Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma

G. van Gorkom*, B. Gijsbers, E.J. Ververs, A. El Molla, C. Sarodnik, C. Riess, W. Wodzig, G. Bos, C. Van Elssen

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

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/10(8) 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.
Original languageEnglish
Article number134
Number of pages12
JournalAntioxidants
Volume11
Issue number1
DOIs
Publication statusPublished - 1 Jan 2022

Keywords

  • ascorbic acid
  • vitamin C
  • peripheral blood mononuclear cells
  • PERFORMANCE LIQUID-CHROMATOGRAPHY
  • VITAMIN-C
  • LYMPHOCYTES
  • COMPONENTS

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