Modeling and Optimizing of Effective Factors on Kinetic Spectrophotometric Determination of Vitamin B12

A new, accurate, sensitive, and reliable kinetic spectrophotometric method for the determination of vitamin B₁₂ in biological samples and pharmaceutical formulations has been developed. A central composite design (CCD) of the response surface methodology was used in our research to investigate the effect of reaction time and temperature and the concentration of reagents including orange G, bromate, and sulfuric acid on the assay of vitamin B₁₂. Vitamin B₁₂ shows a strong inhibitory effect on the oxidation of orange G by bromate in acidic media. The wide linear dynamic range, low detection limit, and short time analysis introduce the proposed method as a new strategy for the determination of vitamin B₁₂ in biological and pharmaceutical samples. Also, linear polynomial mathematical models were obtained for the determination of vitamin B₁₂. Under optimum experimental conditions, the calibration curve was linear over the range 1.0–200.0 µg/mL. The limit of detection was 0.56 µg/mL. Statistical comparison of the results with the reference methods shows excellent agreement and indicates no significant difference in accuracy and precision.