Determination of methotrexate in spiked human urine using SERS-active sorbent

This report is dedicated to determination of anticancer drug methotrexate (MTX) in human urine using surface-enhanced Raman spectroscopy (SERS). Aluminum oxide loaded with silver nanoparticles (AO-Ag) was proposed as SERS-active sorbent and used for solid-phase extraction (SPE) of the analyte and its SERS-based determination (SPE-SERS protocol). MTX has strong SERS signal only in alkaline media that challenges its determination in urine due to strong background signal caused by creatinine. The application of SPE step enables to purify and concentrate the analyte making MTX determination possible. Also, the application of the same material for SPE pretreatment and SERS analysis enables to simplify and speed-up the protocol. The protocol was developed and tested using artificially spiked samples of human urine collected during different time of day to account deviating composition of the urine matrix. The use of dilution step of the analyte-containing urine was proposed prior SPE-SERS protocol to reduce the difference between morning-time- and daytime-collected urine achieving maximal reliability of the analysis. Additional physicochemical study was performed to estimate an influence of the primary intrinsic urine components (salts, urea, creatinine) and their mixtures on the analytical signal. Final protocol enables MTX determination in human urine within 20-300 μg mL^-1 range of concentrations with satisfactory precision (11-19% RSD), accuracy (97-104% apparent recovery), and limit of detection (4.2 μg mL^-1). Accounting that the analysis requires less than 15 min and portable Raman spectrometer, the protocol seems to be promising for therapeutic drug monitoring in hospitals to identify poor MTX clearance in a timely manner and minimize adverse effects of therapy. Graphical Abstract.