Improving LC–MS sensitivity through increases in chromatographic performance: Comparisons of UPLC–ES/MS/MS to HPLC–ES/MS/MS

Recent technological advances have made available reverse phase chromatographic media with a 1.7 micron particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed ultra performance liquid chromatography (UPLC), offers significant theoretical advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometers capable of high-speed acquisitions. This paper explores the differences in LC/MS performance by conducting a side-by-side comparison of UPLC for several methods previously optimized for HPLC-based separation and quantification of multiple analytes with maximum throughput. In general, UPLC produced significant improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC, which were found to be analyte-dependent, were as large as 10- fold and improvements in method speed were as large as 5- fold under conditions of comparable peak separations. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak widths and from a separation of diastereomers not possible using HPLC. Overall, the improvements in LC/MS method sensitivity, speed, and resolution provided by UPLC show that further advances can be made in analytical methodology to add significant value to hypothesis-driven research.