Direct activation of microglia by ß-glucosylceramide causes phagocytosis of neurons that exacerbates Gaucher disease.

Gaucher disease (GD) is the most common lysosomal storage disease caused by recessive mutations in the degrading enzyme of ß-glucosylceramide (ß-GlcCer). However, it remains unclear how ß-GlcCer causes severe neuronopathic symptoms, which are not fully treated by current therapies. We herein found that ß-GlcCer accumulating in GD activated microglia through macrophage-inducible C-type lectin (Mincle) to induce phagocytosis of living neurons, which exacerbated Gaucher symptoms. This process was augmented by tumor necrosis factor (TNF) secreted from activated microglia that sensitized neurons for phagocytosis. This characteristic pathology was also observed in human neuronopathic GD. Blockade of these pathways in mice with a combination of FDA-approved drugs, minocycline (microglia activation inhibitor) and etanercept (TNF blocker), effectively protected neurons and ameliorated neuronopathic symptoms. In this study, we propose that limiting unrestrained microglia activation using drug repurposing provides a quickly applicable therapeutic option for fatal neuronopathic GD.

Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS.

Quantitative information on blood metabolites can be used in developing advanced medical strategies such as early detection and prevention of disease. Monitoring bioactive lipids such as steroids, bile acids, and PUFA metabolites could be a valuable indicator of health status. However, a method for simultaneously measuring these bioactive lipids has not yet been developed. Here, we report a LC/MS/MS method that can simultaneously measure 144 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal by methanol precipitation and purification of bioactive lipids by solid-phase extraction improved the recovery of the targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods for a wide range of bioactive lipid analyses. Using the developed method, we studied the plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The developed method of bioactive lipid analysis can be applied to health monitoring and disease biomarker discovery in precision medicine.

Delta-6 desaturase FADS2 is a tumor-promoting factor in cholangiocarcinoma.

Cholangiocarcinoma is a fatal disease with limited therapeutic options. We screened genes required for cholangiocarcinoma tumorigenicity and identified FADS2, a delta-6 desaturase. FADS2 depletion reduced in vivo tumorigenicity and cell proliferation. In clinical samples, FADS2 was expressed in cancer cells but not in stromal cells. FADS2 inhibition also reduced the migration and sphere-forming ability of cells and increased apoptotic cell death and ferroptosis markers. Lipidome assay revealed that triglyceride and cholesterol ester levels were decreased in FADS2-knockdown cells. The oxygen consumption ratio was also decreased in FADS2-depleted cells. These data indicate that FADS2 depletion causes a reduction in lipid levels, resulting in decrease of energy production and attenuation of cancer cell malignancy.

Predicting Retention Time in Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography High-Resolution Tandem Mass Spectrometry (Unified-HILIC/AEX/HRMS/MS) for Comprehensive Structural Annotation of Polar Metabolome.

The accuracy of the structural annotation of unidentified peaks obtained in metabolomic analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be enhanced using retention time (RT) information as well as precursor and product ions. Unified-hydrophilic-interaction/anion-exchange liquid chromatography high-resolution tandem mass spectrometry (unified-HILIC/AEX/HRMS/MS) has been recently developed as an innovative method ideal for nontargeted polar metabolomics. However, the RT prediction for unified-HILIC/AEX has not been developed because of the complex separation mechanism characterized by the continuous transition of the separation modes from HILIC to AEX. In this study, we propose an RT prediction model of unified-HILIC/AEX/HRMS/MS, which enables the comprehensive structural annotation of polar metabolites. With training data for 203 polar metabolites, we ranked the feature importance using a random forest among 12,420 molecular descriptors (MDs) and constructed an RT prediction model with 26 selected MDs. The accuracy of the RT model was evaluated using test data for 51 polar metabolites, and 86.3% of the ΔRTs (difference between measured and predicted RTs) were within ±1.50 min, with a mean absolute error of 0.80 min, indicating high RT prediction accuracy. Nontargeted metabolomic data from the NIST SRM 1950-Metabolites in frozen human plasma were analyzed using the developed RT model and in silico MS/MS prediction, resulting in a successful structural estimation of 216 polar metabolites, in addition to the 62 identified based on standards. The proposed model can help accelerate the structural annotation of unknown hydrophilic metabolites, which is a key issue in metabolomic research.

Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay.

C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors.

Archaeal Glycerolipids Are Recognized by C-Type Lectin Receptor Mincle.

Recently, various metabolites derived from host microbes have been reported to modulate the immune system, with potential involvement in health or diseases. Archaea, prokaryotic organisms, are present in the human body, but their connection with the host is largely unknown when compared to other microorganisms such as bacteria. This study focused on unique glycerolipids from symbiotic methanogenic archaea and evaluated their activities toward an innate immune receptor. The results revealed that archaeal lipids were recognized by the C-type lectin receptor Mincle and induced immune responses. A concurrent structure-activity relationship study identified the key structural features of archaeal lipids required for recognition by Mincle. Subsequent gene expression profiling suggested qualitative differences between the symbiotic archaeal lipid and the pathogenic bacteria-derived lipid. These findings have broad implications for understanding the function of symbiotic archaea in host health and diseases.

Structural Analysis of Intracellular Lipid Radicals by LC/MS/MS Using a BODIPY-Based Profluorescent Nitroxide Probe.

Free radical-mediated lipid peroxidation (LPO) induces the formation of numerous lipid radicals, which contribute to the development of several oxidative diseases. To understand the mechanism of LPO in biological systems and the significance of these radicals, identifying the structures of individual lipid radicals is imperative. In this study, we developed an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and a profluorescent nitroxide probe, N-(1-oxyl-2,2,6-trimethyl-6-pentylpiperidin-4-yl)-3-(5,5-difluoro-1,3-dimethyl-3H,5H-5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-7-yl)propanamide (BDP-Pen), for the detailed structural analysis of lipid radicals. The MS/MS spectra of BDP-Pen-lipid radical adducts showed product ions and thus allow the prediction of the lipid radical structures and individual detection of isomeric adducts. Using the developed technology, we separately detected the isomers of arachidonic acid (AA)-derived radicals generated in AA-treated HT1080 cells. This analytical system is a powerful tool for elucidating the mechanism of LPO in biological systems.

Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells.

Effective tumor immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute a specialized microenvironment that excludes T cells from the vicinity of cancer cells, and its underlying mechanisms are still poorly understood. DOCK2 is a Rac activator critical for migration and activation of lymphocytes. We herein show that cancer-derived cholesterol sulfate (CS), a lipid product of the sulfotransferase SULT2B1b, acts as a DOCK2 inhibitor and prevents tumor infiltration by effector T cells. Using clinical samples, we found that CS was abundantly produced in certain types of human cancers such as colon cancers. Functionally, CS-producing cancer cells exhibited resistance to cancer-specific T-cell transfer and immune checkpoint blockade. Although SULT2B1b is known to sulfate oxysterols and inactivate their tumor-promoting activity, the expression levels of cholesterol hydroxylases, which mediate oxysterol production, are low in SULT2B1b-expressing cancers. Therefore, SULT2B1b inhibition could be a therapeutic strategy to disrupt tumor immune evasion in oxysterol-non-producing cancers. Thus, our findings define a previously unknown mechanism for tumor immune evasion and provide a novel insight into the development of effective immunotherapies.

Chronic hyperglycemia reduces the expression of intercellular adhesion molecules and increases intercellular hyperpermeability in the periodontal epithelium.

