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Related Experiment Video

Updated: Jul 12, 2026

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Published on: April 11, 2014

Dehydroascorbic acid quantification in human plasma: Simultaneous direct measurement of the ascorbic

P-C Violet¹, N Munyan¹, H F Luecke²

¹Molecular and Clinical Nutrition Section, Digestive Diseases Branch, Intramural Research Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA, 20892.

|November 26, 2024

Summary

A new UPLC/MS/MS method accurately measures both ascorbic acid (vitamin C) and dehydroascorbic acid in human plasma. This breakthrough enables better understanding of vitamin C"s role in health and disease.

Keywords:

Ascorbic acid Dehydroascorbic acid Human plasma Mass spectrometry Unispray Vitamin C

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Last Updated: Jul 12, 2026

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

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Published on: April 11, 2014

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

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Published on: February 27, 2020

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

Published on: June 30, 2020

Area of Science:

Biochemistry
Analytical Chemistry
Human Physiology

Background:

Ascorbic acid (AA) and dehydroascorbic acid (DHA) form a critical biological redox couple.
Current analytical methods cannot simultaneously quantify both AA and DHA, hindering research.
Advances in understanding vitamin C
s physiological and pathophysiological roles are limited by this analytical gap.

Purpose of the Study:

To develop and validate a novel UPLC/MS/MS method for the simultaneous, independent quantification of AA and DHA in human plasma.
To overcome the limitations of existing techniques in measuring the AA/DHA couple.

Main Methods:

Ultra-Performance Liquid Chromatography coupled with tandem Mass Spectrometry (UPLC/MS/MS) was employed.
Isotopically labeled internal standards were used for both AA and DHA to ensure precision and accuracy.
A simplified plasma preparation protocol involving only two steps was utilized.

Main Results:

The UPLC/MS/MS method demonstrated high accuracy and reproducibility with low inter-assay variation (<10%).
Lower limits of quantification were 16 nM for both AA and DHA, with linearity over a 100-fold concentration range (R² > 0.99).
Analysis of human plasma samples revealed mean concentrations of 56 ± 14 μmol/L for AA and 6 ± 2 μmol/L for DHA.

Conclusions:

This pioneering UPLC/MS/MS method provides a robust and accurate tool for measuring the AA/DHA couple in human plasma.
The developed method facilitates further research into the physiological and pathophysiological implications of vitamin C redox cycling.