Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques.

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 µm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

Determination of nonpolar and polar lipid classes in human plasma, erythrocytes and plasma lipoprotein fractions using ultrahigh-performance liquid chromatography-mass spectrometry.

A novel normal-phase (NP) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method is developed for a separation and quantitation of nonpolar lipid classes occurring in human plasma, erythrocytes and plasma lipoprotein fractions. The baseline class separation of cholesteryl esters (CE), cholesterol, triacylglycerols (TG), regioisomers of 1,2- and 1,3-diacylglycerols (DG) and 1-monoacylglycerols (1-MG) is achieved using an optimized hexane - 2-propanol-acetonitrile mobile phase within 18min for all nonpolar lipid classes or only 9min excluding monoacylglycerols not detected in studied samples. The determination of individual nonpolar lipid classes is performed by the response factor approach and the use of dioleoyl ethylene glycol as a single internal standard. Polar lipid classes, such as phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidylcholines (PC), sphingomyelins (SM) and lysophosphatidylcholines (LPC), are separated by hydrophilic interaction liquid chromatography (HILIC) using 5mmol/L aqueous ammonium acetate-methanol-acetonitrile gradient within 13minutes. The quantitation of polar lipid classes is done by a similar approach as for nonpolar lipid classes, but a different internal standard (sphingosyl PE d17:1/12:0) is used. The complementary information on fatty acyl profiles after the transesterification of the total lipid extract is obtained by gas chromatography with flame ionization detection (GC/FID). The applicability of developed methodology for fast and comprehensive characterization of blood lipidome is illustrated on samples of human plasma, erythrocytes, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) fractions.

Identification of new phase II metabolites of xanthohumol in rat in vivo biotransformation of hop extracts using high-performance liquid chromatography electrospray ionization tandem mass spectrometry.

Polyphenolic compounds occurring in hop extracts and their phases I and II metabolites formed during in vivo rat biotransformation have been analyzed using HPLC/MS/MS with electrospray ionization (ESI). Two main groups of polyphenolics are present in the hops, i.e., xanthohumol related compounds and so called alpha- and beta-bitter acids (humulones and lupulones). In our study, hybrid quadrupole-time-of-flight (QqTOF) analyzer is used for the identification of both natural phenolics and their metabolites due to the possibility of accurate mass measurements in full scan and tandem mass spectra supported by MS(n) data obtained with the ion trap analyzer. Both ESI polarity modes are used for the determination of molecular weights based on [M+H](+) and [M-H](-) ions in the full scan spectra and the structural information in subsequent tandem mass spectra. The emphasis is given on the elemental composition determination of individual metabolites based on accurate masses typically better than 5ppm even with the external calibration. Advanced software tools are used for the metabolite identification using the comparison of the blank chromatogram with the real incubation sample together with the software prediction and detection of possible metabolites. Chromatograms of rat incubations are also compared with chromatograms of pure rat feed, rat feed enriched with hop extracts and the placebo experiment. More than ten compounds originating from the hops are identified in rat feces, two of them belong to phase I metabolites and five compounds are phase II metabolites.

Orthogonality of silver-ion and non-aqueous reversed-phase HPLC/MS in the analysis of complex natural mixtures of triacylglycerols.

The goal of this work is the study of possibilities of two basic separation modes used in the analysis of complex triacylglycerol (TG) samples of plant oils and animal fats, i.e. non-aqueous reversed-phase (NARP) and silver-ion HPLC coupled with atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The orthogonality of both separation modes is tested for complex TG mixtures containing fatty acids (FAs) with different acyl chain lengths, different number, positions and geometry of double bonds (DBs) and different regioisomeric positions of FAs on the glycerol skeleton. The retention in NARP mode is governed by the equivalent carbon number, while the retention in silver-ion chromatography increases with the increasing number of DBs with a clear differentiation between cis- and trans-FAs. Moreover, silver-ion mode enables at least the partial resolution of regioisomeric TG mixtures including cis-/trans-regioisomers, as illustrated on two examples of randomization mixtures. Off-line 2D coupling of both complementary modes (NARP in the first dimension and silver-ion in the second dimension) yields the superior chromatographic selectivity resulting in the highest number of identified TGs ever reported for studied samples. Off-line 2D chromatograms are processed with the home-made software providing various ways of data visualization.

Statistical evaluation of triacylglycerol composition in plant oils based on high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry data.

