A Reversed-phase High Performance Liquid Chromatography (HPLC) method for bio-analysis of Methotrexate

Methotrexate (MTX) is a chemotherapeutic agent used in the treatment of many disorders, including autoimmune diseases and cancers. The availability of a reliable analysis method for drug assay in biological fluids of interest is a prerequisite for all pharmacokinetic studies in humans or animal models. Considering the complex matrices of the biological specimens, as well as the low concentrations of the majority of the drugs in biological fluids, the development of an available yet sensitive method for the bioanalytical studies is often a challenging issue. For drug assay in aqueous, plasma, animal brain, and liver tissue environments in a concentration range of 25-600 ng/ml, a reverse phase high performance liquid chromatography (RP-HPLC) was developed. System suitability tests indicated an acceptable analytic separation efficiency, and peak shapes prove high selectivity of the method. Limit of detection (LOD) and limit of quantification (LOQ) were 10 ng/ml and 25ng/ml, which reflects method sensitivity. Regression analysis showed a linear correlation between area under the curve (AUC) of peaks and corresponding MTX concentrations. The within-day and between-day precision and accuracy were both in acceptable ranges. Recovery index of the method for median concentration (200 ng/ml) was about 74%. The developed method is in accordance with the acceptable criteria of analytical method validation. The sensitivity of the method in all the tested matrices makes the method suitable in terms of detection and quantitation of low concentration samples throughout the study. Moreover, the assay method has a fairly short run-time and lacks any significant interference.