Exploring the Potential of Chemically Matched Fragments as Internal Standards for Quantitative SERS with Panobinostat

Surface-enhanced Raman spectroscopy (SERS) has great potential for therapeutic drug monitoring (TDM) due to its high sensitivity; however, achieving accurate and robust quantitative data remains challenging. The most effective approach for quantitative SERS is to use internal standards (IS). Isotopologues are particularly effective; however, these or closely related analogues of the target compound are often unavailable. We have addressed this problem by using fragments of the target compound as the IS. Here, 2-methylindole (2-MI) was identified as the most suitable fragment for Panobinostat (Pano). Tests with an enhancing colloid that degraded significantly over a 4-week period showed that the 2-MI IS allowed changes in the absolute intensity of the Pano signal over time to be corrected and that the standard error achieved with the degrading enhancing medium matched that of results obtained with the fresh colloid. The calibration range could be extended by using either low (4 × 10^-7 M) or high (10^-5 M) concentrations of 2-MI, both of which gave linear log/log Pano calibration curves (R² = 0.993 and 0.998, respectively) although these curves had different slopes. In contrast, thiophenol (TP), a nonchemically matched IS, gave reasonable results at a low (10^-6 M) concentration (R² = 0.942) but completely failed at a high (10^-5 M) concentration due to surface saturation. These findings can be rationalized using a model where, at submonolayer concentrations, both the target and IS adsorb to the nanoparticle surface in proportion to their concentrations; however, at higher concentrations, competition for active sites on surface alters the relative intensities observed. This fragment-based approach significantly increases the availability of chemically matched IS for large target molecules and therefore significantly broadens the range of compounds where robust quantitative SERS analysis is possible.