关键词: 反相液相色谱, 亲水相互作用液相色谱, 代谢物覆盖度, 代谢谱分析, 双模式分离

Abstract:

As one of the youngest “omics” disciplines, metabolomics, more specifically, metabolite profiling, enables the comprehensive quantitative analysis of a primary metabolome. At present, comprehensive metabolite profiling analysis is a challenging task owing to the greatly different physicochemical properties of metabolites and the complexity of sample matrices, especially given the wide variety of biological samples. Consequently, the development of a multimodal separation method with higher metabolite coverage than single-mode chromatography is necessary for comprehensive metabolite profiling analysis and biomarker discovery.

Reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) are used to analyze nonpolar and polar metabolites, respectively. HILIC and RPLC separation results are commonly combined to achieve comprehensive metabolite profiling analysis. However, this practice leads to overlapping datasets and extended analysis times. Alternatively, multiple LC systems can be coupled to generate combined HILIC and RPLC separation results. In this study, we developed an alternating HILIC/RPLC-tandem mass spectrometry (MS/MS) dual-mode separation system consisting of one autosampler, two high-pressure LC pumps, a HILIC column, an RP column, a 6-port 2-position switching valve, a 10-port 2-position switching valve, and a mass spectrometer for the comprehensive and high-throughput analysis of polar and nonpolar metabolites.

In this configuration, the autosampler is always on the same flow path as the mass spectrometer, whereas the HILIC and RP columns are switched in and out of the main flow path. At the initial stage of the analysis, the autosampler is connected to a HILIC column, followed by the mass spectrometer. While gradient elution is performed on the HILIC column, the RP column is equilibrated using the second high-pressure pump. After HILIC separation, the 6-port 2-position and 10-port 2-position switching valves are switched simultaneously. At this stage of the analysis, the autosampler is connected to the RP column, followed by the mass spectrometer, while the HILIC column is equilibrated. This process is repeated for the subsequent samples. Because the equilibration time of one column overlaps with the gradient elution of the other column, the total analysis time can be reduced by approximately 40%. This system was used to analyze 14 representative endogenous metabolites, including four common types: organic acids, amino acids, choline, and lysophospholipids. The alternating HILIC/RPLC-MS/MS dual-mode separation system had high metabolite coverage and could analyze all 14 representative metabolites simultaneously. By contrast, only 10 of the 14 metabolites could be analyzed using the HILIC-only system. The HILIC-only system failed to analyze organic acids. On the other hand, the RP-only system failed to analyze hydrophilic amino acids, choline, and lysophospholipids. These findings indicate that only 6 of the 14 representative metabolites could be analyzed by the RP-only system. Method validation was conducted to ensure the accuracy of the proposed method. Fourteen representative metabolites were spiked into a urine matrix for validation. All 14 metabolites exhibited linear responses, with correlation coefficients greater than 0.99. The limits of detection (LODs) were in the range of 0.02-42.86 ng/mL, and the limits of quantification (LOQs) were in the range of 0.08-142.86 ng/mL. The average recoveries at three spiked levels were in the range of 85.2%-113.9%. The intra- and inter-day precisions (RSDs) were less than 10%. The proposed instrument configuration and dual-mode method are practical and easy to use for metabolite profiling research in clinical applications such as disease diagnosis, drug development, and biomarker discovery. Furthermore, the proposed instrument configuration and method could potentially be used to analyze other complex samples in proteomics and glycomics research.

Key words: reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography, metabolite coverage, metabolic profiling, dual-mode separation