High-Performance Liquid Chromatography Determination of Methotrexate in Plasma

This article describes a simple and fast high-performance liquid chromatography method for the determination of methotrexate (MTX) in plasma. Samples were collocted from children receiving high-dose MTX at shafa Hospital (Ahvaz University of Medical sciences, Ahvaz, iran) at various times after the end of eachinfusion. Plasma was deproteinized with trichloroacetic acid and the supernatant was injected into a 250×4.6 mm octadecylsilane column. Mobile phase was made of TRIS-phosphate buffer (pH 5.7): methanol: acetonitrile (82:11:7) with a flow rate of 1.8 ml/min. Ultraviolet detection was done at 313 nm and at ambient temperature. Para-aminoacetophenone was used as internal standard. Methotrexate and internal standard retention times were 4.4 and 6.5 minutes, respectively. Results showed that reproducibility (precision) of method within a day was 2.6 to 6 percent and between days was 5.5 to 9.5 percent. The recovery of the method was between 61.5 and 72.7 percent. The quantitation limit of the method for methotrexate was 0.1 µM. This method is suitable for quantitation of methotrexate after infusion of high doses of this drug and has good accuracy, precision and quantiatation limit.