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

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Nutritional Science

Background:

• Ascorbic acid (vitamin C) is crucial for antioxidant defense and enzymatic functions.
• Vitamin C deficiency and hypovitaminosis are common in certain patient groups, leading to misdiagnosed symptoms.
• Accurate quantification of vitamin C is essential for clinical diagnosis and management.

Purpose of the Study:

• To develop and validate a robust and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying total ascorbic acid in human plasma.
• To assess the prevalence of vitamin C deficiency and hypovitaminosis in a patient population using the developed method.

Main Methods:

• Plasma samples were collected in EDTA, kept on ice, and processed within two hours to preserve ascorbic acid.
• Dehydroascorbic acid was reduced to ascorbic acid using tris(2-carboxyethyl)phosphine (TCEP).
• Ascorbic acid was separated by reversed-phase chromatography and detected using LC-MS/MS in negative ion mode.

Main Results:

• The method achieved a short run time of 4.6 minutes with high precision (intra-assay 1.8–2.2%, inter-assay 3.2–5.7%).
• The limit of quantification was 1.1 μmol/L, adequate for identifying deficiency (<11.4 μmol/L).
• Retrospective analysis of 547 samples revealed 10% deficiency and 20% hypovitaminosis; males had lower vitamin C levels than females.

Conclusions:

• The validated LC-MS/MS method provides accurate and efficient total ascorbic acid measurement in clinical settings.
• A significant percentage of patients exhibit suboptimal vitamin C levels, necessitating increased clinical awareness.
• The findings support targeted screening and nutritional interventions for vitamin C deficiency and hypovitaminosis.