Lipidomic differentiation between human kidney tumors and surrounding normal tissues using HILIC-HPLC/ESI-MS and multivariate data analysis.

The goal of this work is the comparison of differences in the lipidomic compositions of human cell lines derived from normal and cancerous breast tissues, and tumor vs. normal tissues obtained after the surgery of breast cancer patients. Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC/ESI-MS) using the single internal standard approach and response factors is used for the determination of relative abundances of individual lipid species from five lipid classes in total lipid extracts of cell lines and tissues. The supplementary information on the fatty acyl composition is obtained by gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters. Multivariate data analysis (MDA) methods, such as nonsupervised principal component analysis (PCA), hierarchical clustering analysis (HCA) and supervised orthogonal partial least-squares discriminant analysis (OPLS-DA), are used for the visualization of differences between normal and tumor samples and the correlation of similarity between cell lines and tissues either for tumor or normal samples. MDA methods are used for differentiation of sample groups and also for identification of the most up- and downregulated lipids in tumor samples in comparison to normal samples. Observed changes are subsequently generalized and correlated with data from tumor and normal tissues of breast cancer patients. In total, 123 lipid species are identified based on their retention behavior in HILIC and observed ions in ESI mass spectra, and relative abundances are determined. MDA methods are applied for a clear differentiation between tumor and normal samples both for cell lines and tissues. The most upregulated lipids are phospholipids (PL) with a low degree of unsaturation (e.g., 32:1 and 34:1) and also some highly polyunsaturated PL (e.g., 40:6), while the most downregulated lipids are PL containing polyunsaturated fatty acyls (e.g., 20:4), plasmalogens and ether lipids. Copyright © 2016 John Wiley & Sons, Ltd.

Negative-ion hydrophilic liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) method has been optimized for the quantitative analysis of ganglioside (GM3) and other polar lipid classes, such as sulfohexosylceramides (SulfoHexCer), sulfodihexosylceramides (SulfoHex2Cer), phosphatidylglycerols (PG), phosphatidylinositols (PI), lysophosphatidylinositols (LPI), and phosphatidylserines (PS). The method is fully validated for the quantitation of thestudied lipids in kidney normal and tumor tissues of renal cell carcinoma (RCC) patients based on the lipid class separation and the coelution of lipid class internal standard with the species from the same lipid class. The raw data are semi-automatically processed using our software LipidQuant and statistically evaluated using multivariate data analysis (MDA) methods, which allows the complete differentiation of both groups with 100% specificity and sensitivity. In total, 21 GM3, 28 SulfoHexCer, 26 SulfoHex2Cer, 10 PG, 19 PI, 4 LPI, and 7 PS are determined in the aqueous phase of lipidomic extracts from kidney tumor tissue samples and surrounding normal tissue samples of 20 RCC patients. S-plots allow the identification of most upregulated (PI 40:5, PI 40:4, GM3 34:1, and GM3 42:2) and most downregulated (PI 32:0, PI 34:0, PS 36:4, and LPI 16:0) lipids, which are primarily responsible for the differentiation of tumor and normal groups. Another confirmation of most dysregulated lipids is performed by the calculation of fold changes together with T and p values to highlight their statistical significance. The comparison of HILIC/ESI-MS data and matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) data confirms that lipid dysregulation patterns are similar for both methods. Graphical abstract ᅟ.

Lipidomics is a rapidly expanding research field in which multiple techniques are utilized to quantitate the hundreds of chemically distinct lipids in cells and determine the molecular mechanisms through which they facilitate cellular function. Recent developments in electrospray ionization mass spectrometry (ESI/MS) have made possible, for the first time, the precise identification and quantification of alterations in a cell's lipidome after cellular perturbations. This review provides an overview of the essential role of ESI/MS in lipidomics, presents a broad strategy applicable for the generation of lipidomes directly from cellular extracts of biological samples by ESI/MS, and summarizes salient examples of strategies utilized to conquer the lipidome in physiologic signaling as well as pathophysiologically relevant disease states. Because of its unparalleled sensitivity, specificity, and efficiency, ESI/MS has provided a critical bridge to generate highly accurate data that fingerprint cellular lipidomes to facilitate insight into the functional role of subcellular membrane compartments and microdomains in mammalian cells. We believe that ESI/MS-facilitated lipidomics has now opened a critical door that will greatly increase our understanding of human disease.

