A simple, sensitive, selective rapid spectrophotometric method has been developed for the determination of post synaptic ?1- Adrino receptor antagonist Methotrexate in pure form and pharmaceutical formulations based on the comparative method of analysis and recovery studies like reductive coupling reaction and diazotization with1,10-PT,2,2-BP, FC reagent, NaNO2/HCl and k3FeCN6/FeCl3 at PH_4.0 which is extractable at 430 nm,480nm,680nm,440nm & 680nm. Beer’s law is obeyed in the concentration ranges 0.5 -3 µg/ml,1-6 µg/ml,0.4-2 µg/ml1-4 µg/ml and 0.1 -0.5 µg/ml . The developed method was applied directly and easily for the analysis of the Pharmaceutical formulations and R.S.D was found to be 0.46%,1.5%,0.6%,0.88% and 0.89% and Recovery 99.92 ± 0.12 , 99.19 ± 0.12, 99.81 ± 0.12 100.03 ± 0.72 and 99.95 ± 0.15 respectively. The method was completely validated and proven to be rugged. The interferences of the other ingredients and excipients were not observed. The repeatability and the performance of the proved method were established by point and internal hypothesis and through recovery studies.
Introduction
Methotrexate is an antineoplastic anti-metabolite that inhibits folic acid reductase, disrupting DNA synthesis and cell replication. Despite existing HPLC and electrochemical methods, there is no UV-Visible spectrophotometric method for analyzing technical-grade and formulation-based Methotrexate using color complex formation.
Objective
To develop and validate simple, accurate, and reproducible UV-Visible spectrophotometric methods for the quantitative estimation of Methotrexate based on colored complex and diazotization reactions involving its primary amine functional group.
Materials and Methods
A. Preparation of Standard Solutions
Stock solution: 1 mg/ml Methotrexate in DMSO.
Working solutions: Prepared by stepwise dilution to desired concentrations (e.g., 100 µg/ml).
B. Reagents Used
Analytical grade reagents were prepared in distilled water:
1,10-Phenanthroline (0.01M)
Ferric chloride (0.003M)
Ortho phosphoric acid (0.2M/0.02M)
2,2-Bipyridine (0.01M)
K?Fe(CN)?, HCl (1N), Sodium carbonate (20%)
Folin-Ciocalteu reagent (1:2 dilution)
Sodium nitrite (0.1%)
C. Developed Spectrophotometric Methods
Method 1: 1,10-Phenanthroline Complex
MTX reacted with 1,10-Phenanthroline and FeCl?, heated at 100°C for 15 min.
Acidified with ortho phosphoric acid.
Orange-red chromogen measured at 430 nm.
Method 2: 2,2-Bipyridine Complex
MTX reacted with 2,2-Bipyridine and FeCl?, heated at 100°C for 15 min.
Acidified similarly.
Orange chromogen measured at 480 nm.
Method 3: Folin–Ciocalteu Reaction
MTX reacted with sodium carbonate and Folin-Ciocalteu reagent.
Incubated for 20 min.
Blue chromogen measured at 680 nm.
Conclusion
In the present study, an analytical method was developed for quantitative analysis of drug in its pure and formulations.The experimental results demonstrated a properly conducted validation study of UV-Vis spectrophotometric method for analytical determination of MTX .The suggested methodologies showed high specificity, linearity, precision, accuracy, robustness with low limit of detection and quantification, which demonstrates the reliability required for recovery of the MTX in these drug delivery systems. The analytical methods were sufficiently simple, rapid, and suitable and may be safely used in the quantitative analysis of MTX .
References
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