normal healthy Dutch adults were found in the range 20-80 mumol/l.