Methotrexate (MTX)  is one of the oldest and currently used drugs. It is an antimetabolite of  folic acid, and a specific inhibitor of dihydrofolate reductase (DHFR), having an indirect and direct effect on other molecular targets,  influencing on DNA replication and cell proliferation. MTX has a wide use in the treatment of tumor and autoimmunological diseases. This drug at the same time demonstrates a range of  disadvantages, characteristic  for low molecular compounds such as fast metabolism and fast excretion from an organism, as well as adverse biodystribution and a  low selectivity of therapeutic use. Solving these problems involves the binding process of MTX with macromolecular carriers resulting in enhancement of the delivery, selectivity and improvement of pharmacological  properties of MTX. Research on MTX conjugates with natural and synthetic polymers such as dextrans, albumin, fibrinogen,  polyethylene glycol and other is in progress. During a study on potential carrier for MTX a fast and precise method is needed to determine the total amount of the drug bound and free in investigated preparation. In this paper, two simple, cheap and fast methods regarding quantification of methotrexate in the conjugated preparations are presented. In both methods sodium bicarbonate solution was used as  a solvent. For both methods basic parameters such as linearity, range of the method, limit of detection (LOD) and limit of quantification (LOQ), reproducibility and  recovery  were validated. The method for analysis of   a total MTX in preparations was based on absorption spectrophotometry. Validation was performed by measuring absorbance at 372 nm of the sodium bicarbonate solution. Curve describing drug concentration against absorption had a linear character in the range of 0,60µg/ml-20µg/ml. The method  for free methotrexate determination was based on size exclusion chromatography and UV-VIS detection at the wavelength of  302 nm. Superdex^® Peptide column (150 x 4.6 mm) and a mobile phase 0,1M sodium bicarbonate  flow rate of 400µl/min was used. In a free drug determination method a curve had a linear character in the range of  1,00-100µg/ml. LOD of the method was 0,15µg/ml, and LOQ was 0,50µg/ml.

This project is co-financed by the European Union as part of the European Social Fund. This project was supported by National Science Centre, Poland (N N302 098434).

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