Non-aqueous reversed-phase-HPLC
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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.
- MeSH
- hmotnostní spektrometrie metody MeSH
- oleje rostlin chemie MeSH
- stříbro chemie MeSH
- triglyceridy analýza MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- oleje rostlin MeSH
- stříbro MeSH
- triglyceridy MeSH
Enantiomers of triacylglycerols (TAGs) containing any combination of very long chain fatty acids (VLCFAs) and/or very long chain polyunsaturated fatty acids (VLCPUFAs) with diolein, dilinolein and didocosahexaenoin were synthesized. Gradient non-aqueous reversed-phase high-performance liquid chromatography/high resolution atmospheric pressure chemical ionization-tandem mass spectrometry (NARP-HPLC/HRMS2-APCI) and chiral liquid chromatography were used for the separation and identification of molecular species of these TAGs. Further, NARP-LC and chiral LC were used to separate natural mixtures of TAGs obtained from four natural sources, i.e. ximenia oil (Ximenia americana), green alga (Botryococcus braunii), breweŕs yeast (Saccharomyces pastorianus) and a dinoflagellate (Amphidinium carterae). The ratio of regioisomers and enantiomers in individual samples was determined and a hypothesis has been confirmed on the biosynthetic pathway of natural TAGs, which is based on the preferential representation of VLCFAs and VLCPUFAs in the sn-1 position of the glycerol backbone.
- Klíčová slova
- Enantiomeric separation, Non-aqueous reversed-phase-HPLC, Triacylglycerols, Very long chain fatty acids, Very long-chain polyunsaturated fatty acids,
- MeSH
- atmosférický tlak MeSH
- Chlorophyta chemie metabolismus MeSH
- chromatografie s reverzní fází MeSH
- Dinoflagellata chemie metabolismus MeSH
- kyseliny mastné neesterifikované chemie MeSH
- nenasycené mastné kyseliny chemie MeSH
- Saccharomyces chemie metabolismus MeSH
- stereoizomerie MeSH
- tandemová hmotnostní spektrometrie MeSH
- triglyceridy analýza chemie izolace a purifikace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kyseliny mastné neesterifikované MeSH
- nenasycené mastné kyseliny MeSH
- triglyceridy MeSH
Triacylglycerols (TAGs) containing cyclofatty acids (cycloFAs) from two oilseeds of Sterculia foetida and Hydnocarpus wightiana were analysed using both reversed-phase (RP18) and chiral phase columns. TAGs were identified using high-resolution electrospray ionization mass spectrometry in the positive ion mode. Fifty-five molecular species of TAGs have been identified in sterculic oil, 27 of which contained at least one cyclopropenyl-FA (e.g., malvalic or sterculic acids). The structures of regioisomers and enantiomers were determined for five major TAGs with cyclopropenyl-FAs. One hundred thirty-six TAGs were identified in chaulmoogra oil, 71 of which contained at least one cyclopentenyl-FA (e.g., gorlic, chaulmoogric, and hydnocarpic acids, etc.). Furthermore, in three molecular species, regioisomers and enantiomers were identified using HPLC on a chiral phase column. Eight molecular species of TAGs were prepared through organic synthesis to facilitate the identification of enantiomers. Retention times of fatty acid-containing triacylglycerols with one ring and one double bond are very similar to triacylglycerols with a dienoic fatty acid, but elution times are shorter. For example, dimalvaloylpalmitate elutes earlier than dilinoleylpalmitate. The order of elution of TAGs on the chiral column differs. In TAGs with 2 degrees of unsaturation (ring and double bond, e.g. PStP-StPP-PPSt), the order of elution is symmetric-asymmetric-asymmetric TAGs. TAGs with 4 degrees of unsaturation (one ring and three double bonds or two rings and two double bonds) present a different pattern. When TAGs contain two rings and two double bonds, the order of elution TAGs is asymmetric-symmetric-asymmetric (StStP-StPSt-PStSt); when TAGs contain a ring and 3 double bonds, the elution order is symmetric-asymmetric-asymmetric TAGs (OStO-StOO-OOSt). In species with a higher degree of unsaturation (e.g., 5), the elution order of the TAGs is asymmetric-asymmetric-symmetric (e.g. CCO-OCC-COC).
- Klíčová slova
- Chaulmoogra oil, Cyclic fatty acids, Enantiomeric separation, Non-aqueous reversed-phase-HPLC, Sterculic seeds, Triacylglycerols,
- MeSH
- chromatografie s reverzní fází MeSH
- cyklopropany analýza MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- kyseliny mastné mononenasycené analýza MeSH
- mastné kyseliny analýza chemie izolace a purifikace MeSH
- oleje rostlin chemie MeSH
- Sterculia chemie MeSH
- stereoizomerie MeSH
- triglyceridy chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chaulmoogra oil MeSH Prohlížeč
- cyklopropany MeSH
- kyseliny mastné mononenasycené MeSH
- malvalic acid MeSH Prohlížeč
- mastné kyseliny MeSH
- oleje rostlin MeSH
- sterculic acid MeSH Prohlížeč
- triglyceridy MeSH
Blackcurrant (Ribes nigrum) seed oil is rich in alpha- and gamma-linolenic acids, the latter in particular being of potential use in medicine. The enzymatic hydrolysis of the oil was carried out in supercritical carbon dioxide using lipase Lipozyme as catalyst and changes in the composition of acylglycerols were recorded. Mono-, di-, and triacylglycerols and free fatty acids were separated by non-aqueous high-performance liquid chromatography in reversed phase mode and detected by UV diode array and 1H NMR detectors. Lipozyme was found to exert low specificity to individual fatty acids in the hydrolysed oil.
