A sustainable automated chromatographic method to measure methotrexate in human serum: Fitting with the ten principles of green sample preparation

An accessible eco-friendly chromatographic methodology was established by integrating flow injection analysis (FIA) with on-line solid phase extraction (SPE) into a conventional high performance liquid chromatography (HPLC) equipped with fluorescence detection (FLD) (FIA-SPE-HPLC-FLD) for quantifying methotrexate (MTX) in untreated serum samples. The FIA-SPE-HPLC-FLD method incorporated a cerium (IV) trihydroxy hydroperoxide (CTHPO) packed column for the derivatization of MTX into highly fluorescent derivatives. This step was followed by on-line SPE of matrix proteins with a protein-coated C8 column (PC-C8-column). For the CTHPO-column, a carrier stream of potassium dihydrogen phosphate (0.01 M, pH 3.4) was used, which also functioned as the extraction mobile phase for PC-C8-column. Automated SPE relied on the principle of size exclusion liquid chromatography performed under non-denaturing conditions. In measuring stage, the fluorescent derivatives were transferred to the TSK gel ODS-80 TM, analytical column, by employing a mixture of acetonitrile and potassium dihydrogen phosphate (0.01 M, pH 6.0) in a 10:90 (v:v) ratio. Quantification was conducted using excitation and emission wavelengths of 367 nm and 463 nm, respectively. Both the CTHPO- andPC-C8-columns exhibited substantial effectiveness in chemically modifying the analyte and extracting proteins. Validation of the FIA-SPE-HPLC-FLD method demonstrated good linearity across a concentration range of 5-1000 ng/mL MTX, with recovery percentages between 96.86 % and 98.15 %, indicating minimal matrix effect. The FIA-SPE-HPLC-FLD method aligns with 9 out of the 10 principles of GSP, highlighting its sustainability for evaluating lower concentrations of MTX in clinical human serum samples.