An Efficient Reversed-Phase High-Performance Liquid Chromatography-Based Approach for the Determination of Methotrexate in Biological Fluids
Ali M. Lakho S.A. Akylbekov N. Alexey D. Zairov R.
February 2025Pleiades Publishing
Journal of Analytical Chemistry
2025#80Issue 2358 - 363 pp.
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.
CNS lymphoma , methotrexate , RP-HPLC , serum sample
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Department of Chemistry, Faculty of Science, University of Karachi, Karachi, 75270, Pakistan
Alexander Butlerov Institute of Chemistry, Kazan Federal University, Kazan, 420008, Russian Federation
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sindh, Sindh, 71000, Pakistan
Korkyt Ata Kyzylorda University, Kyzylorda, 120014, Kazakhstan
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center of RAS, Kazan, 420088, Russian Federation
Department of Chemistry
Alexander Butlerov Institute of Chemistry
Department of Pharmaceutical Chemistry
Korkyt Ata Kyzylorda University
Arbuzov Institute of Organic and Physical Chemistry
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