Quantitative Online Liquid Chromatography–Surface-Enhanced Raman Scattering (LC-SERS) of Methotrexate and its Major Metabolites

The application of Raman spectroscopy as a detection method coupled with liquid chromatography (LC) has recently attracted considerable interest, although this has currently been limited to isocratic elution. The combination of LC with rapidly advancing Raman techniques, such as surface-enhanced Raman scattering (SERS), allows for rapid separation, identification and quantification, leading to quantitative discrimination of closely eluting analytes. This study has demonstrated the utility of SERS in conjunction with reversed-phase liquid chromatography (RP-LC), for the detection and quantification of the therapeutically relevant drug molecule methotrexate (MTX) and its metabolites 7-hydroxy methotrexate (7-OH MTX) and 2,4-diamino-N(10)-methylpteroic acid (DAMPA) in pure solutions and mixtures, including spikes into human urine from a healthy individual and patients under medication. While the RP-LC analysis developed employed gradient elution, where the chemical constituents of the mobile phase were modif...