Development and validation of an LC-MS/MS method for the analysis of total ascorbic acid capacity in plasma

A robust and fast LC-MS/MS method for quantifying total ascorbic acid (vitamin C) in plasma was developed and validated. Ascorbic acid is vital for antioxidant defense and enzymatic processes. Deficiency and hypovitaminosis remain prevalent in specific patient groups and can result in overlooked or misattributed clinical symptoms. To preserve ascorbic acid after venapuncture, EDTA blood samples were kept on ice and processed within two hours. Dehydroascorbic acid was reduced to ascorbic acid using tris(2-carboxyethyl)phosphine (TCEP). Ascorbic acid was separated by reversed-phase chromatography and detected by tandem mass spectrometry in negative ion mode. Method validation followed standard protocols. Retrospective analysis of 547 patient samples, analyzed with this method, was performed to assess the incidence of vitamin C deficiency and hypovitaminosis.A TCEP concentration of 15 mM was sufficient. Ascorbic acid eluted at 1.08 min, with a total runtime of 4.6 min. Intra- and inter-assay imprecision (three concentrations) ranged from 1.8 to 2.2% and 3.2–5.7%, respectively. The limit of quantification was 1.1 μmol/L, suitable for detecting deficiency (<11.4 μmol/L). Among the tested patients, 10% were deficient and 20% had hypovitaminosis. Males had ~11 μmol/L lower vitamin C levels than females, independent of age.This validated LC-MS/MS method enables accurate assessment of total ascorbic acid in clinical settings. With a significant proportion of patients showing suboptimal vitamin C levels, increased awareness is needed to support targeted screening and nutritional interventions. • A streamlined sample-stabilization workflow, fully reducing dehydroascorbic acid. • Fast reversed-phase separation and negative-ion MRM detection. • Rigorous validation: enabling confident detection of clinical deficiency. • Clinical impact: 10% of patients were deficient and 20% had hypovitaminosis.