Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.

Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

Exploring the Impact of Organic Solvent Quality and Unusual Adduct Formation during LC-MS-Based Lipidomic Profiling.

Liquid chromatography-mass spectrometry (LC-MS) is the key technique for analyzing complex lipids in biological samples. Various LC-MS modes are used for lipid separation, including different stationary phases, mobile-phase solvents, and modifiers. Quality control in lipidomics analysis is crucial to ensuring the generated data's reliability, reproducibility, and accuracy. While several quality control measures are commonly discussed, the impact of organic solvent quality during LC-MS analysis is often overlooked. Additionally, the annotation of complex lipids remains prone to biases, leading to potential misidentifications and incomplete characterization of lipid species. In this study, we investigate how LC-MS-grade isopropanol from different vendors may influence the quality of the mobile phase used in LC-MS-based untargeted lipidomic profiling of biological samples. Furthermore, we report the occurrence of an unusual, yet highly abundant, ethylamine adduct [M+46.0651]+ that may form for specific lipid subclasses during LC-MS analysis in positive electrospray ionization mode when acetonitrile is part of the mobile phase, potentially leading to lipid misidentification. These findings emphasize the importance of considering solvent quality in LC-MS analysis and highlight challenges in lipid annotation.

Short-Term Stability of Serum and Liver Extracts for Untargeted Metabolomics and Lipidomics.

Thermal reactions can significantly alter the metabolomic and lipidomic content of biofluids and tissues during storage. In this study, we investigated the stability of polar metabolites and complex lipids in dry human serum and mouse liver extracts over a three-day period under various temperature conditions. Specifically, we tested temperatures of -80 °C (freezer), -24 °C (freezer), -0.5 °C (polystyrene box with gel-based ice packs), +5 °C (refrigerator), +23 °C (laboratory, room temperature), and +30 °C (thermostat) to simulate the time between sample extraction and analysis, shipping dry extracts to different labs as an alternative to dry ice, and document the impact of higher temperatures on sample integrity. The extracts were analyzed using five fast liquid chromatography-mass spectrometry (LC-MS) methods to screen polar metabolites and complex lipids, and over 600 metabolites were annotated in serum and liver extracts. We found that storing dry extracts at -24 °C and partially at -0.5 °C provided comparable results to -80 °C (reference condition). However, increasing the storage temperatures led to significant changes in oxidized triacylglycerols, phospholipids, and fatty acids within three days. Polar metabolites were mainly affected at storage temperatures of +23 °C and +30 °C.

Optimization of Mobile Phase Modifiers for Fast LC-MS-Based Untargeted Metabolomics and Lipidomics.

Liquid chromatography-mass spectrometry (LC-MS) is the method of choice for the untargeted profiling of biological samples. A multiplatform LC-MS-based approach is needed to screen polar metabolites and lipids comprehensively. Different mobile phase modifiers were tested to improve the electrospray ionization process during metabolomic and lipidomic profiling. For polar metabolites, hydrophilic interaction LC using a mobile phase with 10 mM ammonium formate/0.125% formic acid provided the best performance for amino acids, biogenic amines, sugars, nucleotides, acylcarnitines, and sugar phosphate, while reversed-phase LC (RPLC) with 0.1% formic acid outperformed for organic acids. For lipids, RPLC using a mobile phase with 10 mM ammonium formate or 10 mM ammonium formate with 0.1% formic acid permitted the high signal intensity of various lipid classes ionized in ESI(+) and robust retention times. For ESI(-), the mobile phase with 10 mM ammonium acetate with 0.1% acetic acid represented a reasonable compromise regarding the signal intensity of the detected lipids and the stability of retention times compared to 10 mM ammonium acetate alone or 0.02% acetic acid. Collectively, we show that untargeted methods should be evaluated not only on the total number of features but also based on common metabolites detected by a specific platform along with the long-term stability of retention times.