Spectrophotometric Methods for the Determination of Methotrexate in Pharmaceutical Formulations

Abstract

Three visible spectrophotometric methods (A-C) for the determination of Methotrexate (MTX) in pharmaceutical formulations are described. Method A is based on the reduction of Fe(III) to Fe(II) by the drug which forms a coloured complex (λ_max: 510 nm) with 1,10-phenanthroline (PHEN). Method B involves the reduction of Folin-Ciocalteu (F-C) reagent by the drug and the reduced species produced possesses a characteristic intense blue colour (λ_max: 770 nm). Method C is based on the coupling reaction through the involvement of an aromatic secondary amino group in p-N-methylaminobenzoylglutamic acid (released through reduction of the drug with zinc and HCl) with 1,2-naphthaquinone-4-sulfonic acid sodium salt (NQS) to form a coloured product (λ_max: 500 nm). Regression analysis of a Beer's plot showed good correlation in the concentration ranges of 1.0–8.0, 2.0–14.0 and 25.0–300.0 μg.ml⁻¹ for methods A,B and C, respectively. No interference was observed from the additives and the applicability of the methods was tested by analysing the pharmaceutical formulations containing methotrexate. Recoveries were 98.9–101.1%.

Keywords:

• Visible spectrophotometry
• 1,10-phenanthroline
• Folin-Ciocalteu reagent
• 1-2-naphthaquinone-4-sulfonic acid sodium
• nucleophilic substitution