A shotgun lipidomics approach that allowed the analysis of eight lipid classes directly from crude extracts of the soil bacterium Sinorhizobium meliloti is presented. New MS-MS transitions are reported for the analysis of monomethylphosphatidylethanolamines, dimethylphosphatidylethanolamines, and three bacterial non-phosphorus-containing lipid classes [sulfoquinovosyldiacylglycerols, ornithines, and diacylglyceryl-(N,N,N-trimethyl)-homoserines]. Unique MS-MS transitions allowed the analysis of isomeric species from various lipid classes without chromatography. Analyses required small sample amounts and minimal preparation; thus, this methodology has excellent potential to be used as a screening tool for the analysis of large numbers of samples in functional genomics studies. FA distributions within lipid classes of S. meliloti are described for the first time, and the relative positions of fatty acyl substituents (sn-1, sn-2) in phospholipids are presented. FA distributions in diacylglyceryl-(N,N,N-trimethyl)-homoserines were identical to those of phospholipids, indicating a common biosynthetic origin for these lipids. The method was applied to the analysis of mutants deficient in the PhoB regulator protein. Increased lipid cyclopropanation was observed in PhoB-deficient mutants under P(i) starvation.

Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile contaminants and naturally occurring toxins in foods. However, polar compounds, such as hydrophilic polypeptides and quaternary ammonium salts, are often not satisfactorily separated by RPLC and present a challenge for analytical scientists. In this study, hydrophilic interaction liquid chromatography (HILIC), on an amide-based stationary phase in combination with electrospray ionization (ESI) tandem mass spectrometry (MS-MS), is successfully employed to simultaneously separate polar mushroom toxins, including amanitins and phallotoxins, which are cyclic oligopeptides and muscarine, a quaternary ammonium compound, in mushrooms. The sensitivity of different ionization modes is studied, and the positive ionization mode is found to provide a more sensitive and effective tool for the unambiguous identification of the concerned polar toxins because of their characteristic fragmentation patterns. The properties of the mobile phase are also found to have significant impacts on the separation. At a high acetonitrile (ACN) concentration, hydrophilic interaction dominates, and all analytes under study demonstrate a much higher affinity with the stationary phase. The addition of methanol (MeOH) as a modifier could further enhance the HILIC separation for amanitins, phallotoxins, and muscarine. Valley-to-valley separation is achieved upon the optimatizatiqn of the mobile phase (comprising of ACN, MeOH, and ammonium formate buffer at pH approximately 3.5) and the solvent gradient. HILIC coupled with ESI-MS-MS is demonstrated to be a novel technique for the simultaneous separation and confirmatory analysis of the concerned polar toxins by providing an environment of solubility and retention that could not be achieved through the use of RPLC.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

How it looks in America

In this paper, we propose tools for studying biophysical characteristics of a person by processing polygraph charts. We present the results produced by two methods of multivariate data analysis: the principal component analysis (PCA) and projection on latent structures (PLS). Data of biophysical state of a person during the polygraph testing - galvanic skin response (GSP), photoplethysmogram, blood pressure, and other biophysical characteristics – are processed and analyzed.It is shown that load diagrams are the preferable method to identify the relationship between the biophysical characteristics of a person. The principal component analysis provides an opportunity to assess minor changes in biophysical characteristics of a person and to take into account situational significant identifiers. They can attest to the fact that a possibly significant physical-chemical process runs in a human body.It is demonstrated that the projection on latent structures should be used to develop regression models that can constantly be used in polygraph testing.It is concluded that proposed methods of multivariate data analysis can help in an investigation of biophysical characteristics of a person using polygraph data. Also, the proposed methods are able to detect possible malfunctions in the responsible physical-chemical processes. The obtained results enable solving practical problems of information security matters, for example, the identification of an insider.DOI 10.14258/izvasu(2018)1-05

Quantitative determination of metformin, saxagliptin and 5-hydroxy saxagliptin simultaneously by hydrophilic interaction liquid chromatography - electrospray ionization mass spectrometry and its application to a bioequivalence study with a single-pill combination in human

Lipidomic profile in three species of dinoflagellates (Amphidinium carterae, Cystodinium sp., and Peridinium aciculiferum) containing very long chain polyunsaturated fatty acids