- MeSH
- diglyceridy analýza izolace a purifikace MeSH
- hydrolýza MeSH
- kinetika MeSH
- kyseliny linolenové chemie metabolismus MeSH
- lipolýza * MeSH
- mastné kyseliny analýza chemie MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- oleje rostlin chemie metabolismus MeSH
- oxid uhličitý chemie MeSH
- referenční standardy MeSH
- superkritická fluidní chromatografie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1,2-dilinoleoylglycerol MeSH Prohlížeč
- blackcurrant seed oil MeSH Prohlížeč
- diglyceridy MeSH
- kyseliny linolenové MeSH
- mastné kyseliny MeSH
- oleje rostlin MeSH
- oxid uhličitý MeSH
Lipid-like compounds containing a dimethylarsinoyl group, i.e. Me2As(O)-, have been identified by liquid chromatography/inductively coupled plasma mass spectrometry (LC/ICP-MS) and non-aqueous reversed-phase high-performance liquid chromatography (positive and/or negative high-resolution tandem electrospray ionization mass spectrometry (NARP-HPLC/HR-ESI+(-)-MS/MS) from three strains of green algae of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta). The algae were cultivated in a medium containing 10 g arsenic/L, i.e. 133.5 mmol/L of Na2HAsO4.7H2O. After extraction by methyl-tert-butyl ether (MTBE), total lipids were analyzed by ICP-MS or ESI-MS without any further separation or fractionation. A total of 39 molecular species of arsenic triacylglycerols (AsTAG), 15 arsenic phosphatidylcholines (AsPC), 8 arsenic phosphatidylethanolamines (AsPE), 6 arsenic phosphatidylinositols (AsPI), 2 arsenic phosphatidylglycerols (AsPG) and 5 unknown lipids (probably ceramides) were identified. The structures of all molecular species were confirmed by tandem MS. Dry matter of the individual strains contained different amounts of total arsenolipids, i.e. C. elongata CCALA 427 (0.32 mg/g), C. onubensis (1.48 mg/g), C. elongata S3 (2.13 mg/g). On the other hand, there were only slight differences between strains in the relative abundances of individual molecular species. Possible biosynthesis of long-chain lipids with the end group Me2As(O) has also been suggested.
- Klíčová slova
- Arsenolipids, Coccomyxa elongata, Coccomyxa onubensis, Dimethylarsinoyl long chains, Green alga, Inductively coupled plasma mass spectrometry (ICP-MS), Non-aqueous reversed-phase high-performance liquid chromatography (NARP-HPLC), Positive/negative high-resolution tandem electrospray ionization mass spectrometry (HR-ESI-MS/MS), Trebouxiophyceae,
- MeSH
- arsenikové přípravky chemie izolace a purifikace metabolismus MeSH
- Chlorella chemie metabolismus MeSH
- lipidy chemie izolace a purifikace MeSH
- molekulární struktura MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- arsenikové přípravky MeSH
- lipidy MeSH
Gradient elution reversed-phase high-performance liquid chromatography (RP-HPLC) was used for the determination of compounds occurring during the production of biodiesel from rapeseed oil. Individual triacylglycerols (TGs), diacylglycerols, monoacylglycerols and methyl esters of oleic, linoleic and linolenic acids and free fatty acids were separated in 25 min using a combined linear gradient with aqueous-organic and non-aqueous mobile phase steps: 70% acetonitrile+30% water in 0 min, 100% acetonitrile in 10 min, 50% acetonitrile+50% 2-propanol-hexane (5:4, v/v) in 20 min and 5 min final hold-up. Another method with a non-aqueous linear mobile phase gradient [from 100% methanol to 50% methanol+50% 2-propanol-hexane (5:4, v/v) in 15 min] was used for fast monitoring of conversion of rapeseed oil triacylglycerols to fatty acid methyl esters and for quantitation of residual TGs in the final biodiesel product. Sensitivity and linearity of various detection modes (UV detection at 205 nm, evaporative light scattering detection and mass spectrometric detection) were compared. The individual sample compounds were identified using coupled HPLC-atmospheric pressure chemical ionization mass spectrometry in the positive-ion mode.
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.