The statistical evaluation of triacylglycerol profiles in plant oils based on high-performance liquid chromatography mass spectrometry (HPLC/MS) analysis enables the differentiation of various plant oils on the basis of the multidimensional data matrix. A data set of 93 oil samples from 60 varieties of plants composed from 355 triacylglycerols is evaluated using the principal component analysis. Analyzed samples are resolved in the principal component analysis plot, and similarities among some types of plant oils are visualized by the formation of clusters. The authentication of plant oils is tested with model samples of olive oil adulterated with sunflower oil at different concentration levels. Our HPLC/MS method using the statistical multivariate data analysis of a large data matrix enables a clear identification of adulterated olive oils already from 1% of added sunflower oil as an adulterant.

Regioisomeric characterization of triacylglycerols using silver-ion HPLC/MS and randomization synthesis of standards.

Silver-ion normal-phase high-performance liquid chromatography (HPLC) provides a superior separation selectivity for lipids differing in the number and position of double bonds in fatty acid chains including the resolution of triacylglycerol (TG) regioisomers under optimized conditions. Our silver-ion HPLC method is based on the coupling of three columns in the total length of 75 cm and a new mobile phase gradient consisting of hexane-acetonitrile-2-propanol which provides better resolution and also reproducibility in comparison to previously used mobile phases. In our work, the chemical interesterification (randomization) of single-acid TG standards is used for the generation of regioisomeric series of TGs, because it provides a random distribution of fatty acids in TGs at well-defined concentration ratios. The baseline separation of regioisomeric TG pairs containing up to three double bonds and the partial separation of TG regioisomers with four to seven double bonds are reported for the first time. Our silver-ion high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is applied for the regioisomeric characterization of complex samples of plant oils and animal fat, where the results clearly demonstrate different preference of sn-2 occupation in plants (mainly unsaturated fatty acids) versus animal fat (mainly saturated fatty acids).

Triacylglycerols profiling in plant oils important in food industry, dietetics and cosmetics using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Optimized non-aqueous reversed-phase high-performance liquid chromatography method using acetonitrile-2-propanol gradient elution and the column coupling in the total length of 45 cm has been applied for the high resolution separation of plant oils important in food industry, dietetics and cosmetics. Positive-ion atmospheric pressure chemical ionization mass spectrometry is used for the unambiguous identification and also the reliable quantitation with the response factors approach. Based on the precise determination of individual triacyglycerol concentrations, the calculation of average parameters important in the nutrition is performed, i.e. average carbon number, average double bond number, relative concentrations of essential, saturated, monounsaturated and polyunsaturated fatty acids. Results are reported in the form of both chromatographic fingerprints and tables containing relative concentrations for all triacylglycerols and fatty acids in individual samples. In total, 264 triacylglycerols consisting of 28 fatty acids with the alkyl chain length from 6 to 26 carbon atoms and 0 to 4 double bonds have been identified in 26 industrial important plant oils.

Characterization of Ni(II) complexes of Schiff bases of amino acids and (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide using ion trap and QqTOF electrospray ionization tandem mass spectrometry.

This work demonstrates the application of electrospray ionization mass spectrometry (ESI-MS) using two different mass analyzers, ion trap and hybrid quadrupole time-of-flight (QqTOF) mass analyzer, for the structural characterization of Ni(II) complexes of Schiff bases of (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide with different amino acids. ESI enables the determination of molecular weight on the basis of rather simple positive-ion ESI mass spectra containing only protonated molecules and adducts with sodium or potassium ions. Fragmentation patterns are characterized by tandem mass spectrometric experiments, where both tandem mass analyzers provide complementary information. QqTOF data are used for the determination of elemental composition of individual ions due to mass accuracies always better than 3 ppm with the external calibration, while multistage tandem mass spectra obtained by the ion trap are suitable for studying the fragmentation paths. The novel aspect of our approach is the combination of mass accuracies and relative abundances of all isotopic peaks in isotopic clusters providing more powerful data for the structural characterization of organometallic compounds containing polyisotopic elements. The benefit of relative and absolute mean mass accuracies is demonstrated on the example of studied Ni(II) complexes.

Quantitation of triacylglycerols from plant oils using charged aerosol detection with gradient compensation.

Quantitative analysis of triacylglycerols (TGs) in plant oils and animal fats by normalization of peak areas can lead to erroneous results due to the large response differences with common HPLC detectors between the various TGs. The charged aerosol detector (CAD), that generates an almost universal response for non-volatile compounds, was combined with non-aqueous reversed-phase HPLC (NARP-HPLC) to develop a simple quantitative method, without need for RFs, for the analysis of complex natural TG mixtures from plant oils. Two 25 cm Hypersil ODS columns, connected in series, and a mobile phase gradient composed of acetonitrile, 2-propanol and hexane were used. Mobile phase compensation was applied, by mixing of the column effluent with the inversed gradient delivered by a second HPLC pump, for the suppression of the response dependency of the analytes on the mobile phase composition. Calibration curves of 16 saturated (from C7:0 to C22:0) and 3 unsaturated (C18:1, C18:2, C18:3) single-acid TG standards were measured and their RFs were compared with a previously described method using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The variation in response of the most common TGs (containing fatty acid chains from 12 to 19 carbons) could be reduced to less than 5% making the combination of NARP-HPLC with CAD and mobile phase compensation an adequate tool for fast quantitative analysis of TGs in common plant oils.

