Non-aqueous reversed-phase-HPLC
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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.
- 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
Predmetom práce bolo doplnenie niektorých experimentálne stanovených fyzikálno-chemických deskriptorov 1-[3-(3-propoxyfenylkarbamoyloxy)-2-hydroxypropyl]-4-(3-trifluórmetylfenyl)piperazíniumchloridu (pracovne označeného ako 8e), účinnej, relatívne vysokolipofilnej zlúčeniny proti netuberkulóznemu M. kansasii My 235/80. Identita látky 8e bola potvrdená 1H- a 13C-NMR spektrami, spektrometriou v IR oblasti ako aj elementárnou analýzou. Výstup z hmotnostnej spektrometrie potvrdil existenciu molekulového iónu [M + H+]+. Čistota hodnotenej zlúčeniny 8e bola overená pomocou adsorpčnej chromatografie na tenkej vrstve, stabilita jej vodných a metanolových roztokov bola hodnotená pri pôsobení UV/VIS žiarenia. Hodnoty niektorých základných fyzikálno-chemických charakteristík molekuly 8e stanovených v redchádzajúcich publikáciách (pKa, log Pexp) boli korelované s konštantami, ktoré prislúchajú niektorým v praxi používaným antimykobakteriálne aktívnym molekulám (izoniazid, pyrazínamid, kyselina para -aminosalicylová, etiónamid, streptomycín). Pre stanovenie obsahu molekuly 8e sa použila RP-HPLC (reversed-phase HPLC) metóda vnútorného štandardu a UV/VIS spektrofotometria pri vlnovej dĺžke 238 nm (vodné prostredie) a 244 nm (prostredie metanolu).
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 1H- and 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
- atypické mykobakteriální infekce farmakoterapie imunologie MeSH
- blízká infračervená spektroskopie MeSH
- chromatografie kapalinová MeSH
- farmaceutická chemie metody MeSH
- fenylkarbamáty analýza MeSH
- fyzikální chemie metody MeSH
- hmotnostní spektrometrie MeSH
- magnetická rezonanční spektroskopie MeSH
- piperaziny analýza MeSH
- spektrofotometrie MeSH
Predmetom publikácie je komplexné spektrálne a fyzikálno- chemické hodnotenie mono[{3-[4-(2-etoxyetoxy)- benzoyloxy]-2-hydroxypropyl}-terc-butylamónium] fumarátu, potenciálneho ultrakrátko pôsobiaceho blokátora ß1-adrenergných receptorov. Totožnosť hodnotenej zlúčeniny (pracovne označenej ako UPB-2) bola potvrdená 1H-, 13C-NMR a IR spektrami. V rámci stanovenia základných fyzikálno-chemických charakteristík bola určená hodnota teploty topenia, rozpustnosť v spektre rozpúšťadiel, čistota (adsorpčná chromatografia na tenkej vrstve), povrchová aktivita (nepriama Traubeho stalagmometrická metóda), acidobázické charakteristiky (hodnota pKa stanovená alkalimetrickou titráciou), hodnoty log ε (spektrofotometricky v UV/ /VIS oblasti), ako aj hodnotenie vplyvu kyslého a zásaditého prostredia na stabilitu UPB-2. Ďalšími experimentálne stanovenými parametrami boli lipohydrofilné deskriptory určené pomocou RP-HPLC (log k’), shake flask metódou stanovené hodnoty rozdeľovacích koeficientov Pexp (resp. log Pexp) v rôznych rozdeľovacích systémoch. Na základe log Pexp-údajov bola predpovedaná schopnosť UPB-2 prechádzať cez hematoencefalickú bariéru. Pre stanovenie obsahu UPB-2 bola aplikovaná RP-HPLC (reversed-phase HPLC) metóda vnútorného štandardu a UV/VIS spektrofotometria pri vlnovej dĺžke 260 nm (vodné prostredie) a 258 nm (prostredie metanolu).
