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Updated: Jun 7, 2026

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Development of a multi-source blue light irradiation platform for photometric quantification of ascorbic acid via
Ghufran K Allawi1,2, Turkey N S1
1Department of Chemistry, College of Science, University of Baghdad Baghdad Iraq nagham.turkey@sc.uobaghdad.edu.iq.
Abstract:
A novel and meticulously optimised continuous flow injection analysis (CFIA) protocol was developed for the spectrophotometric quantification of ascorbic acid using a locally engineered optical system. The detection unit incorporated eight blue light-emitting diodes (LEDs) arranged in a matrix geometry distributed across two angular regions: 0-90° and 0-180°, while dual solar cells served as photonic detectors. Spectral measurements were specifically conducted at 0-180°, yielding enhanced absorbance stability and signal quality. Based on a redox-induced quenching mechanism, the system utilises a chromogenic FeSCN2+ complex, formed in situ through the controlled mixing of ferric (Fe3+) and thiocyanate (SCN-) ions under strictly maintained physical and chemical conditions. Upon interaction at the Y-point junction, injected ascorbic acid reduces the complex, yielding a quantifiable decrease in absorbance intensity proportional to its concentration. A linear calibration range was established from 0.5 to 40 mmol L-1 (r 2 = 0.9986), LOD of 0.05 mmol L-1 (1.3209 μg per sample) (S/N = 3) and LOQ of 0.131 mmol L-1 (S/N = 10). Method precision was evaluated at two concentrations, 12 and 30 mmol L-1, showing intra-day RSD% < 1% and inter-day RSD% < 2%, affirming excellent repeatability and method reliability. Validation was extended to three commercial pharmaceutical preparations containing 500 mg of ascorbic acid using the standard addition method. Comparative assessment against UV spectrophotometry and HPLC, with statistical evaluation via Student's t-test and one-way ANOVA, confirming no significant differences among techniques (t_cal < t_tab; F_cal < F_tab, α = 0.05), with recovery values between 97.13% and 103.6%.
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