Pemetrexed (PMX) is a new antifolate drug utilizied in the treatment of pleural mesothelioma and non-small cell lung cancer. The mechanism of its action is based on its ability to inhibit the activity of enzymes involved in purine and pyrimidine synthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamideribonucleotideformyl transferase (GARFT). Due to its low molecular weight PMX reveals a number of disadvantages that are typical for biologically active small molecules, such as enhanced metabolism and excretion from an organism, as well as adverse biodistribution profile. One of the possible solutions for these problems is to bind PMX with macromolecular carriers what may result in improved selectivity and pharmacological properties of PMX. During a study on potential carrier for PMX a fast and precise method is needed to determine the bounded and free drug comprised in investigated conjugates preparations. Two simple and time-efficient methods for quantification of pemetrexed in the polysaccharide conjugates are presented. The method for the analysis of a total amount of PMX in different conjugates was based on a spectrophotometry absorption. Validation was performed by measuring the absorbance of the standard solution in 0,1M sodium bicarbonate at 225nm. The curve representing drug concentration against absorption had a linear character in the range of 4.697-46.97µM. The reproducibility of this method was between 0.8544 and 5.488%. The recovery of this method was between 96.23 and 101.7%. The limit of a quantitative method  was 1.070µM. The method for quantitative analysis of unbound PMX was based on size exclusion chromatography and UV-VIS detection at the wavelength of  225nm. Superdex® Peptide column (150 x 4.6 mm) and a mobile phase 0.1M sodium bicarbonate with a  flow rate of 0.4ml/min were applied. In the free drug determination method, the curve had a linear character in the range of  4.461-223.0μM. The reproducibility and precision of a presented method was 0.3761 to 2.452%. The recovery of the method was between 93.18 and 104.5%. The limit of a quantitative method was 2.897μM.

This project was supported by the statutory fund of Laboratory of Biomedical Chemistry (Institute of Immunology and Experimental Therapy Polish Academy of Sciences).

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