Dehydroascorbic acid quantification in human plasma: Simultaneous direct measurement of the ascorbic acid/dehydroascorbic acid couple by UPLC/MS-MS

• Published in: Redox biology Vol. 78; p. 103425
• Main Authors: Violet, P.-C., Munyan, N., Luecke, H.F., Wang, Y., Lloyd, J., Patra, K., Blakeslee, K., Ebenuwa, I.C., Levine, M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2024; Elsevier
• Subjects: Ascorbic acid; Ascorbic Acid - blood; Chromatography, High Pressure Liquid - methods; Dehydroascorbic acid; Dehydroascorbic Acid - blood; Human plasma; Humans; Mass spectrometry; Reproducibility of Results; Research Paper; Tandem Mass Spectrometry - methods; Unispray; Vitamin C; Dehydroascorbic acid; Unispray; Ascorbic acid; Human plasma; Mass spectrometry; Vitamin C
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2024.103425

Ascorbic acid (AA, vitamin C) and dehydroascorbic acid (DHA) constitute a biological couple. No technique can accurately, independently, and simultaneously quantify both members of the couple in animal and human samples, thereby constraining advances in physiology and pathophysiology. Here we describe a new UPLC/MS/MS method to measure both compounds directly and independently in human plasma. Lower limits of quantification were 16 nM, with linear coefficients >0.99 over a 100-fold concentration range. The method was stable and reproducible with <10 % injection-to-injection variation. Use of isotopic labeled internal standards for both compounds ensured precision and accuracy. Plasma preparation required only 2 steps. In plasma samples from 14 anonymized subjects who met criteria for blood donation, mean concentrations were 6±2 μmol/L (mean ± SD) and 56 ± 14 μmol/L for DHA and AA respectively, with (DHA)/(AA + DHA) ratio of 9.8 %. This method represents a pioneering approach to measuring the AA/DHA couple in human plasma. •Novel LC-MS method for quantifying dehydroascorbic acid in human plasma.•Simultaneous, direct, and independent quantification of AA/DHA redox couple.•Streamlined 2-step sample preparation.•Lower quantification limit 16 nM; linear co-efficient >0.99 over 100-fold concentration range.•Enhanced sensitivity and specificity using 13C internal standards.