Abstract

SIMULTANEOUS METHOD DEVELOPMENT AND VALIDATION OF NEW RP-HPLC AND UV-VISIBLE SPECTROPHOTOMETRIC ESTIMATION OF METHOTREXATE AND FOLIC ACID IN BULK & PHARMACEUTICAL DOSAGE FORMS

Korivi Ramanjaneyulu, K. Thejomoorthy*, Ch Saibabu

ABSTRACT

Methotrexate is an folic acid antagonist and Folic acid is an expectoration improver and mucolytic agent. Seven new, simple, accurate and precise UV-Spectrophotometric (Four methods), colorimetric(One methods) and one RP-HPLC method have been developed and validated according to ICH guidelines for the simultaneous estimation of Methotrexate and Folic acid in their combined dosage form. First method is based on simultaneous estimation using two wavelengths, 243nm (λmax of METHO) and 299nm (λmax of FOLIC). The second method is based on Q-absorption Ratio method using two wavelengths 278nm (λmax of METHO) and 299nm (Isoabsorptive point). The third method involves the use of first order derivative technique, here 239 nm, the zero crossing point of METHO, 295 nm, the zero crossing point of FOLIC. The fourth method involves the use of dual wave length techniquemethotrexate at 235nm, 247nm and folic acid at 286nm, 313nm. Colorimetry: The first method involves the use of MBTH reagent for estimation of METHO using wavelength, 622 nm. RP-HPLC Method: the system was used with C18 column and the detection was made at 306nm in the UV region. Mobile phase consisted of ACN:phosphate buffer (3:97) at a flow rate of 0.6ml/min with linearity of 5-25μg/ml for METHO and 2-10μg/ml for FOLIC. The Rt values were 6.8 and 5.12 min for METHO and FOLIC respectively. All the methods were validated and all the parameters were found to be within the acceptance criteria.

Keywords: Methotrexate, Validation, Folic acid, HPLC, Analysis.