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

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/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.

Low Levels of Vitamin C during Pregnancy; a Risk Marker of Progression of Diabetic Retinopathy in Type 1 Diabetic Women?

“…Tubes were centrifuged and plasma was removed and deproteinized by the addition of 6% perchloric acid. The samples were kept at −80 °C until analysis and assayed by HPLC [ 27 ], an accepted gold standard for this measurement [ 28 ].…”

Conductive Polymer/Graphene‐based Composites for Next Generation Energy Storage and Sensing Applications

“…Among the commonly applied high-performance liquid chromatography, spectrophotometric techniques, and chemical sensors, electrochemical sensors seem to be highly promising in detection of various analytes. [79] The compact size and high selectivity and sensitivity of electrochemical detection setups, which are a specific type of chemical sensors, make them suitable candidates for detecting various analytes. [80] Moreover, the principle of electrochemical detection is facile and covers the measurement of electric currents generated during redox reactions on the working electrode surface immersed in the solution of the analyte to be detected.…”

“…In the last few decades, scientists have been developing novel techniques of analyte detection to meet environmental requirements and provide highly sensitive detection solutions for diagnosing several serious diseases. Among the commonly applied high‐performance liquid chromatography, spectrophotometric techniques, and chemical sensors, electrochemical sensors seem to be highly promising in detection of various analytes [79] . The compact size and high selectivity and sensitivity of electrochemical detection setups, which are a specific type of chemical sensors, make them suitable candidates for detecting various analytes [80] .…”

Synthesis of MoO3−x nanosheets and its application in ascorbic acid detection of fruit and vegetables

“…Various new methods for the detection of AA content in fruit and vegetables have emerged. The main detection methods of AA are high performance liquid chromatography (Klimczak et al., 2015; van Gorkom et al., 2022; Zuo et al., 2015), fluorescence (Flu) (Lyu et al., 2019; Zhang et al., 2017), and ultraviolet (UV) spectrophotometry (Mazhabi et al., 2018). All these methods are highly precise and accurate, but the environmentally polluting reagents are commonly used.…”