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Updated: Jun 23, 2026
11:56
A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
A simple and sensitive assay for ascorbate using potassium ferricyanide as spectroscopic probe reagent
1College of Chemistry and Environmental Science, Henan Normal University, Jianshe Road No. 46, Xinxiang, Henan 453007, People's Republic of China.
Analytical Biochemistry
|May 21, 2009
Summary
A new assay accurately measures ascorbate (vitamin C) using potassium ferricyanide. This rapid, inexpensive method is reliable for various samples, including biological fluids and food, with high precision.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biomedical Science
Background:
- Ascorbate (vitamin C) is essential for numerous biological functions.
- Accurate and efficient methods for ascorbate determination are crucial in research and diagnostics.
- Existing assays may be costly, time-consuming, or lack robustness.
Purpose of the Study:
- To develop a rapid, inexpensive, and reliable spectroscopic assay for ascorbate quantification.
- To validate the assay's performance and assess its applicability to diverse sample types.
- To investigate ascorbate and ascorbate phosphate uptake in osteoblasts.
Main Methods:
- Utilized potassium ferricyanide as a spectroscopic probe reagent.
- Employed a spectrophotometric method monitoring the formation of soluble Prussian blue at 735 nm.
- Determined ascorbate concentration based on absorbance and established a linear regression equation.
Main Results:
- Established a linear relationship between ascorbate concentration and absorbance (A=-0.01911+0.16208C).
- Achieved an apparent molar absorption coefficient of 2.85 x 10^4 L/mol·cm.
- Demonstrated assay robustness across various samples including orange juice, fruits, and swine plasma.
- Observed similar intracellular ascorbate plateau levels for ascorbate and ascorbate phosphate in osteoblasts at 24h.
Conclusions:
- The developed assay is rapid, economical, and reliable for ascorbate determination.
- The assay exhibits high precision with uncertainties around 5%.
- It is suitable for analyzing ascorbate in diverse biological and food matrices, including cellular uptake studies.
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