BACKGROUND/AIMS: Hyperglycemia in diabetes is closely associated with periodontal disease progression. This study aimed to investigate the effect of hyperglycemia on the barrier function of gingival epithelial cells as a cause of hyperglycemia-exacerbated periodontitis in diabetes mellitus. METHODS: The abnormal expression of adhesion molecules in gingival epithelium in diabetes was compared between db/db and control mice. To study the effects of hyperglycemia on interepithelial cell permeability, the mRNA and protein expressions of adhesion molecules were investigated using a human gingival epithelial cell line (epi 4 cells) in the presence of either 5.5 mM glucose (NG) or 30 mM glucose (HG). Immunocytochemical and histological analyses were performed. We also studied HG-related intracellular signaling to assess abnormal adhesion molecule expression in the cultured epi 4 cells. RESULTS: The results of the proteomic analysis implied the abnormal regulation of cell-cell adhesion, and mRNA and protein expression assessments revealed the significant downregulation of Claudin1 expression in the gingival tissues of db/db mice (p < .05 vs control). Similarly, the mRNA and protein expressions of adhesion molecules were lower in epi 4 cells cultured under HG conditions than in those cultured under NG conditions (p < .05). Three-dimensional culture and transmission electron microscopy revealed reduced thickness of the epithelial cell layers with no flattened apical cells and heterogeneously arranged intercellular spaces among adjacent epi 4 cells under the HG. These results were consistent with the increased permeability of epi 4 cells under the HG relative to that of cells under the NG. This abnormal expression of intercellular adhesion molecules under the HG was related to the increased expression of receptors for advanced glycation end products (AGEs) and oxidative stress relative to that seen under the NG, along with stimulation of ERK1/2 phosphorylation in epi 4 cells. CONCLUSIONS: High glucose-induced impairment of intercellular adhesion molecule expression in gingival epithelial cells was related to the intercellular permeability of gingival cells, representing a possible link to hyperglycemia-related AGE signaling, oxidative stress, and ERK1/2 activation.

Differential effect of canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice.

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

Improved 2,3-Butanediol Production Rate of Metabolically Engineered Saccharomyces cerevisiae by Deletion of RIM15 and Activation of Pyruvate Consumption Pathway.

Saccharomyces cerevisiae is a promising host for the bioproduction of higher alcohols, such as 2,3-butanediol (2,3-BDO). Metabolically engineered S. cerevisiae strains that produce 2,3-BDO via glycolysis have been constructed. However, the specific 2,3-BDO production rates of engineered strains must be improved. To identify approaches to improving the 2,3-BDO production rate, we investigated the factors contributing to higher ethanol production rates in certain industrial strains of S. cerevisiae compared to laboratory strains. Sequence analysis of 11 industrial strains revealed the accumulation of many nonsynonymous substitutions in RIM15, a negative regulator of high fermentation capability. Comparative metabolome analysis suggested a positive correlation between the rate of ethanol production and the activity of the pyruvate-consuming pathway. Based on these findings, RIM15 was deleted, and the pyruvate-consuming pathway was activated in YHI030, a metabolically engineered S. cerevisiae strain that produces 2,3-BDO. The titer, specific production rate, and yield of 2,3-BDO in the test tube-scale culture using the YMS106 strain reached 66.4 ± 4.4 mM, 1.17 ± 0.017 mmol (g dry cell weight h)-1, and 0.70 ± 0.03 mol (mol glucose consumed)-1. These values were 2.14-, 2.92-, and 1.81-fold higher than those of the vector control, respectively. These results suggest that bioalcohol production via glycolysis can be enhanced in a metabolically engineered S. cerevisiae strain by deleting RIM15 and activating the pyruvate-consuming pathway.

Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography Mass Spectrometry (Unified-HILIC/AEX/MS): A Single-Run Method for Comprehensive and Simultaneous Analysis of Polar Metabolome.

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.

Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity.

Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3ß-hydroxy-5-cholenoic acid (3ß-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3ß-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3ß-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3ß-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion.

Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants.

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype.

Lipid Profiling of Serum and Lipoprotein Fractions in Response to Pitavastatin Using an Animal Model of Familial Hypercholesterolemia.

Statins are widely used for the treatment of atherosclerotic cardiovascular diseases. They inhibit cholesterol biosynthesis in the liver and cause pleiotropic effects, including anti-inflammatory and antioxidant effects. To develop novel therapeutic drugs, the effect of blood-borne lipid molecules on the pleiotropic effects of statins must be elucidated. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for hypercholesterolemia, are suitable for the determination of lipid molecules in the blood in response to statins because their lipoprotein metabolism is similar to that of humans. Herein, lipid molecules were investigated by lipidome analysis in response to pitavastatin using WHHLMI rabbits. Various lipid molecules in the blood were measured using a supercritical fluid chromatography triple quadrupole mass spectrometry. Cholesterol and cholesterol ester blood concentrations decreased by reducing the secretion of very low density lipoproteins from the liver. Independent of the inhibition effects of cholesterol biosynthesis, the concentrations of some lipids with anti-inflammation and antioxidant effects (phospholipid molecules with n-6 fatty acid side chains, lysophosphatidylcholines, phosphatidylethanolamine plasmalogens, and ceramide molecules) were significantly altered. These findings may lead to further investigation of the mechanism of statin action.