- MeSH
- aerosoly * MeSH
- kalibrace MeSH
- oleje rostlin chemie MeSH
- referenční standardy MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- spektrofotometrie ultrafialová MeSH
- triglyceridy analýza MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aerosoly * MeSH
- oleje rostlin MeSH
- triglyceridy MeSH
Correlations between the separation selectivity in aqueous and non-aqueous reversed-phase systems and in normal-phase LC systems were investigated for samples containing different numbers of two repeat structural elements. Such samples are best separated in "orthogonal" two-dimensional chromatographic systems, showing selectivity for one type of the repeat structural element only in the first dimension and for the other structural element only in the second dimension. The number of resolved compounds improves as the degree of orthogonality of the separation systems increases with decreasing correlation between the selectivities for the sample structural distribution in the two dimensions. Orthogonal systems with non-correlated selectivities for each repeat structural element provide the highest number of separated peaks and regular arrangement of the peaks over the two-dimensional retention space according to the individual structural element distribution and the best use of the available peak capacity. Fully orthogonal systems are difficult to find in practice. Partially orthogonal system with correlated selectivities for one structural type distribution, but with one system non-distinguishing the distribution for the other structural element are still useful for the two-dimensional separations. The correlations between the selectivities for repeat regular structural increments were employed to evaluate the suitability of phase systems for two-dimensional HPLC separations. The selectivity correlation in various reversed-phase and normal-phase systems was evaluated for two sample types: (1) Various RP columns show significantly inversely correlated selectivities for acyl lengths and numbers of double bonds distribution, but the differences in the double bond selectivity can be used for practical separations of triacylglycerols with the same equivalent carbon numbers. (2) Synthetic EO-PO block (co)oligomers with two-dimensional distribution of oxyethylene and oxypropylene monomer units were separated according to the two distribution types using on-line two-dimensional reversed-phase-normal-phase LC with a C18 column in the first dimension and an aminopropyl silica column in the second dimension.
- MeSH
- chromatografie kapalinová metody MeSH
- polymery chemie MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- polymery MeSH
Non-aqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with atmospheric pressure chemical ionization (APCI) was used for separation of triacylglycerols from five strains of haptophyte algae (genera Coccolithophora, Isochrysis, and Prymnesium). This study describes the separation and identification of C18 polyunsaturated triacylglycerols containing stearidonic and octadecapentaenoic fatty acids, including their regioisomers. Salinity affects the proportion of saturated and unsaturated fatty acids. The biosynthesis of C18 polyunsaturated triacylglycerols was found to be very stereospecific and to depend on the salinity of cultivation media, asymmetric regioisomers predominating at low salinity (sn-OpOpSt and/or PoStSt) and symmetric ones at high salinity (sn-OpStOp and or StPoSt).
- Klíčová slova
- Coccolithophyceae, Haptophyte algae (genera Coccolithophora, Isochrysis and Prymnesium), Liquid chromatography-mass spectrometry, Octadecapentaenoic acid, Salinity, Stearidonic acid,
- MeSH
- Haptophyta chemie genetika MeSH
- mastné kyseliny analýza MeSH
- molekulární struktura MeSH
- nenasycené mastné kyseliny chemie MeSH
- omega-3 mastné kyseliny chemie MeSH
- salinita MeSH
- stereoizomerie MeSH
- triglyceridy analýza MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Německo MeSH
- Názvy látek
- mastné kyseliny MeSH
- nenasycené mastné kyseliny MeSH
- omega-3 mastné kyseliny MeSH
- stearidonic acid MeSH Prohlížeč
- triglyceridy MeSH
The purpose of the paper was the completing of the experimentally estimated physicochemical descriptors spectrum of 1-[3-(3-propoxyphenylcarbamoyloxy)-2-hydroxypropyl]-4-(3-trifluoromethylphenyl)piperazinium chloride (labelled as 8e), an effective, highly lipophilic compound against non-tuberculous M. kansasii My 235/80. The identity of the structure 8e was verified by 1Hand 13C-NMR spectral data, IR spectrometry and by elemental analysis as well. The readout from mass spectrometry confirmed an existence of the molecular ion [M + H+]+. The purity of the evaluated compound 8e was checked by absorption thin-layer chromatography, the stability of its aqueous and methanolic solutions was investigated under UV/VIS light. The values of some physicochemical descriptors assigned to 8e, which had been previously published (pKa, log Pexp), were correlated with the constants associated with some antimycobacterially active molecules which are commonly used in therapeutical practice (isoniazid, pyrazinanide, para-aminosalicylic acid, ethionamide, streptomycine). For the content determination of the molecule of 8e, RP-HPLC (reversed-phase HPLC) method with an internal standard and UV/VIS spectrophotometry at the wavelength of 238 nm (aqueous medium) and at 244 nm (methanolic medium) were used.
- MeSH
- antituberkulotika chemie MeSH
- magnetická rezonanční spektroskopie MeSH
- netuberkulózní mykobakterie účinky léků MeSH
- piperaziny chemie MeSH
- spektrofotometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- Názvy látek
- 1-(3-(3-propoxyphenylcarbamoyloxy)-2-hydroxypropyl)-4-(3-trifluoromethylphenyl)piperazinium chloride MeSH Prohlížeč
- antituberkulotika MeSH
- piperaziny MeSH
