Analytical method development and validation for iron and ascorbic acid in ferrous bisglycinate effervescent tablets by colorimetry and reverse phase-high-performance liquid chromatography

Introduction: The new ferrous bisglycinate effervescent formulation is a combination of iron and ascorbic acid where iron is in complex form with glycine. No analytical method was reported so far for the analysis of iron and ascorbic acid in effervescent tablets. Hence, an attempt was made to develop and validate the colorimetric and RP-HPLC method. Objective: To develop and validate a colorimetric and RP-HPLC analytical method for ascorbic acid and iron by optimizing the concentration of sulphuric acid and volume of sodium acetate to breakdown ferrous bisglycinate complex. Methods: Colorimetric method for estimation of total iron: The wave length used was 510 nm with diluent-1 as 0.5 M sulfuric acid and 1.2 M sodium acetate 5 mL. The diluent-2 was Milli Q-water. 10 mL of 1,10 phenanthroline (1 mg/mL) used as complexing agent. Analytical method development for ascorbic acid using HPLC: A reversed phase-high-performance liquid chromatography method was developed for ascorbic acid employing Inertsil octadecylsilyl column (250 mm ×4.6 mm; 5 μm) 20 mM potassium dihydrogen phosphate (pH - 2.5) and methanol (97:03) was used as mobile phase. An isocratic program was followed using 0.3 M hydrochloric acid with 5% ortho phosphoric acid as diluents with 1 ml/min flowrate. Results: The validated data of colorimetric method showed that the method was linear over a range of 0.625-3.75 μg/mL (r² = 0.999), accurate and precise.Validated data showed that the method was linear over a range of 4.0-60.0 μg/mL (r² = 0.999). The accuracy of the method ranged from 100% to 102%. The relative standard deviation value for method precision was 0.8%. Conclusion: The proposed method is simple and more economical in comparison with other reported methods discussed in literature and it has all the potential for routine quality control analysis.