Estimation of 5-fluorouracil by high-performance liquid chromatography reversed-phase validated method
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The main intention of the current work is the development of a suitable, simple, precise, reproducible, and accurate high-performance liquid chromatography reversed-phase validated method which can be used for the estimation of 5-Fluorouracil.The estimation was done by high-performance liquid chromatography (HPLC) method. In this suitable stationary phase and optimum composition of the mobile phase was selected which provides good resolution and short run time for the estimation of 5-Fluorouracil (5-FU). Stationary phase used was Nucleodur C18 column (Reverse phase, 250mm × 4.6mm i.d., particle having 5 micron size) and mobile phase consists of combination of Ortho-Phosphoric Acid (OPA) (0.5%) and methanol having ratio 95:5, v/v were used in an isocratic mode of elution. The mobile phase used with a flow rate of 0.8 mL/min and volume of the injection for 5-FU was 20μL. The eluent observed at 266nm for measurement of 5-FU. The validation of this method was carried out with the help of various parameters such as sensitivity, selectivity, system suitability, precision (inter-day and intra-day), accuracy, and linearity according to International Conference on Harmonization guidelines i.e. ICH Q2 (R1). The 5-FU showing retention time at 7.2 min. The responses showing linearity in the concentration range between 2–10μg/mL with correlation coefficient 0.99. The % mean recovery of 5-FU was calculated at three different levels i.e Lower Quality control Concentration (LQC), Middle Quality control Concentration (MQC), High Quality control Concentration (LQC) whose results falls within the range i.e. 95% to 105%, which indicates the accuracy of this method. The % relative standard deviation (RSD) precision (intraday and intermediate) at 3 different levels was < 2% which indicated the precision of this method. LOD i.e. Limit of Detection and LOQ i.e. Limit of Quantification was found to be 0.870277 and 2.637202 respectively for 5-FU.
5-Fluorouracil, RP-HPLC, ICH Guidelines, System suitability, Accuracy, Precision.