High-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry and gas chromatography-flame ionization detection characterization of Delta5-polyenoic fatty acids in triacylglycerols from conifer seed oils.

Edible conifer seeds can serve as a source of triacylglycerols (TGs) with unusual Delta5 unsaturated polymethylene interrupted fatty acids (UPIFAs), such as cis-5,9-octadecadienoic (taxoleic), cis-5,9,12-octadecatrienoic (pinolenic), cis-5,11-eicosadienoic (keteleeronic) and cis-5,11,14-eicosatrienoic acids (sciadonic). Conifer seed oils from European Larch (Larix decidua), Norway Spruce (Picea abies) and European Silver Fir (Abies alba) have been analyzed by non-aqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with atmospheric pressure chemical ionisation (APCI)-MS detection. The influence of different positions of double bonds in Delta5-UPIFAs on the retention and fragmentation behavior is described and used for the successful identification of TGs in each oil. TGs containing Delta5-UPIFAs have a higher retention in comparison with common TGs found in plant oils with single methylene interrupted Delta6(9)-FAs and also significantly changed relative abundances of fragment ions in APCI mass spectra. Results obtained from HPLC/MS analyses are supported by validated GC/FID analyses of fatty acid methyl esters after the transesterification. The total content of Delta5-UPIFAs is about 32% for European Larch, 27% for Norway Spruce and 20% for European Silver Fir. In total, 20 FAs with acyl chain lengths from 16 to 24 carbon atoms and from 0 to 3 double bonds have been identified in 64 triacylglycerols from 3 conifer seed oils.

Quantitation of triacylglycerols in plant oils using HPLC with APCI-MS, evaporative light-scattering, and UV detection.

The main constituents of plant oils are complex mixtures of TGs differing in acyl chain lengths, number and positions of double bonds, and regioisomerism. A non-aqueous reversed-phase HPLC method with acetonitrile-2-propanol gradient and 30 + 15 cm NovaPak C18 columns makes possible an unambiguous identification of the highest number of TGs ever reported for these oils, based on positive-ion APCI mass spectra. A new approach to TG quantitation is based on the use of response factors with three typical detection techniques for that purpose (APCI-MS, evaporative light-scattering detection, and UV at 205 nm). Response factors of 23 single-acid TGs (saturated TGs from C7 to C22, 7 unsaturated TGs), 4 mixed-acid TGs, diolein and monoolein are calculated from their calibration curves and related to OOO. Due to differences between saturated and unsaturated acyl chains, the use of response factors significantly improves the quantitation of TGs. 133 TGs containing 22 fatty acids with 8-25 carbon atoms and 0-3 double bonds are identified and quantified in 9 plant oils (walnut, hazelnut, cashew nut, almond, poppy seed, yellow melon, mango, fig, date) using HPLC/APCI-MS with a response factor approach. Average parameters and relative fatty acid concentrations are calculated with both HPLC/APCI-MS and GC/ FID.

Characterization of triacylglycerol and diacylglycerol composition of plant oils using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Triacylglycerols (TGs) and diacylglycerols (DGs) in 16 plant oil samples (hazelnut, pistachio, poppy-seed, almond, palm, Brazil-nut, rapeseed, macadamia, soyabean, sunflower, linseed, Dracocephalum moldavica, evening primrose, corn, amaranth, Silybum arianum) were analyzed by HPLC-MS with atmospheric pressure chemical ionization (APCI) and UV detection at 205 nm on two Nova-Pak C18 chromatographic columns connected in series. A single chromatographic column and non-aqueous ethanol-acetonitrile gradient system was used as a compromise between the analysis time and the resolution for the characterization of TG composition of five plant oils. APCI mass spectra were applied for the identification of all TGs and other acylglycerols. The isobaric positional isomers can be distinguished on the basis of different relative abundances of the fragment ions formed by preferred losses of the fatty acid from sn-1(3) positions compared to the sn-2 position. Excellent chromatographic resolution and broad retention window together with APCI mass spectra enabled positive identification of TGs containing fatty acids with odd numbers of carbon atoms such as margaric (C17:0) and heptadecanoic (C17:1) acids. The general fragmentation patterns of TGs in both APCI and electrospray ionization mass spectra were proposed on the basis of MSn spectra measured with an ion trap analyzer. The relative concentrations of particular TGs in the analyzed plant oils were estimated on the basis of relative peak areas measured with UV detection at 205 nm.