Abstract

SIMULTANEOUS ESTIMATION OF FOLIC ACID AND METHOTREXATE IN TABLET DOSAGE FORM BY HIGHPERFORMANCE LIQUID CHROMATOGRAPHY

Kamlesh Kashinath Saindane*, Harshada Talapadatur, Ujwala Parab, Vijay Kumar Munipalli, Raman Mohan Singh and Bharti Fegade

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Abstract

A new, simple, accurate, fast, economic, and precise Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Folic acid (FA) and Methotrexate (MTX) in a tablet’s dosage form. The chromatographic separation was performed on the Hemochrom Intsil C18 (250 mm × 4.6 mm i.d., 5 μm particle size) column and a mobile phase comprising of Buffer (50mM Potassium dihydrogen phosphate, KH2PO4) and Methanol in the ratio of (60:40 % v/v) and adjust the pH 7.2 with KOH. The mobile phase is used as diluent. The flow rate was kept at 1 ml/min and detection was carried out at 270 nm. The retention time was found to be about 2.6 min for FA and 4.8 min for MTX. The parameters such as accuracy, precision, linearity, ruggedness, robustness and LOD, LOQ were used for validating the developed method according to ICH guidelines. The method was linear over a concentration range of 5-30 μg/ml with a regression coefficient of 0.9999 for FA and a concentration range of 37.5-225 μg/ml with a regression coefficient of 0.9998 for MTX, respectively. The limit of Detection and Limit of Quantitation values was found to be 0.04 μg/ml and 0.11 μg/ml for FA and 0.28 μg/ml and 0.85 μg/ml for MTX, respectively. The percentage RSD of every parameter was found within the limit. The method is selective and specific, it was successively applied in pharmaceutical dosage formulation with potential application in the pharmaceutical analysis of FA and MTX in an individual tablet dosage form.

Keywords: Folic Acid, Methotrexate, RP-HPLC, Method Development, Validation.