Development and Validation of a Simultaneous RP-HPLC Method for the Estimation of Ferrous Fumarate and Ascorbic Acid for Pharmaceutical Quality Control

Abstract Our aim was to develop an accurate, precise, and reliable RP-HPLC method for estimating ferrous fumarate and ascorbic acid in pro-liposomes. The aim was to make the analytical method simple, environmentally friendly, and accurate. To measure ferrous fumarate (FF) and ascorbic acid (AA), an isocratic method was used in an HPLC, with 220 and 245 nm detection, using phosphate buffer (pH 3.0) and methanol (95:5). It was found that AA and FF were retained for 6.4 and 8.3 min, respectively. A linear relationship was observed between 0.1 and 50 μg/mL for both ferrous fumarate (r2 = 0.994) and ascorbic acid (r2 = 0.999). The resolution between the two peaks ≥2, tailing factor (TF) ≤2, and theoretical plate count >2000 were evaluated and recorded as system suitability parameters. The LOD and LOQ for FF were calculated to be 0.52 and 1.58 μg/mL, respectively, whereas the same for AA were calculated to be 0.17 and 0.53 μg/mL, respectively. The validation method was tested under various conditions, including flow rate, injection volume, column temperature, and mobile phase ratio. Additionally, the optimized method was successfully used to estimate the entrapment efficiency for FF and AA in the developed pro-liposomes, which were found to be 92 and 65%, respectively.