Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible
method to measure AA status in the human body. Serum AA concentrations do not
correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for
clinical application is lacking. This present study describes a method to measure AA in the peripheral
blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The
method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs
and plasma were performed with HPLC with HILIC separation and UV detection. The sample
preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European
Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation.
A highly precise execution of the method was found with intra- and inter-assay variations at a
maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108
cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not
present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for
the quantitative determination of AA within PBMCs and plasma from human blood.
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