For quite of time, supercritical fluid chromatography (SFC) is expected to make a big revolution in separation technologies. The technique is well known for developing fast and ultrafast high ...efficiency separation, improving the analytical method greenness. The evolution of SFC technologies (i.e., instrumentations, stationary phases) have shifted dramatically trends in SFC application from the chiral and pharmaceutical analysis toward new diverse application fields including drugs and bioactive compounds, metabolomics, environment, and food science analysis. Moreover, the applications have been greatly expanded toward the analysis of polar and very polar metabolites, resulting in certain successes. This review focuses on the most of SFC related studies during 2020–2021, covering the new development of SFC technologies, recent trends in application and method developments, and more importantly discusses the upcoming perspective regarding the art of supercritical fluid separation.
•SFC towards the greener chromatography.•New instrumental and stationary phase developments boost the SFC-method performance.•Modelling takes the vibe in many SFC challenges.•More UC applications are expected in the upcoming.
Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by ...large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment.
The gas chromatography/mass spectrometry (GC/MS)-based metabolomics requires a two-step derivatization procedure consisting of oximation and silylation. However, due to the incomplete derivatization ...and degeneration of the metabolites, good repeatability is difficult to obtain during the batch derivatization, as the time between completing the derivatization process and GC analysis differs from sample to sample. In this research, we successfully obtained good repeatability for the peak areas of 52 selected metabolites by sequential derivatization and interval injection, in which the oximation and silylation times were maintained at constant values. In addition, the derivatization times and amount of reagents employed were varied to confirm that the optimal derivatization conditions differed for the various metabolites. In conventional batch derivatization, six metabolites, viz. glutamine, glutamic acid, histidine, alanine, asparagine, and tryptophan, exhibited fluctuations in their peak areas. Indeed, we found that for all six metabolites these differences originated from the silylation process, while the variations for glutamine and glutamic acid were related to the oximation process.
•Analytical method is developed using sequential derivatization and interval GC injection.•Sequential and batch derivatization are compared using a mixture of the 52 metabolites.•Derivatization times and amount of derivatization reagents are investigated.•In conventional batch derivatization, 6 metabolites exhibited fluctuations in their peak areas.
One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal ...method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites retention times (RTs) = 0–12.8 min and AEX-dominant separation for polar anionic metabolites (RTs = 12.8–26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.
Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for ...biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.—Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba.
•SFC/MS-specific optimization of analytical conditions brought high sensitivity.•Optimum values of MS parameters in SFC/MS were different from that of LC/MS.•High sensitive analysis was possible in ...SFC/MS than with LC/MS.•High sensitivity of SFC/MS contributed to good recovery in real sample analysis.•SFC and LC have complementarity in recovery owing to different separation pattern.
Supercritical fluid chromatography/mass spectrometry (SFC/MS) has great potential in high-throughput and the simultaneous analysis of a wide variety of compounds, and it has been widely used in recent years. The use of MS for detection provides the advantages of high sensitivity and high selectivity. However, the sensitivity of MS detection depends on the chromatographic conditions and MS parameters. Thus, optimization of MS parameters corresponding to the SFC condition is mandatory for maximizing performance when connecting SFC to MS. The aim of this study was to reveal a way to decide the optimum composition of the mobile phase and the flow rate of the make-up solvent for MS detection in a wide range of compounds. Additionally, we also showed the basic concept for determination of the optimum values of the MS parameters focusing on the MS detection sensitivity in SFC/MS analysis. To verify the versatility of these findings, a total of 441 pesticides with a wide polarity range (logPow from −4.21 to 7.70) and pKa (acidic, neutral and basic). In this study, a new SFC-MS interface was used, which can transfer the entire volume of eluate into the MS by directly coupling the SFC with the MS. This enabled us to compare the sensitivity or optimum MS parameters for MS detection between LC/MS and SFC/MS for the same sample volume introduced into the MS. As a result, it was found that the optimum values of some MS parameters were completely different from those of LC/MS, and that SFC/MS-specific optimization of the analytical conditions is required. Lastly, we evaluated the sensitivity of SFC/MS using fully optimized analytical conditions. As a result, we confirmed that SFC/MS showed much higher sensitivity than LC/MS when the analytical conditions were fully optimized for SFC/MS; and the high sensitivity also increase the number of the compounds that can be detected with good repeatability in real sample analysis. This result indicates that SFC/MS has potential for practical use in the multiresidue analysis of a wide range of compounds that requires high sensitivity.
