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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Ascorbic acid determination with an automated enzymatic procedure
W Lee1, S M Roberts, R F Labbe
1Department of Laboratory Medicine, Harborview Medical Center and University of Washington, Seattle 98104-2499, USA.
Clinical Chemistry
|January 1, 1997
Summary
Automated plasma ascorbic acid (vitamin C) measurement is now faster and more precise using a new assay on the Roche Fara analyzer. This method offers improved throughput compared to HPLC, with reliable results for antioxidant nutrient analysis.
Area of Science:
- Clinical Chemistry
- Biochemistry
- Analytical Chemistry
Background:
- Ascorbic acid (AA), a vital antioxidant nutrient, requires accurate plasma concentration monitoring.
- Existing methods like HPLC for AA determination can be time-consuming and less precise.
- There is a need for rapid, automated, and reliable methods for plasma AA analysis.
Purpose of the Study:
- To develop and validate a new automated procedure for determining plasma ascorbic acid concentration.
- To utilize a Roche Fara centrifugal analyzer for high-throughput AA measurement.
- To compare the new method's performance against conventional techniques.
Main Methods:
- Plasma deproteinization using metaphosphoric acid.
- Enzymatic oxidation of ascorbic acid to dehydroascorbic acid using AA oxidase.
- Chromogenic coupling with o-phenylenediamine and spectrophotometric measurement at 340 nm.
- Assay validation including linearity, precision, and interference testing.
Main Results:
- The automated procedure demonstrated significantly faster throughput than HPLC methods.
- Results showed good correlation with conventional methods, indicating high accuracy.
- The assay exhibited excellent linearity beyond the typical reference range (26.1–84.6 micromol/L).
- Improved precision was observed compared to existing techniques.
- Severe hemolysis was identified as the only significant interference.
Conclusions:
- The developed automated method provides a rapid, precise, and reliable means for plasma ascorbic acid determination.
- This new procedure is suitable for routine clinical laboratory use, enhancing antioxidant nutrient monitoring.
- The method offers advantages in speed and precision over traditional HPLC assays.
