Moving past the 3 Rs towards a broader framework: Urinary methotrexate determination using a recyclable oxidant by capillary zone electrophoresis

This study seeks to demonstrate the importance of green analytical chemistry in the context of sustainable development, with a particular emphasis on the “3Rs” framework—Reduce, Reuse, and Recycle. A green pre-capillary derivatization (G-Pre-Cap-D) was implemented for capillary zone electrophoresis (CZE) coupled with fluorescence detection (FLD) (G-Pre-Cap-D-CZE-FLD) to quantify methotrexate (MTX). Cerium (IV) trihydroxy hydroperoxide (CTHPO) was applied as solid-phase reagent (SPR) for chemical modification of MTX. The proposed approach's sustainability strategy provides new insights into utilization of CTHPO as a reactivable eco-friendly derivatization SPR. It prioritizes the minimization of environmental waste, which relies on the efficient reactivation of spent SPR. Furthermore, the reactivating process was executed in an eco-conscious manner within a closed system using hydrogen peroxide. G-Pre-Cap-D of MTX was achieved by exposing the analyte to CTHPO-SPR for chemical modification into highly fluorescent derivatives. Optimal derivatization conditions were established by mixing MTX with CTHPO-SPR in 0.03 M potassium dihydrogen phosphate solution (pH 3.4) at 65°C with continuous stirring for 2 min. CZE analysis was then carried out on a 80 cm× 75 µm (i.d.) fused silica capillary using 0.1 M dipotassium hydrogen phosphate (pH 10) at 12 kV. The migrated derivatives were quantified at 463 and 367 nm for emission and excitation wavelengths, respectively. Method validation demonstrated good linearity, with accuracy and precision values ranging from 88.7% to 94.8% and 4.09% to 7.49%, respectively. Ultimately, the G-Pre-Cap-D-CZE-FLD method was successfully applied for the quantification of urinary MTX in human samples, demonstrating its practicality and sustainability.