The goal of metabolomics analyses is a comprehensive and systematic understanding of all metabolites in biological samples. Many useful platforms have been developed to achieve this goal. Gas ...chromatography coupled to mass spectrometry (GC/MS) is a well-established analytical method in metabolomics study, and 200 to 500 peaks are routinely observed with one biological sample. However, only ~100 metabolites can be identified, and the remaining peaks are left as "unknowns".
We present an algorithm that acquires more extensive metabolite information. Pearson's product-moment correlation coefficient and the Soft Independent Modeling of Class Analogy (SIMCA) method were combined to automatically identify and annotate unknown peaks, which tend to be missed in routine studies that employ manual processing.
Our data mining system can offer a wealth of metabolite information quickly and easily, and it provides new insights, particularly into food quality evaluation and prediction.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Supercritical Fluid Chromatography (SFC), a high-throughput separation technique, has been widely applied as a promising routine method in pharmaceutical, pesticides, and metabolome analysis in the ...same way as conventional liquid chromatography and gas chromatography. Unified chromatography (UC), an advanced version of SFC, which applied gradient elution with mobile phase changing continuously from supercritical to subcritical and to liquid states, can further extend the SFC applications. UC mostly applying the popular mobile phase of 95%:5%/Methanol:Water with additives allows to analyze many hydrophilic compounds. However, many of phosphorylated metabolites or multi carboxylic acids show very poor peak shapes or even can't be eluted under UC conditions, thus hampering the UC's metabolome coverage. In this study, we proposed the first proof-of-concept of UC/HILIC, a novel strategy to extend the current UC metabolome coverage by employing an aqueous gradient right after the UC gradient on a single packed column in a single measurement. The proposed method showed significant improvement regarding the chromatographic performance and metabolome coverage, while still maintaining the precision and high throughput in comparison with conventional UC methods.
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•Development of advanced supercritical fluid chromatography methodology.•Unified chromatography coupling with hydrophilic chromatography.•Incorporation of HILIC-like mobile phase in SFC for improving the metabolome coverage.•Simultaneous analysis of a wide range of compounds from lipids to highly hydrophilic metabolites.
Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic substances that are mainly generated during heating in food; therefore, the European Union (EU) has regulated the amount of benzoapyrene and ...PAH4 in various types of food. In addition, the Scientific Committee on Food of the EU and the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives have recommended that 16 PAHs should be monitored. Since coffee beverages and dark beer are roasted during manufacture, monitoring these 16 PAHs is of great importance. On the other hand, supercritical fluid chromatography (SFC) is a separation method that has garnered attention in recent years as a complement for liquid and gas chromatography. Therefore, we developed a rapid high-sensitivity analytical method for the above-mentioned 16 PAHs in coffee beverages and dark beer involving supercritical fluid chromatography/atmospheric pressure chemical ionization-mass spectrometry (SFC/APCI-MS) and simple sample preparation. In this study, we developed a novel analytical technique that increased the sensitivity of MS detection by varying the back-pressure in SFC depending on the elution of PAHs. In addition, analysis of commercially available coffee and dark beer samples in Japan showed that the risk of containing the 16 PAHs may be low.
•Simultaneous analysis of 16 EU priority PAHs using SFC/APCI/MS is developed.•New sensitive technique by varying the back pressure is developed.•Sample preparation is simple in coffee beverages and dark beer.•SFC analysis of 16 PAHs can be performed in 14 min.
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