Applicability of wide-pore columns in reversed-phase liquid chromatography for human blood lipid profiling

• First study of pore size influence on the separation of complex lipids • Equal performance of wide-pore Waters BEH C 18 columns for discovery lipidomics applications compared to narrow-pore Waters BEH C 18 despite smaller surface area and carbon loading • Conformation-dependent effects may impact the separation of different lipid classes and deuterated lipid standards on particles with different pore sizes For the resolution of isomer-rich lipid extracts, it is a common practice during RPLC method development to evaluate column efficiency and mobile phase composition. However, further gains can be made by tuning other parameters, such as the microstructure of the packing material. Today, lipid separations are mostly performed with columns packed with particles having narrow pore sizes (80-120 Å). This is unsurprising, given their expected hydrodynamic radius of lipids up to of 23 Å. However, specific investigations of pore size influence on lipid separations are scarce. Therefore, a systematic comparison of 150 mm columns packed with C 18 particles having pore sizes of 130 and 300 Å was performed with respect to selectivity, peak capacity and chromatographic resolution. An RPLC/HRMS-based lipidomic analysis was developed on 150 mm columns. Similar loadability, selectivity, sensitivity and average peak capacity were demonstrated by 300 Å column compared to its narrow-pore counterpart. Overall, current experimental results indicate that wide-pore columns are equally applicable for RPLC/HRMS-based discovery lipidomics and may be even beneficial for the analysis of lipids with high structural complexity. In addition, these columns appear to be better suitable for high-throughput analysis due to lower lipid retention via smaller surface area.