Method for Structural Determination of Lipid-Derived Radicals.

Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we propose a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis.

In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells.

Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations.

Intracellular metabolite ß-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity.

Sensing and reacting to tissue damage is a fundamental function of immune systems. Macrophage inducible C-type lectin (Mincle) is an activating C-type lectin receptor that senses damaged cells. Notably, Mincle also recognizes glycolipid ligands on pathogens. To elucidate endogenous glycolipids ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified a lipophilic component that activates reporter cells expressing Mincle. Mass spectrometry and NMR spectroscopy identified the component structure as ß-glucosylceramide (GlcCer), which is a ubiquitous intracellular metabolite. Synthetic ß-GlcCer activated myeloid cells and induced production of inflammatory cytokines; this production was abrogated in Mincle-deficient cells. Sterile inflammation induced by excessive cell death in the thymus was exacerbated by hematopoietic-specific deletion of degrading enzyme of ß-GlcCer (ß-glucosylceramidase, GBA1). However, this enhanced inflammation was ameliorated in a Mincle-deficient background. GBA1-deficient dendritic cells (DCs) in which ß-GlcCer accumulates triggered antigen-specific T-cell responses more efficiently than WT DCs, whereas these responses were compromised in DCs from GBA1 × Mincle double-deficient mice. These results suggest that ß-GlcCer is an endogenous ligand for Mincle and possesses immunostimulatory activity.

Improved quantitation of lipid classes using supercritical fluid chromatography with a charged aerosol detector.

Quantitatively and rapidly analyzing lipids is necessary to elucidate their biological functions. Herein, we developed a quantitative method for various lipid classes using supercritical fluid chromatography (SFC) coupled with a charged aerosol detector (CAD), providing high-throughput data analysis to detect a large number of molecules in each lipid class as one peak. Applying the CAD was useful for analyzing lipid molecules in the same lipid class with a constant response under the same mobile phase composition. First, we optimized the washing method for the diethylamine column, achieving baseline separation of lipid classes while maintaining good peak shapes. In addition, the CAD conditions (organic solvent evaporation and numerical correction of the CAD data) were optimized to improve the signal-to-noise ratio. We used an internal standard (ceramide phosphoethanolamine d17:1-12:0), which did not coelute with the lipid classes and showed high extraction efficiency. Based on a quantitative analysis of HepG2 cells, the concentration of lipid classes detected by CAD was adequate compared with that obtained by triple-quadrupole MS (QqQMS) in a previous study because the deviations of the concentrations were 0.6- to 2.3-fold. These results also supported the quantitative performance of SFC-QqQMS developed in our previous report.

Comparison of Retention Behavior between Supercritical Fluid Chromatography and Normal-Phase High-Performance Liquid Chromatography with Various Stationary Phases.

The retention behavior of a wide variety of stationary phases was compared in supercritical fluid chromatography (SFC) and normal-phase high-performance liquid chromatography (NP-HPLC). We also attempted to elucidate the retention behavior in SFC by investigating the selectivity of the different stationary phases. SFC separation conditions with polar stationary phases, such as silica gel (SL) and diol (Diol) phases, operate via adsorptions that include hydrophilic and ionic interactions similar to those in NP-HPLC. Moreover, non-polar stationary phases, such as pentabromophenyl (PBr), pyrenylethyl (PYE), and octadecyl (C18), could be used despite the non-polar mobile phase conditions, because the dispersion and π-π interactions were stronger in SFC than in HPLC. These results reflect the selectivity of the stationary phase and its retention factor, thus providing useful information for the selection of appropriate stationary phases for particular analytes.