An Efficient Reversed-Phase High-Performance Liquid Chromatography-Based Approach for the Determination of Methotrexate in Biological Fluids

Abstract

In this work, a reversed-phase high-performance liquid chromatography method is reported for the determination of methotrexate (MTX), an anticancer drug. The method was found to be simple, sensitive, accurate, and precise. It was validated for the determination of the drug in human serum samples. For this purpose, spiking of the drug and internal standard (IS) was performed in drug-free plasma to assess the method’s applicability. p-Aminoacetophenone was employed as an IS. Protein precipitation was carried out using 2 M trichloroacetic acid, followed by centrifugation. MTX and IS were isolated using a C18 analytical column. The interference of the method was evaluated using blank plasma from six different subjects, and no interference was observed. The method demonstrated a linear response in the range of 300–20 000 ng/mL, with limits of detection and quantification of 1.82 and 6.07 ng/mL, respectively. The method was validated according to FDA and ICH guidelines Q2(R2), with intra- and inter-day accuracy and precision values within allowable limits. Using this method, ≥50% recovery of the drug was achieved from human blood plasma.