•An LC×LC method was developed for the analysis of wine constituents and contaminants.•Triple quadrupole MS was employed for sensitive targeted pesticide determination.•Orthogonality metrics were calculated for the full and shift gradient approaches. A novel system for comprehensive two-dimensional liquid chromatography coupled to a triple quadrupole mass spectrometer is described for the analysis of wine components. The first dimension consisted of a 250-mm microbore cyano column utilizing 5μm diameter particles, interfaced to a 50-mm superficially-porous particle C18 column with 2.7μm diameter particles. Both columns were operated under reversed-phase conditions. Correlation between the two chromatographic separation modes was decreased by designing a 60-s shift gradient program in the second dimension, and the increase in orthogonality was evaluated quantitatively utilizing a number of orthogonality metrics. The system was employed for the analysis of a red wine sample, without preliminary clean-up procedures, and a total of 43 polyphenols were separated and identified. Comparison with a one-dimensional LC system showed a large increase in the number of identified components with the two-dimensional system. Optimized multiple reaction monitoring experiments allowed for the determination of trans-resveratrol, which is one of the most active antioxidant component of wine, and for monuron, a plant protection product (herbicide) of interest to regulatory agencies. The estimated limits of detection and of quantification were 0.3μgL−1 and 1μgL−1, respectively, well below the minimum detection limit (10μgL−1) set by current regulation.