Evaluation of column length and particle size effect on the untargeted profiling of a phytochemical mixture by using UHPLC coupled to high-resolution mass spectrometry

Liquid chromatography coupled to high-resolution mass spectrometry is the technique of choice for the untargeted profiling of food matrices. Despite the high potential of high-resolution mass spectrometry, when dealing with complex mixtures, an efficient separation technique is also needed. The novel core-shell chromatographic columns packed with sub-2 μm sized particles are claimed to show very good resolution. However, the analytes retention can be significantly altered when working under ultra-high performance chromatographic conditions. In this work, an evaluation of four chromatographic systems, with either a single or two in-series Kinetex™ C₁₈ columns, either packed with 2.6 or 1.7 μm particles, is presented for the targeted analysis of a standard mixture and the untargeted analysis of a strawberry extract. An ultra-high performance chromatographic system coupled via an electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer was used. From the extensive comparison, a surprising result was obtained, namely, that the system identifying the largest number of features was the one with two in-series connected columns with the larger particle size. The inconsistency among the theoretical assumptions and the applicative findings points out the importance of an extensive chromatographic evaluation for the comprehensive untargeted profiling of complex real samples.