Multivariate data analysis for Raman imaging of a model pharmaceutical tablet

In the context of this thesis, an advanced nano-flow liquid chromatography (nLC) – nanoelectrospray (NSI) - mass spectrometry (MS) system was developed for a global (i.e. not lipid class- or species-restricted) lipidomics approach. In order to grant broad range analyses of different lipid classes and species, the loading- and sample preparation procedures were particularly optimized. A direct sample injection with the resuspension mixture of BuOH:IPA:H2O 8:23:69 + 5 mM phosphoric acid (8Bu+) thereby proved to be suitable for global lipidomics experiments. In this regard, the addition of phosphoric acid to the samples strongly enhanced the general detectability of lipids with terminal phosphate groups, which were found to be hardly analyzable under standard reversed-phase LC conditions. The robustness of the nLC-MS method was then evaluated based on repetitive analyses of a yeast extract that was spiked with standards. Over a measuring period of two days, high retention time stabilities were achieved for all analyzed lipids. The bulk of phospholipids could be stably detected with relative standard deviations below 10 %, while the MS-response was less stable for late eluting very hydrophobic lipids. Using normalization approaches, these deviations could nevertheless be reduced with structurally- or retention time-related standards. A direct sensitivity comparison to a corresponding narrow-bore HPLC platform revealed an increase of 2-3 orders of magnitude for the MS response by application of the nLC system that conformed with an increase of up to two orders of magnitude for the linear dynamic range. Sub-femtomol traces of individual lipids (< nM concentrations) could thus be detected with the nLC-MS platform in both positive and negative mode analyses. In order to permit a thorough comparison of the lipidome coverage, a semi-autonomous workflow was developed that allowed an accurate identification of individual (molecular) lipid species. The presumably simple yeast (Saccharomyces cerevisiae) lipidome was then selected as a model for the in-deep evaluation of the lipidome coverage. The direct comparison of the yeast phospholipidome to the corresponding narrow-bore HPLC revealed a more than three-fold increase in lipid identifications on molecular species level when identical sample concentrations were comparatively analyzed with both LC systems. All in all, 935 lipid species, with each comprising multiple molecular species isomers, were identified across 30 analyzed lipid classes from 4 lipid categories in yeast. Even though this thesis uncovered more endogenous yeast lipids than any reference study, a detailed analyses of isomeric signals further indicates that also novel lipid classes, such as headgroup-methylated PS variants, and oxidized lipids can be found in natural yeast lipid extracts. Overall, this thesis shows that the insights into the complexity of a lipidome can be strongly deepened by using an nLC-MS system for a global lipidomics experiments.

Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.

Aging changes the lipidome and mitochondrial function in a sex-dependent manner, yet their associations remain poorly understood. Twenty-four younger (7M/17F) and forty-three older (21M/22F) adults underwent blood draws and skeletal muscle biopsies for this cross-sectional investigation. Plasma lipidomic profiling was performed via liquid chromatography-tandem mass spectrometry, while peak mitochondrial O2 utilization (OXPHOS) and hydrogen peroxide (H2O2) emission were assessed using high-resolution respirometry. Plasma lipidomic analysis annotated 535 lipid species across 28 different lipid classes. Lipid-age associations were identified in four lipid classes for both sexes with twelve lipid classes demonstrating sex-specific associations, including triglycerides (TG), carnitines (CAR), and fatty acids (FA). For lipid-OXPHOS interactions, the primary lipid class and species associated with higher OXPHOS exclusively in males were ceramides (CER) and dimethyl cholesterol esters (dimethyl-CE), while TG were the primary lipid species associated with impaired OXPHOS in females. For lipid-H2O2 interactions, the primary lipid class and species associated with higher H2O2 were methyl desmosteryl esters (methyl-DE), methyl cholesterol esters (methyl-CE), FA and TG in males whereas females exhibited 10 (CE, SM, LPC, dihexosylceramides (Hex2Cer), LPE, PI, HexCer, LPI, CAR, and hexosyl-N-acetylneuraminyl-ceramides (Hex2NeuAcCer)) lipid classes associated exclusively with H2O2 emission. These findings establish novel age- and sex-specific relationships between age-related changes in plasma lipids and skeletal muscle mitochondrial function, revealing distinct lipid signatures for respiration and H2O2 emission in males and females.