A Reversed-phase High Performance Liquid Chromatography (HPLC) method for bio-analysis of Methotrexate
Methotrexate (MTX) is a chemotherapeutic agent used in 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 while 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 were indicating a method with acceptable analytic separation efficiency and peak shape proving method’s selectivity. Limit of detection (LOD) and limit of quantification (LOQ) determined to be 10 ng/ml and 25ng/ml, which reflect method sensitivity. Regression analysis showed a linear correlation between area under curve (AUC) of peaks and corresponding MTX concentrations. The within-day and between-day precision and accuracy was both in acceptable ranges. Recovery index of method for median concentration (200 ng/ml) is also about 74%.
The developed method was accorded to the acceptable criteria of analytical method validation. The sensitivity of the method in all the tested matrices made the method suitable in terms of detection and quantitation of low concentration samples throughout the study. Also, the assay method had fairly short run-time and lacks any significant interference.
Methotrxate monograph. In: Galichet LY, editor. Clarke's Analysis of Drugs and Poisons. 3th ed. London: Pharmaceutical Press; 2005.
Rider BJ. Methotrexate monograph. In: Dowd FJ, et al., editors. X pharm. Amsterdam: Elsevier Inc.; 2007.
Gerstner ER, Batchelor TT. Primary Central Nervous System Lymphoma. In: Norden AD, Reardon DA, Wen PYC, editors. Primary Central Nervous System Tumors; Pathogenesis and Therapy. New York: Humana Press; 2011. p. 333-54.
Olson J, Blakeley J, Grossman S, Weingart J, Rashid A, Supko J. Differences in the distribution of methotrexate into high grade gliomas following intravenous administration as monitored by microdialysis, are associated with blood-brain-barrier integrity. ASCO Meeting Abstracts. 2006; 24:1548.
Hamidi M, Azadi A, Rafiei P, Ashrafi H. A Pharmacokinetic Overview of Nanotechnology-Based Drug Delivery Systems: An ADME-Oriented Approach. Crit Rev Ther Drug Carr Syst. 2013; 30:435–467
Azadi A, Rouini MR, Hamidi M. Neuropharmacokinetic evaluation of methotrexate-loaded chitosan nanogels. Int J Biol Macromol., 2015; 79: 326-335.
Azadi A, Hamidi M, Rouini M-R. Methotrexate-loaded chitosan nanogels as ‘Trojan Horses’ for drug delivery to brain: Preparation and in vitro/in vivo characterization. Int J Biol Macromol. 2013; 62:523-30.
Azadi A, Hamidi M, Khoshayand MR, Amini M, Rouini MR. Preparation and optimization of surface-treated methotrexate-loaded nanogels intended for brain delivery. Carbohydr Polym. 2012;90:462-471.
United States Pharmacopoeia. 25th ed. Micromedex; 2005.
Sadray S, Rezaee S, Rezakhah S. Non-linear heteroscedastic regression model for determination of methotrexate in human plasma by high-performance liquid chromatography. J Chromatogr B. 2003; 787: 293-302.
Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER). 2001.
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