Fast HPLC-ECD analysis of ascorbic acid, dehydroascorbic acid and uric acid.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Accurate quantification of ascorbic acid (AA) and uric acid (UA) in human plasma is crucial for understanding various physiological and pathological processes.
• Existing methods may lack the sensitivity, speed, or efficiency required for routine clinical analysis.

Purpose of the Study:

• To develop and validate a robust and rapid high-pressure liquid chromatography-electrochemical detection (HPLC-ECD) method for simultaneous determination of AA and UA in human plasma.
• To compare the performance of traditional HPLC with ultra-HPLC (UHPLC) for this analysis.

Main Methods:

• Development of a reverse-phase HPLC-ECD method utilizing homogentisic acid as an internal standard.
• Analysis of ascorbic acid (AA), uric acid (UA), and dehydroascorbic acid (DHAA) in human plasma samples.
• Comparison of analytical performance between conventional HPLC and UHPLC systems.

Main Results:

• The UHPLC method achieved a detection limit of 0.05 ng for both AA and UA, which is two times lower than conventional HPLC.
• UHPLC analysis reduced run times by fourfold and generated less solvent waste.
• Both HPLC and UHPLC methods demonstrated good accuracy and precision, with intra- and inter-day coefficients of variation (CVs) below 7% for AA and UA.

Conclusions:

• The developed UHPLC-ECD method provides a sensitive, rapid, and accurate approach for quantifying AA and UA in human plasma.
• This method offers significant advantages over conventional HPLC in terms of sensitivity, speed, and reduced waste generation.
• The validated assay is suitable for routine clinical laboratory analysis and biochemical research.