Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma
Gwendolyn van Gorkom,
Birgit Gijsbers,
Erik-Jan Ververs,
Ahmed El Molla,
Cindy Sarodnik,
Celine Riess,
Will Wodzig,
Gerard Bos,
Catharina Van Elssen
Affiliations
Gwendolyn van Gorkom
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Birgit Gijsbers
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Erik-Jan Ververs
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Ahmed El Molla
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Cindy Sarodnik
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Celine Riess
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Will Wodzig
Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Gerard Bos
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Catharina Van Elssen
Department of Internal Medicine, Division of Hematology, GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
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.
