Atsuki Matsubara

High throughput and exhaustive analysis of diverse lipids by using supercritical fluid chromatography-mass spectrometry for metabolomics.

We have developed an analytical system that enables the simultaneous rapid analysis of lipids with varied structures and polarities through the use of supercritical fluid chromatography-mass spectrometry (SFC-MS). The separation conditions for SFC (column, modifier, back pressure, etc.) and the detection conditions for mass spectrometry (ionization method, parameters, etc.) were investigated to develop a simultaneous analytical method for lipid mixtures that included phospholipids, glycolipids, neutral lipids, and sphingolipids. When cyanopropylated silica gel-packed column was used for the separation, all lipids were successfully detected and the analysis time was less than 15 min. The use of an octadecylsilylated column resulted in separation, which was dependent on the differences in the unsaturation of the fatty acid side chains and isomer separation. This system is a powerful tool for studies on lipid metabolomics because it is useful not only as a fingerprinting method for the screening of diverse lipids but also for the detailed profiling of individual components.

Metabolic profiling of lipids by supercritical fluid chromatography/mass spectrometry.

This review describes the usefulness of supercritical fluid chromatography (SFC) for the metabolic profiling of lipids. First, non-targeted lipid profiling by SFC/MS is described. The use of SFC/MS allows for high-throughput, exhaustive analysis of diverse lipids, and hence, this technique finds potential applications in lipidomics. Development of a polar lipid profiling method with trimethylsilyl (TMS) derivatization widens the scope of applicability of SFC/MS. SFC is a high-resolution technique that is suitable for non-targeted profiling aimed at the simultaneous analysis of many components. Next, targeted lipid profiling by SFC/MS is described. SFC is useful for the separation of lipids, such as carotenoids and triacylglycerols, which have numerous analogs with similar structures. In addition, SFC/MS shows the maximum efficiency for the target analysis of lipids in a biological sample that includes many matrices. Finally, a high-resolution, high-throughput analytical system based on SFC/MS is stated to be suitable for lipidomics because it is useful not only for the screening of lipid mixtures (as a fingerprint method) but also for the detailed profiling of individual components.

Application of supercritical fluid chromatography/mass spectrometry to lipid profiling of soybean.

A metabolomics technology for lipid profiling based on supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS) was applied to analyze lipids of soybean. Principal component analysis (PCA) was used to discriminate twelve soybean cultivars according to their suitability for different processed foods such as natto, tofu, edamame, and nimame. By PCA assay, triacylglycerol (TAG) was found as the main variable for discrimination of soybean cultivars. Therefore, a high-throughput and high-resolution TAG profiling method by SFC/MS was developed to more effective discrimination. By investigating several columns, three Chromolith Performance RP-18e columns connected in series were chosen as the most effective column for TAG profiling. Diverse TAGs were separated effectively for 8 min without purification. Additionally, each TAG was identified successfully by the programmed cone voltage fragmentation even without MS/MS analysis and any standard sample.

Highly sensitive and accurate profiling of carotenoids by supercritical fluid chromatography coupled with mass spectrometry

We attempted to establish a high-speed and high-resolution profiling method for a carotenoid mixture as a highly selective and highly sensitive detection method; the analysis was carried out by supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS). When an octadecyl-bonded silica (ODS) particle-packed column was used for separation, seven carotenoids including structural isomers were successfully separated within 15 min. This result indicated not only improved separation but also improved throughput compared to the separation and throughput in RP-HPLC. The use of a monolithic ODS column resulted in additional improvement in both the resolution and the throughput; the analysis time was reduced to 4 min by increasing the flow rate. Furthermore, carotenoids in biological samples containing the complex matrices were separated effectively by using several monolithic columns whose back pressure was very low. The mass spectrometer allowed us to perform a more sensitive analysis than UV detection; the detection limit of each carotenoid was 50 pg or below. This is the first report of carotenoid analysis carried out by SFC-MS. The profiling method developed in this study will be a powerful tool for carrying out accurate profiling of biological samples.

High-throughput phospholipid profiling system based on supercritical fluid extraction–supercritical fluid chromatography/mass spectrometry for dried plasma spot analysis

The dried blood spots (DBS) and dried plasma spots (DPS) analysis is available as a diagnostic tool for genetic diseases and is useful for the screening of biomarkers. In this study, a high-throughput analytical system based on supercritical fluid extraction-supercritical fluid chromatography with tandem mass spectrometry (SFE-SFC/MS/MS) was constructed for phospholipids profiling of DPS. This system is able to simultaneously perform extraction and separation, allowing phospholipids that have common polar head groups to be analyzed. Phospholipids in only 3 μl of plasma can be extracted in 5 min and analyzed within 15 min using this system. A total of 134 phospholipids, including phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, phosphatidylethanolamine and lysophosphatidylethanolamine, were annotated, and 74 phospholipids were analyzed with good repeatability. The SFE-SFC/MS/MS, which is able to perform high-throughput lipid profiling analysis for clinical diagnosis and drug discovery, may also be suitable for the screening of biomarkers.

Highly sensitive and rapid profiling method for carotenoids and their epoxidized products using supercritical fluid chromatography coupled with electrospray ionization-triple quadrupole mass spectrometry.

