Dehydroascorbic acid quantification in human plasma: Simultaneous direct measurement of the ascorbic acid/dehydroascorbic acid couple by UPLC/MS-MS
P.-C. Violet,
N. Munyan,
H.F. Luecke,
Y. Wang,
J. Lloyd,
K. Patra,
K. Blakeslee,
I.C. Ebenuwa,
M. Levine
Affiliations
P.-C. Violet
Molecular 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
N. Munyan
Molecular 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
H.F. Luecke
Intramural Research Division, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA, 27709
Y. Wang
Molecular 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
J. Lloyd
Laboratory of Bioorganic Chemistry, Intramural Research Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA, 20892
K. Patra
Molecular 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
K. Blakeslee
Principal Technical Support Specialist, Waters Mid Atlantic District Office, Waters Corporation, Columbia, MD, 21046, USA
I.C. Ebenuwa
Molecular 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
M. Levine
Molecular 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; Corresponding author.
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.