The present paper aims at a complex spectral and physicochemical evaluation of mono[{3-[4-(2-ethoxyethoxy)- benzoyloxy]-2-hydroxypropyl}-tert-butylammonium] fumarate, the potential ultra-short acting blocker of ß1-adrenergic receptors. The identity of the evaluated compound (labelled as UPB-2) was confirmed by 1H-, 13C-NMR and IR spectral data as well. The estimated physicochemical parameters included melting point data, solubility in various media, purity checking (adsorption thin-layer chromatography), surface activity determination (non- -direct Traube stalagmometric method), acidobasic characteristics (pKa value determination by alkalimetric titration), log ε values estimation (spectrophotometrically in UV/VIS region) and a study of the influence of acidic and alkaline media towards the stability of UPB-2. Other experimentally estimated values were lipohydrophilic descriptors using RP- -HPLC (log k’) and the log Pexps in various lipohydrophilic media by the shake flask method. Based on the log Pexp readouts, the ability to permeate across the brain-blood barrier was predicted. For the content determination of UBP-2 the RP-HPLC (reversed-phase HPLC), the method of an internal standard and UV/VIS spectrophotometry at the wavelength of 260 nm (aqueous medium) and at 258 nm (methanolic medium) was applied.
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).
- 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
Fatty acid diesters of long-chain 1,2-diols (1,2-DDE), or type II wax diesters, were analyzed in the vernix caseosa of a newborn girl. 1,2-DDE were isolated from the total lipid extract by the semipreparative TLC using plates coated with silica gel. Chromatographic separation of the 1,2-DDE molecular species was achieved on the non-aqueous reversed-phase HPLC with two Nova-Pak C18 columns connected in series (a total length of 45cm) and using an acetonitrile-ethyl acetate gradient. 1,2-DDE eluted from the column in the order of their equivalent chain number. The analytes were detected as ammonium adducts by an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionization source. Their structures were elucidated using tandem mass spectrometry with MS, MS(2) and MS(3) steps in a data-dependent mode. More than two thousand molecular species of 1,2-DDE were identified in 141 chromatographic peaks. The most abundant 1,2-DDE were monounsaturated lipids consisting of a C22 diol and a C18:1 fatty acid together with C16:0, C14:0 or C15:0 fatty acids. The positions of double bonds were characterized by the fragmentation of [M+C3H5N](+) formed in the ion source.
High chromatographic resolution of wax esters (WEs) was achieved by non-aqueous reversed-phase liquid chromatography on a Nova-Pak C18 column by optimising the acetonitrile/ethyl acetate mobile phase gradient. The retention behaviour of WEs was studied in this chromatographic system. The WEs eluted according to their equivalent carbon number (ECN) values; within the group of WEs with the identical ECN, the most unsaturated species tended to elute first. The isobaric WEs with different positions of the ester moiety were separated from each other whenever the lengths of the chains were sufficiently different. The methyl-branched esters eluted at shorter retention times than the straight-chained analogues, and the resolution among methyl-branched WEs depended on the position of the branching. The analytes were detected by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) using data-dependent scanning. WEs provided simple full-scan spectra with abundant protonated molecules and low-intensity fragments. Collision-induced dissociation (CID) promoted identification of the WE molecular species. The responses of WEs were found to be dependent on the number of double bonds and on the alkyl-chain length; the limits of the detection ranged from 20micromol/L to 200nmol/L. The HPLC/APCI-MS was applied for the analysis of the WEs isolated from honeycomb beeswax, jojoba oil and human hair. Good agreement between reported results and the literature data was achieved, with several novel polyunsaturated WEs also being found.
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.
Fatty acid esters of long-chain hydroxy fatty acids or (O-acyl)-hydroxy fatty acids (OAHFAs) were identified for the first time in vernix caseosa and characterized using chromatography and mass spectrometry. OAHFAs were isolated from the total lipid extract by a two-step semipreparative TLC. The general structure of OAHFAs was established using high-resolution and tandem mass spectrometry of intact lipids and their transesterification and derivatization products. Two isomeric lipid classes were identified: O-acyl esters of ω-hydroxy fatty acids (ωOAHFA) and O-acyl esters of α-hydroxy fatty acids (αOAHFAs). To the best of our knowledge, αOAHFAs have never been detected in any biological sample before. Chromatographic separation and identification of OAHFAs species were achieved using non-aqueous reversed-phase HPLC coupled to electrospray ionization hybrid linear ion trap-Orbitrap mass spectrometry. The lipid species were detected as deprotonated molecules, and their structures were elucidated using data-dependent fragmentation in the negative ion mode. More than 400 OAHFAs were identified in this way. The most abundant ωOAHFAs species were 28:0/ω-18:2, 29:0/ω-18:2, 30:0/ω-18:2, 32:0/ω-18:2, and 30:0/ω-18:3, while αOAHFAs comprised saturated species 21:0/α-24:0, 22:0/α-24:0, 23:0/α-24:0, 24:0/α-24:0, and 26:0/α-24:0. OAHFAs were estimated to account for approximately 0.04% of vernix caseosa lipids. Graphical Abstract.
Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.
