Eco-friendly and Rapid Ultra-high Performance Liquid Chromatography for Simultaneous Determination of Methotrexate and Folic Acid in Human Urine

Ultra-high performance liquid chromatography (UHPLC) appears recently to have the potential to replace the less environment-friendly analytical techniques. In the current work, a new eco-friendly, simple and rapid UHPLC method has been developed for the simultaneous determination of MTX and FA in human urine. The UHPLC analysis has been carried out on a Titan C18 column (100x2.1 mm, 1.9 μm) u s i n g 6 7 . 0 m m o l L 1 . p h o s p h a t e b u f f e r ( p H = 7 . 4 ) a s a m o b i l e p h a s e i n a n isocratic mode of elution at a flow rate of 1.0 mL min-1 and column temperature at 37C. Analytes have been rapidly and efficiently separated within 2.0 min. For comparative studies, the quantitative first derivative UV spectrophotometric determinations (first UVDS) have been performed at 250 nm of MTX and 270 nm of FA under ambient temperature. The p r o p o s e d methodologies have been successfully validated, indicating their reliability to be applied to the analysis of real samples. The relative standard deviations (RSDs%) of the proposed methods were less than 1% proving the precision of the measurements. The linearity concentrations of FA and MTX have been ranged from 5 to 500 and 50 to 2000 ng mL, respectively. Sensitivities of the proposed UHPLC and first UVDS methods are almost comparable and both the methods are suitable for the analysis of MTX and FA in human urine. Further applicability of UHPLC method and first UVDS has been demonstrated by the quantification of MTX and FA in patient urine samples. Urine samples have been collected from 1 to 24 h after administration and concentrations of analytes have been recorded within interval times. Observed MTX urine concentration levels of studied patients were all less than 114 ng mL-1 after 18 h reflecting the absence of a high risk for developing MTX toxicity. Therefore, the current UHPLC-UV method has salient features such as friendly environment, inexpensive, fast and without tedious sample pretreatment for MTX and FA analysis in human urine over the hitherto reported procedures.