Epoxy carotenoids, which are products of carotenoid oxidation, are potential oxidative stress markers. However, it is difficult to profile epoxy carotenoids owing to their small amount and difficulty in their separation from hydroxy carotenoids. In this study, a high-performance analytical system based on supercritical fluid chromatography (SFC) coupled with tandem mass spectrometry (MS/MS) was developed for the simultaneous analysis of carotenoids and epoxy carotenoids. SFC is an effective separation technique for hydrophobic compounds, by which major carotenoids in human serum and their epoxidation products can be analyzed within 20 min. The use of MS/MS increased the sensitivity; the detection limit for each carotenoid was of the sub-fmol order. When the constructed method was applied to biological samples such as human serum and low-density lipoprotein (LDL), the precise detection of the target carotenoids was disturbed by several isomers. However, highly selective detection of epoxy carotenoids was performed by targeting product ions that were generated with a structure-specific neutral loss of 80Da. Furthermore, the sample volume needed for the analysis was only 0.1ml for the serum, indicating the efficiency of this system in performing small-scale analyses. Using the analytical system developed in this study, highly sensitive and selective analysis of epoxy carotenoids could be performed in a short time. These features show the usefulness of this system in application to screening analysis of carotenoid profiles that are easily modified by oxidative stress.

High-throughput simultaneous analysis of pesticides by supercritical fluid chromatography/tandem mass spectrometry.

Combination techniques such as gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) are commonly used for pesticide residue analysis, but there is no reported method for the simultaneous analysis of multiple pesticides in a sample using a single instrument. Supercritical fluid chromatography (SFC) offers high resolution at high flow rates and various separation modes and hence may aid the rapid simultaneous analysis of pesticide. We developed an SFC/MS/MS method and analyzed 17 pesticides with a wide range of polarities (logP(ow)=-4.6 to 7.05) and molecular weights (112.1-888.6) within 11min using a polar-embedded reversed-phase column. To the best of our knowledge, there is no previous report on the SFC analysis of a wide variety of compounds, including highly hydrophilic ones. By SFC, diquat dibromide (logP(ow)=-4.6), together with cypermethrin (logP(ow)=6.6) and tralomethrin (logP(ow)=5.05), could be detected in the presence of various other pesticides using a single mobile phase. SFC/MS allows for the rapid and simultaneous analysis of low concentrations (ng/L levels) of pesticides that typically need to be analyzed by GC/MS and LC/MS separately.

Development of a polar lipid profiling method by supercritical fluid chromatography/mass spectrometry

We established a high-throughput and high-resolution analytical method based on supercritical fluid chromatography (SFC) coupled with mass spectrometry (MS) for the simultaneous profiling of diverse polar lipids in a mixture. Trimethylsilyl (TMS) derivatization was used for the analysis of ten polar lipids: phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), sphingomyeline (SM), and sphingosine-1-phosphate (S1P). Using the developed method, the peak tailings of PA, PI, LPA, LPI, and S1P improved, and the limit of detection of PG, PI, LPA, LPI, and S1P was enhanced by 12-, 40-, 510-, 39-, and 1490-fold, respectively. Next, in the analysis of sheep plasma, 20 minor species of PI, LPC, LPE, and SM, and 7 molecular species of LPA, LPI, and S1P were additionally analyzed. The relative ratio of the molecular species in each polar lipid was also found by quantification. Finally, the simultaneous and detail profiling of ten polar lipids was successfully performed by SFC/MS applying TMS derivatization. This developed method is particularly applicable to metabolomics, especially for targeting polar lipids.

Development of oxidized phosphatidylcholine isomer profiling method using supercritical fluid chromatography/tandem mass spectrometry.

Phospholipids that contain polyunsaturated fatty acid are easily oxidized by free radicals or oxidants and can yield numerous oxidation species, including positional and structural isomers. However, it is difficult to separate these oxidation products for structural analysis. In this study, a high-resolution separation analytical system based on supercritical fluid chromatography with tandem mass spectrometry (SFC/MS/MS) was established for the separation and identification of oxidized phosphatidylcholine (PC) isomers derived from esterified linoleic acid or arachidonic acid. Separation of oxidatively modified PC containing hydroxy, epoxy and hydroperoxy groups was achieved by SFC. Positional isomers of hydroxides and epoxides were identified based on MS/MS fragment information. To investigate whether this method is applicable to biological samples, we then analyzed oxidized PC isomers from mouse liver. Oxidized isomers, such as hydroxides, hydroperoxides and epoxides, were simultaneously observed. This method may be a powerful tool for providing further insight into how oxidized phospholipids are produced and are correlated with various diseases.

Metabolite analysis by supercritical fluid chromatography

Owing to its favorable properties, such as low viscosity and high diffusivity, a supercritical fluid can be used as the mobile phase in chromatography. Supercritical fluid chromatography (SFC) can provide high-speed and high-resolution separation. Since supercritical carbon dioxide (SCCO(2)), which is generally used as the mobile phase in SFC, is automatically emitted at room temperature, SFC is most commonly used as a preparative method. However, SFC can also be used to perform high-precision biomolecular analysis, especially for hydrophobic metabolites, because of the low polarity of SCCO(2). The use of a mass spectrometer with SFC can widen the scope of application of SFC to bioanalysis. In this review, we summarize practical application of SFC as a tool for the analysis of metabolites in real biological samples.