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

Abstract

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 surfaceenhanced 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 2,4diamino-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 modified stepwise during analysis, this did not overtly interfere with the SERS signals. In addition, the practicability and clinical utility of this approach has also been demonstrated using authentic patients' urine samples. Here, the identification of MTX, 7-OH MTX and DAMPA are based on their unique SERS spectra, providing limits of detection of 2.36, 1.84, and 3.26 μM respectively. Although these analytes are amenable to LC and LC-MS detection an additional major benefit of the SERS approach is its applicability toward the detection of analytes that do not show UV absorption or are not ionised for mass spectrometry (MS)-based detection. The results of this study clearly demonstrate the potential application of online LC-SERS analysis for real-time high-throughput detection of drugs and their related metabolites in human biofluids.

Recent developments in quantitative SERS: Moving towards absolute quantification

“…[85,86]). More recently, an improved on-line LC-SERS system using reversed phase LC, where enhanced analyte separation was possible by programming the mobile phase during elution, showed that it was possible to target and quantitate methotrexate and its major metabolite 7-hydroxy methotrexate in urine from human patients suffering from rheumatoid arthritis [87].…”

Enhancing Disease Diagnosis: Biomedical Applications of Surface-Enhanced Raman Scattering

“…On this premise, to realise the maximum benefits of SERS, there is a need to target known biomarkers identified a priori by other techniques, such as LC-MS or GC-MS, polymerase chain reaction (PCR), etc. As a case in point, Subaihi and coworkers used reversed-phase LC to separate therapeutic drugs in human urine followed by quantitative detection by SERS [28]. Furthermore, specific disease markers can be detected by SERS method, which has been extensively reported by Graham and Faulds and coworkers [23,29].…”

Ultra‐Stable Plasmonic Colloidal Aggregates for Accurate and Reproducible Quantitative SE(R)RS in Protein‐Rich Biomedia

“…[16] As such, SE(R)RS analysis of samples in biological media with colloidal substrates has typically required pre-treatment steps to first remove proteins from the sample,w hich is inconvenient and time-consuming. [17] Alternatively,i ti sa lso possible to first aggregate the colloid with salt to form hot-spots before adding in the sample.H owever,t his narrows the window for SE-(R)RS analysis and adds to the irreproducibility of the measurements. [18] To test the potential of SPAs for SE(R)RS analysis in biological media, samples of adenine dissolved in 3.3 %a lbumin solution were used to provide as imple biologically-relevant test molecule in as olution whose protein content matched that of human serum.…”