RESUMO
High quality chromatographic separation underpins robustness in LC-MS, frequently the analytical method of choice for pharmaceutical drug discovery work. The potential improvements in chromatographic selectivity afforded by serial column coupling (SCC), provide a useful means to enhance the resolution of complex samples. In this work, we present a revised high-throughput form of SCC, in which just two individual mixed phase columns were coupled together and combined with a gradient-optimised, retention-directed ultra-high pressure method to achieve rapid separations, with no further method optimisation necessary. The overall performance was evaluated from an open access DMPK analytical working environment perspective; where in anticipation of bioanalytical or metabolite identification chromatography challenges, or with the knowledge that stronger resolution was required for in-vitro sample analysis, the methodology could be immediately implemented by the analyst. Retention-directed selection of a shallow SCC gradient method was successful in separating peaks throughout the chromatographic window, resulting in a runtime still congruent to high-throughput analyses (3.5 min). In-vitro assay sample interferences were resolved 44-72% of the time, and the overall resolving power for isomeric separations significantly improved against single column comparisons (1.7-fold mean RS improvement). Over a sustained period of time in our laboratory, SCC methods have been used for metabolite identification and bioanalytical samples, where both convenience and effectiveness in solving analytical challenges has been consistently demonstrated. Examples that highlight SCC chromatography, and a guided discussion of the main high-throughput considerations, are included. The technique offers wide applicability, and we would recommend it as a toolbox consideration to the laboratory analyst.
