Use of on-line supercritical fluid extraction-supercritical fluid chromatography/tandem mass spectrometry to analyze disease biomarkers in dried serum spots compared with serum analysis using liquid chromatography/tandem mass spectrometry.

RATIONALE: The analytical stability and throughput of biomarker assays based on dried serum spots (DSS) are strongly dependent on the extraction process and determination method. In the present study, an on-line system based on supercritical fluid extraction-supercritical fluid chromatography coupled with tandem mass spectrometry (SFE-SFC/MS/MS) was established for analyzing the levels of disease biomarkers in DSS. METHODS: The chromatographic conditions were investigated using the ODS-EP, diol, and SIL-100A columns. Then, we optimized the SFE-SFC/MS/MS method using the diol column, focusing on candidate biomarkers of oral, colorectal, and pancreatic cancer that were identified using liquid chromatography (LC)/MS/MS. RESULTS: By using this system, four hydrophilic metabolites and 17 hydrophobic metabolites were simultaneously detected within 15 min. In an experiment involving clinical samples, PC 16:0-18:2/16:1-18:1 exhibited 93.8% sensitivity and 64.3% specificity, whereas PC 17:1-18:1/17:0-18:2 showed 81.3% sensitivity and 92.9% specificity for detecting oral cancer. In addition, assessments of the creatine levels demonstrated 92.3% sensitivity and 78.6% specificity for detecting colorectal cancer. CONCLUSIONS: The results of this study indicate that our method has great potential for clinical diagnosis and would be suitable for large-scale screening. Copyright © 2017 John Wiley & Sons, Ltd.

Supercritical fluid chromatography/Orbitrap mass spectrometry based lipidomics platform coupled with automated lipid identification software for accurate lipid profiling.

We developed a practical analytical system for high-throughput and comprehensive lipid profiling using a supercritical fluid chromatography (SFC) system coupled to an Orbitrap Fourier transform mass spectrometer (Orbitrap FT-MS). Using our SFC method, polar lipid molecular species were separated based on not only their fatty acyl moieties but also their polar head groups, using a single octadecylsilyl (ODS) column. In addition, because automatic data processing software was used for the identification of lipid molecular species, the analysis time including data processing was about a half an hour per sample. A variety of lipid molecular species were detected in mouse plasma, and isomers which often co-elute in reverse phase separation were identified accurately and quantified individually. To the best of our knowledge, this is the first report describing the chromatographic separation of lipids based on both fatty acyl moieties and polar head groups, using a single ODS column. Our results demonstrate that SFC/MS is a powerful tool for the simultaneous analysis of diverse lipid molecular species.

Development of a lipid profiling system using reverse-phase liquid chromatography coupled to high-resolution mass spectrometry with rapid polarity switching and an automated lipid identification software.

Lipidomics requires accurate lipid profiling, which until recently has been challenging at best. In the present study, we developed a practical workflow for high-throughput and exhaustive lipid profiling by combining reverse-phase liquid chromatography coupled to quadrupole orbitrap Fourier transform mass spectrometry, with an automated lipid identification software. This validated method enables highly sensitive and simultaneous analysis of lipids with varying polarities such as glycerophospholipids and sphingophospholipids, by switching the acquisition polarities in mass spectrometry. In addition, it facilitates data-dependent MS(2) analysis targeting the lipid molecular species without any influence from other ions by setting the inclusion list, the target m/z list for the product ion scanning. The m/z values of the target lipid molecular species, stored in the database of Lipid Search software, are added to the inclusion list. Moreover, optimizing the identification conditions of the software for the LC/MS system enables high-throughput and accurate identification of lipid molecular species existing in biological samples. Specifically, LC separation is essential for accurate identification of lipid molecular species that possess some fatty acid chains, because it can be difficult to determine fatty acid chain composition of detected molecular species especially in triacylglycerol compounds in direct infusion mass spectrometry. This method has high reproducibility and can be used for structural analysis even for low-abundance compounds. Using this method, over 400 lipid compounds targeted in this research were detected and identified from a sample of mouse plasma. This result indicates that the LC/MS method in the present study enables efficient lipid profiling.

Current metabolomics: practical applications.

The field of metabolomics continues to grow rapidly over the last decade and has been proven to be a powerful technology in predicting and explaining complex phenotypes in diverse biological systems. Metabolomics complements other omics, such as transcriptomics and proteomics and since it is a 'downstream' result of gene expression, changes in the metabolome is considered to best reflect the activities of the cell at a functional level. Thus far, metabolomics might be the sole technology capable of detecting complex, biologically essential changes. As one of the omics technology, metabolomics has exciting applications in varied fields, including medical science, synthetic biology, medicine, and predictive modeling of plant, animal and microbial systems. In addition, integrated applications with genomics, transcriptomics, and proteomics provide greater understanding of global system biology. In this review, we discuss recent applications of metabolomics in microbiology, plant, animal, food, and medical science.

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.

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.

High-throughput and sensitive analysis of 3-monochloropropane-1,2-diol fatty acid esters in edible oils by supercritical fluid chromatography/tandem mass spectrometry.

We have established a high-throughput and sensitive analytical method based on supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry (QqQ MS) for 3-monochloropropane-1,2-diol (3-MCPD) fatty acid esters in edible oils. All analytes were successfully separated within 9 min without sample purification. The system was precise and sensitive, with a limit of detection less than 0.063 mg/kg. The recovery rate of 3-MCPD fatty acid esters spiked into oil samples was in the range of 62.68-115.23%. Furthermore, several edible oils were tested for analyzing 3-MCPD fatty acid ester profiles. This is the first report on the analysis of 3-MCPD fatty acid esters by SFC/QqQ MS. The developed method will be a powerful tool for investigating 3-MCPD fatty acid esters in edible oils.

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.

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.

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.

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.

Metabolic profiling of ß-cryptoxanthin and its fatty acid esters by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry.

In this study, a high-throughput and high-sensitivity profiling system for ß-cryptoxanthin (ßCX) and ß-cryptoxanthin fatty acid ester (ßCXFA) was constructed by supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry (QqQMS). ßCX and nine ßCXFAs were successfully separated within 20 min using a column packed with octadecylsilyl-bonded silica particles. The limit of detection was 540 fmol for the free form and 32-130 fmol for the esterified forms. These results demonstrate that both the throughput and the sensitivity of this SFC-QqQMS system are considerably higher than those of conventional methods. When this system was applied for the analysis of Citrus unshiu, ßCX and five ßCXFAs were directly detected with much simpler sample pre-preparation. The analysis of other citrus fruits indicated that the ßCXFA profiles varied with their breed variety. Furthermore, gas chromatography-mass spectrometry was used to analyze total fatty acid profiles in C. unshiu, and the results revealed that the profiles of fatty acids located in ßCXFA were distinct. This is the first report on the analysis of ßCX and its fatty acid derivatives by SFC-QqQMS. The profiling system developed in this study will be a powerful tool for investigating xanthophyll fatty acid esters.