L-Methotrexate (L-Mtx) belongs to antineoplastic and antireumatic therapeutic categories. It is an antifolate which produced the first striking, although temporary remission in leukemia and the first cure of a solid tumor, choriocarcinoma. D-Mtx can be present as impurity from racemization of the synthetic product or during the shelf life of the pharmaceutical product, due to improper storage conditions. It was found that the pharmacokinetics of D-Mtx is different from that of L-Mtx. Although D-Mtx exhibits similar DHFR inhibitory effect, yet D-Mtx is much less toxic (40-fold lower LD50) and its antitumor effect is proportionately reduced. Many analytical methods have been reported to analyze Mtx in pharmaceutical formulations and in biological fluids by using LC, capillary zone electrophoresis, spectrophotometric and voltammetric techniques. However, there are only few analytical methods which allow the discrimination between L- and D-enantiomers of Mtx. These methods suffer from long time analysis, pre-step derivatization or unsatisfactory resolution for enantiomeric purity estimation in pharmaceutical formulations. The aim of this work was to develop and validate an HPLC method for the enantioselective analysis of Mtx in pharmaceutical formulations, such as tablets and injections. A simple, sensitive and selective HPLC method for the enantioselective analysis of Mtx was developed and validated by using Chirobiotic TTm stationary phase. Acceptable enantioresolution was achieved using a polar organic mobile phase. Essential steps in method validation were studied: linearity, precision, accuracy, suitability and stability. At wavelength 303 nm, the limits of detection and quantification (S/N= 3; S/N= 10) were found to be 0.9 and 3.8 mg/mL . The separation of 0.2% (m/m) D-Mtx (as limit of quantitation) was successfully obtained with resolution 1.72. Enantiopurity of L-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter and intra-days relative standard deviation £ 1.6%.
METOTREXATE DETERMINATION IN PHARMACEUTICALS BY ENANTIOSELECTIVE HPLC
GOTTI, ROBERTO;BERTUCCI, CARLO;ANDRISANO, VINCENZA
2004
Abstract
L-Methotrexate (L-Mtx) belongs to antineoplastic and antireumatic therapeutic categories. It is an antifolate which produced the first striking, although temporary remission in leukemia and the first cure of a solid tumor, choriocarcinoma. D-Mtx can be present as impurity from racemization of the synthetic product or during the shelf life of the pharmaceutical product, due to improper storage conditions. It was found that the pharmacokinetics of D-Mtx is different from that of L-Mtx. Although D-Mtx exhibits similar DHFR inhibitory effect, yet D-Mtx is much less toxic (40-fold lower LD50) and its antitumor effect is proportionately reduced. Many analytical methods have been reported to analyze Mtx in pharmaceutical formulations and in biological fluids by using LC, capillary zone electrophoresis, spectrophotometric and voltammetric techniques. However, there are only few analytical methods which allow the discrimination between L- and D-enantiomers of Mtx. These methods suffer from long time analysis, pre-step derivatization or unsatisfactory resolution for enantiomeric purity estimation in pharmaceutical formulations. The aim of this work was to develop and validate an HPLC method for the enantioselective analysis of Mtx in pharmaceutical formulations, such as tablets and injections. A simple, sensitive and selective HPLC method for the enantioselective analysis of Mtx was developed and validated by using Chirobiotic TTm stationary phase. Acceptable enantioresolution was achieved using a polar organic mobile phase. Essential steps in method validation were studied: linearity, precision, accuracy, suitability and stability. At wavelength 303 nm, the limits of detection and quantification (S/N= 3; S/N= 10) were found to be 0.9 and 3.8 mg/mL . The separation of 0.2% (m/m) D-Mtx (as limit of quantitation) was successfully obtained with resolution 1.72. Enantiopurity of L-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter and intra-days relative standard deviation £ 1.6%.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
