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 R_S 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.

高质量的色谱分离增强了LC-MS的鲁棒性，而LC-MS通常是药物发现工作中选择的分析方法。串行色谱柱偶联（SCC）色谱法选择性的潜在提高，为提高复杂样品的分离度提供了有用的手段。在这项工作中，我们提出了一种经过修订的高通量SCC形式，其中仅将两个单独的混合相色谱柱耦合在一起，并通过梯度优化的，保留导向的超高压方法进行组合以实现快速分离，而无需进行进一步分离方法优化是必要的。从开放访问DMPK分析工作环境的角度评估了整体性能；在预期进行生物分析或代谢物鉴定色谱法挑战的地方，

浅层SCC梯度方法的保留导向选择成功地分离了整个色谱窗口中的峰，导致运行时间仍与高通量分析（3.5分钟）一致。体外测定中样品干扰的分离时间为44–72％，与单色谱柱比较相比，异构体分离的总体分离能力得到了显着提高（1.7倍的平均R _S改进）。在我们实验室的持续时间内，SCC方法已用于代谢物鉴定和生物分析样品，这些方法在解决分析难题方面既方便又有效。其中包括突出显示SCC色谱的示例以及有关主要高通量考虑因素的指导性讨论。该技术具有广泛的适用性，我们建议实验室分析人员将其作为工具箱考虑因素。