Comprehensive metabolic profiling is a considerable challenge for systems biology since the metabolites in biological samples have significant polarity differences. A heartcutting two-dimensional liquid chromatography−mass spectrometry (2D-LC−MS) method-based polarity partition was established to analyze both the metabolome and lipidome in a single run. Based on the polarity partition strategy, metabolites with high polarity were retained and separated by one-dimensional hydrophilic chromatography, while low-and medium-polarity lipids were collected into a sample loop and injected into two-dimensional reversed-phase chromatography for separation. A simple online dilution strategy realized the online coupling of the 2D-LC−MS, which effectively solved band broadening and peak distortion caused by solvent incompatibility. Moreover, a dual gradient elution procedure was introduced to further broaden the coverage of low-polarity lipids. The metabolites' log P values, which this 2D-LC−MS method could analyze, ranged from −8.79 to 26.86. The feasibility of the 2D-LC−MS system was demonstrated by simultaneous analysis of the metabolome and lipidome in rat plasma related to depression. A total of 319 metabolites were determined within 40 min, including organic acids, nucleosides, carbohydrate derivatives, amino acids, lipids, and other organic compounds. Finally, 44 depression-related differential metabolites were screened. Compared with conventional LC−MS-based methods, the 2D-LC method covered over 99% of features obtained by two conventional methods. In addition, the selectivity and resolution of the hydrophilic metabolites were improved, and the matrix effects of the hydrophobic metabolites were reduced in the developed method. The results indicated that the established 2D-LC system is a powerful tool for comprehensive metabolomics studies.

The online coupling technique of sample preparation with chromatography is a frontier topic in analytical chemistry since it minimizes errors caused by sample loss, shortens analysis time, and reduces solvent consumption. An online pressure change focusing-supercritical fluid selective extraction chromatography (PCF-SFSEC) technique was developed in this study, realizing extraction, purification, separation, and detection in a single run with only microliter-scale samples. The pressure change focusing strategy achieved column-head stacking by decreasing the dissolving capacity of the supercritical fluid, enabling the large volume introduction of extractants into supercritical fluid chromatography without causing peak broadening or distortion. All the extracts could be directly loaded into the chromatography system without split flow. Based on the supercritical fluid selective extraction (SFSE) strategy, the sorbents removed interferences and water from samples, effectively alleviating matrix effects and realizing the direct aqueous sample analysis. The efficiency of online PCF-SFSEC was demonstrated by the enantioselective analysis of 22 chiral drugs in rat plasma, covering eight categories with different pharmacological effects. The entire analysis took 25 min, consuming only 5 μL samples. All analytes in PCF-SFSEC obtained sharp and symmetrical peaks with resolutions higher than 1.0, and 86% had resolutions higher than 1.5. Limits of quantification (LOQs) ranged from 0.0600 to 32.1 μg/L. Recoveries were in the range of 75.8−117.2%. In addition, the developed approach obtained more satisfactory repeatability and significantly reduced matrix effects than conventional methods. The newly established online PCF-SFSEC technique is believed to be a green and powerful tool for the chiral analysis of complex samples.