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Streamlined three step total vitamin C analysis by HILIC-UV for laboratory testing
Michael Fitzpatrick1,2, Paul Bonnitcha1,3,4, Van Long Nguyen1
1Department of Chemical Pathology, NSW Health Pathology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
Clinical Chemistry and Laboratory Medicine
|September 2, 2021
Summary
This study introduces a new hydrophilic interaction chromatography method for measuring vitamin C (ascorbic acid) in plasma. This approach overcomes challenges with analyte stability and improves accuracy in clinical settings.
Area of Science:
- Clinical Chemistry
- Analytical Chemistry
- Chromatography
Background:
- Vitamin C (ascorbic acid) analysis in clinical settings faces challenges due to analyte instability.
- Traditional reverse-phase HPLC methods often exhibit poor retention for ascorbic acid.
Purpose of the Study:
- To describe a novel hydrophilic interaction chromatography (HILIC) method for quantifying total vitamin C in plasma.
- To address the limitations of existing analytical techniques for vitamin C measurement.
Main Methods:
- Utilized hydrophilic interaction chromatography with ultraviolet detection.
- Employed a Waters BEH-Amide column and a mobile phase of 0.005 M potassium phosphate in 80% acetonitrile.
- Separated ascorbic acid and an internal standard under isocratic conditions.
Main Results:
- The method demonstrated excellent precision with a coefficient of variation <5%.
- High accuracy was achieved, with results exceeding 99%.
- Analyte stability after extraction was confirmed to be greater than 24 hours.
Conclusions:
- The developed HILIC-UV method effectively overcomes challenges associated with vitamin C analysis.
- Simple sample preparation enables full automation and rapid analytical run times.
- The assay is well-suited for high-throughput clinical chromatography laboratories.