The present thesis focuses on extension of the analytical capacity of metabolomic workflows to the lower levels of biological dynamic range, while keeping focus on relevant compound families containing metabolites of biological importance. Steroids, fatty acid derivatives and polar signaling molecules may serve as prominent examples. In particular, an analysis of circulating steroidome performed in a cohort of patients diagnosed with different prostate cancer stages in course of current thesis revealed insufficiency of dynamic range of the established analytical methods. Therefore, new developments were focused on maximizing LC performance and analytical sensitivity. Chromatographic investigations included evaluation of different separation modalities and their mechanisms for polar compounds. Next, analytical signal augmentation was achieved by implementation of low flow chromatography methods and their combination with multiplexed electrospray ionization via novel multinozzle silicon chip emitters. Finally, the resulting proof-of-concept methods were applied to a profiling of steroids and complex lipids in biological samples. The outcomes included increased sensitivity (up to 2 orders of magnitude), robust character of the methods, improved metabolite coverage, ease of use and applicability to different biological matrices (cell cultures, human and animal plasmas).