Abstract
Profiling of body fluids is crucial for monitoring and discovering metabolic markers of health and disease and for providing insights into human physiology. Since human urine and plasma each contain an extreme diversity of metabolites, a single liquid chromatographic system when coupled to mass spectrometry (MS) is not sufficient to achieve reasonable metabolome coverage. Hydrophilic interaction liquid chromatography (HILIC) offers complementary information to reverse-phase liquid chromatography (RPLC) by retaining polar metabolites. With the objective of finding the optimal combined chromatographic solution to profile urine and plasma, we systematically investigated the performance of five HILIC columns with different chemistries operated at three different pH (acidic, neutral, basic) and five C18-silica RPLC columns. The zwitterionic column ZIC-HILIC operated at neutral pH provided optimal performance on a large set of hydrophilic metabolites. The RPLC columns Hypersil GOLD and Zorbax SB aq were proven to be best suited for the metabolic profiling of urine and plasma, respectively. Importantly, the optimized HILIC-MS method showed excellent intrabatch peak area reproducibility (CV < 12%) and good long-term interbatch (40 days) peak area reproducibility (CV < 22%) that were similar to those of RPLC-MS procedures. Finally, combining the optimal HILIC- and RPLC-MS approaches greatly expanded metabolome coverage with 44% and 108% new metabolic features detected compared with RPLC-MS alone for urine and plasma, respectively. The proposed combined LC-MS approaches improve the comprehensiveness of global metabolic profiling of body fluids and thus are valuable for monitoring and discovering metabolic changes associated with health and disease in clinical research studies.
Highlights
Abolic profiling of body fluids and are valuable for monitoring and discovering metabolic changes associated with health and disease in clinical research studies
Five different Hydrophilic interaction liquid chromatography (HILIC) columns were examined; two columns were composed of uncharged stationary phases (BEH amide and Hypersil GOLD HILIC), one column was anionic (BEH HILIC), and two were zwitterionic (Syncronis HILIC and ZICHILIC)
Most untargeted metabolomic studies of urine and plasma are performed by reverse-phase liquid chromatography (RPLC)-mass spectrometry (MS) alone and often with suboptimal conditions
Summary
Optimized Analytical Procedures for the Untargeted Metabolomic Profiling of Human Urine and Plasma by Combining Hydrophilic Interaction (HILIC) and Reverse-Phase Liquid Chromatography (RPLC)–Mass Spectrometry*□S. Less than 15% of the LC-MS-based untargeted metabolomic studies performed on biofluids published in 2013 used both HILIC- and RPLC-MS (28 –32) Among these studies, there was no consensus on the analytical procedure (column and chromatographic conditions) that will produce the optimal reproducibility and metabolome coverage for the global analysis of urine and blood. There was no consensus on the analytical procedure (column and chromatographic conditions) that will produce the optimal reproducibility and metabolome coverage for the global analysis of urine and blood In this context, we systematically investigated the performance of a variety of HILIC and RPLC columns under different chromatographic conditions using standard mixtures and biological samples. We investigated the complementarity of HILIC and RPLC in positive and negative electrospray ionization (ESI) modes and found that the optimized HILIC condition greatly expanded the metabolome coverage with 44% and 108% new metabolic features detected, compared with RPLC-MS alone in urine and plasma, respectively
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