Improving the resolution of neuropeptides in rat brain with on‐line HILIC‐RP compared to on‐line SCX‐RP

Abstract

Original PaperImproving the resolution of neuropeptides in rat brain with on-line HILIC-RP compared to on-line SCX-RP Our two already established on-line 2-D LC systems, a strong cation exchange-RP chromatography (SCX-RP) system and a hydrophilic interaction LC (HILIC)-RP 2-D LC system, were compared to explore which system is best suited for our further studies of differences in cerebral neuropeptide expression as a function of hypoxiacaused stress. The same mass spectrometer and database search parameters were applied in both systems. In total, 19 first dimension fractions were collected with the novel on-line HILIC-RP system, including a Hypercarb SPE column that was applied to trap the compounds not retained on a Kromasil C 18 enrichment column. In contrast, six fractions were collected in the SCX-RP method, due to practical limitations of this traditional on-line 2-D LC system. With the on-line HILIC-RP system three times more peaks were detected. It was observed that most of the compounds eluted in the first two fractions in the SCX-RP method, while in the 2-D HILIC-RP method there seemed to be no correlation between peaks detected and fraction number. Thus, from this systematic study it seems that on-line HILIC-RP chromatography is the method of choice for comparative "peptidomics" of cerebral neuropeptides in future studies.

Theory and Practice of UHPLC and UHPLC–MS

“…This was also demonstrated in LC Â LC applications with relatively slow 2 D separations Holm et al, 2005;Li et al, 2011;Mihailova et al, 2008;Pepaj et al, 2006;Vonk et al, 2015;. More recently, however, such trapping functionality has been incorporated into LC Â LC systems with faster 2 D cycle times, and in a system coupling LC and SFC in the first and second dimensions, respectively (Venkatramani et al, 2016).…”

Chromatographic Separation Methods

“…One way to resolve this issue is post -column addition of solvent to modify the polarity of the effl uent from the fi rst column (Louw et al, 2008 ). Another approach is to add a " transitional " SPE column after the fi rst -dimension column to trap and concentrate the analytes before introducing them to the second -dimension column (Mihailova et al, 2008 ).…”

High-efficiency hydrophilic interaction chromatography by coupling 25cm×4.6mm ID×5μm silica columns and operation at 80°C

“…HILIC has become a valuable addition to RP-LC in drug analysis, due to its orthogonality to the latter separation mode [9][10][11][12][13]. HILIC is particularly useful for the analysis of highly polar and ionisable compounds [14][15][16][17][18].…”

“…For the pharmaceutical industry, which will certainly still have a plethora of conventional HPLC instrumentation in its good manufacturing practice (GMP) laboratories, kinetic plots can certainly serve a purpose in directing how conventional instrumentation and columns can be used more appropriately in combination with ETLC and long columns. This can help to optimize the use of conventional particle diameters (5 µm) to yield either maximum efficiency and to compared to sub-2µm particles, or to increased the speed of analysis.Since these developments, a chromatographic technique that has been enjoying recent attention in the pharmaceutical industry is hydrophilic interaction chromatography (HILIC).HILIC has become a valuable addition to RP-LC in drug analysis, due to its orthogonality to the latter separation mode [9][10][11][12][13]. HILIC is particularly useful for the analysis of highly polar 4 and ionisable compounds [14][15][16][17][18].…”