Non-aqueous Reversed-phase High-performance Liquid Research Articles

Analysis of linoleoyl and oleoyl macrogolglycerides by high performance liquid chromatography coupled to the atmospheric pressure photoionization mass spectrometry

Linoleoyl macrogolglyceride (LM) and oleoyl macrogolglyceride (OM) are pharmaceutical ingredients, obtained from corn and apricot kernel oils respectively. This study aims to know the detailed chemical composition of LM and OM, in order to understand their roles in pharmaceutical formulations. These two products were analyzed by non-aqueous reversed phase high-performance liquid chromatography (NARP-HPLC), using Vintage Series KR C18 column (250 × 4.6 mm, 5 µm) and non-aqueous acetonitrile/acetone mixture as mobile phase. The ionization source used was atmospheric pressure photoionization (APPI) and the analyzer was LTQ-Orbitrap® (hybrid analyzer: double linear ion trap coupled to a Fourier transform orbital trap). LM and OM consist of complex mixtures, constituted of mono-(MG), di-(DG) and triglycerides (TG) and mono-(MPEGE) and di PEG-6 esters (DPEGE) of linoleic acid (18:2) for LM and of oleic acid (18:1) for OM. NARP-HPLC-APPI method allowed the separation and the identification of the glyceride classes (MGs, DGs and TGs) and the PEG esters of different chain lengths (PEG-chain lengths of fatty acid moieties and number of units of ethylene oxide), at the same time and in one single run, for both products LM and OM. The comparative study between LM and OM showed that, a higher presence of linoleic esters for LM, and a higher presence of oleic esters for OM.

Arsenolipids in the green alga Coccomyxa (Trebouxiophyceae, Chlorophyta)

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.

Enantiomeric separation of triacylglycerols containing very long chain fatty acids

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.

Determination of ethanol in gasoline by high-performance liquid chromatography

This study employed non-aqueous reversed-phase high-performance liquid chromatography (HPLC) with refractive-index detection and methanol as a mobile phase to quantify the ethanol content in gasoline. The advantages of HPLC are its good separation versatility, high resolution, relatively short analysis time, and automation. Standard samples were prepared to obtain a standard curve and partial validation. Accuracy, precision, linearity, selectivity for methanol (adulterant), and measurement uncertainty were investigated. In the partial validation analysis, the accuracy of the proposed method was identified as its main advantage over the reference method.

Effect of salinity on the fatty acid and triacylglycerol composition of five haptophyte algae from the genera Coccolithophora, Isochrysis and Prymnesium determined by LC-MS/APCI

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).

Synthesis, liquid chromatographic fractionation and partial characterization of polybrominated dibenzofuran congeners

Polybrominated dibenzofurans (PBDFs) are a class of highly toxic environmental contaminants which comprises 135 structurally different congeners. While the gas chromatographic separation and analysis of the most polychlorinated dibenzofurans (PCDFs) are well-documented, comparably little data is currently available in the case of PBDFs. In this study dibenzofuran was brominated to give a mixture of ∼40 PBDFs with one to seven bromine atoms. This synthesis mixture was fractionated by both countercurrent chromatography (CCC) with the solvent system n-hexane/toluene/acetonitrile and non-aqueous reversed-phase high performance liquid chromatography (RP-HPLC) with acetonitrile as the mobile phase. All together 80 consecutive CCC fractions and 40 HPLC fractions were taken and analyzed for PBDFs by gas chromatography coupled to mass spectrometry (GC/MS). CCC and RP-HPLC offered orthogonal separation of the PBDF mixture. As a consequence, selected CCC fractions were further fractionated by RP-HPLC. In this way, eight PBDFs could be isolated and the structures of twelve PBDFs were elucidated by proton magnetic resonance spectroscopy (1H NMR).

Non-polar lipids characterization of Quinoa (Chenopodium quinoa) seed by comprehensive two-dimensional gas chromatography with flame ionization/mass spectrometry detection and non-aqueous reversed-phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection.

A chemical characterization of major lipid components, namely, triacylglycerols, fatty acids and the unsaponifiable fraction, in a Quinoa seed lipids sample is reported. To tackle such a task, non-aqueous reversed-phase high-performance liquid chromatography with mass spectrometry detection was employed. The latter was interfaced with atmospheric pressure chemical ionization for the analysis of triacylglycerols. The main triacylglycerols (>10%) were represented by OLP, OOL and OLL (P = palmitoyl, O = oleoyl, L = linoleoyl); the latter was present in the oil sample at the highest percentage (18.1%). Furthermore, fatty acid methyl esters were evaluated by gas chromatography with flame ionization detection. 89% of the total fatty acids was represented by unsaturated fatty acid methyl esters with the greatest percentage represented by linoleic and oleic acids accounting for approximately 48 and 28%, respectively. An extensive characterization of the unsaponifiable fraction of Quinoa seed lipids was performed for the first time, by using comprehensive two-dimensional gas chromatography with dual mass spectrometry/flame ionization detection. Overall, 66 compounds of the unsaponifiable fraction were tentatively identified, many constituents of which (particularly sterols) were confirmed by using gas chromatography with high-resolution time-of-flight mass spectrometry.

Chromatographic determination of total cholesterol in human lung healthy and cancer tissues of the same patient

Cholesterol is an essential component of mammalian cells, but its role in cancer is unclear. We have determined the total cholesterol content in the healthy and the cancerous lung tissues of the same patient. Tissues of 13 patients (7 women and 6 men, different histological type, different stages of the disease) were obtained during surgical intervention. The samples (0.056–2.004 g) were hydrolyzed in alkaline medium, and the total cholesterol was extracted into n-hexane and simultaneously determined by thin-layer chromatography (TLC) on silica gel high-performance thin-layer chromatography (HPTLC) plates and nonaqueous reversed-phase high-performance liquid chromatography (HPLC), which both proved to be suitable for the determination of cholesterol in lung tissues and gave comparable results. This is the first report about the comparison of the total cholesterol content in the healthy and the cancerous tissue of the same patient. The difference in the results for the total cholesterol from both types of ...

High enantiomeric excess of the flavor relevant 4-alkyl-branched Fatty acids in milk fat and subcutaneous adipose tissue of sheep and goat.

Volatile 4-alkyl-branched fatty acids are characteristic flavor compounds of sheep and goat. Due to the methyl branch, the carbon C-4 represents a stereogenic center with the possible presence of one or both enantiomers in the respective samples. In this study, we used enantioselective gas chromatography to study the enantiomeric composition of 4-methyloctanoic acid (4-Me-8:0) and 4-ethyloctanoic acid (4-Et-8:0) in milk and dairy products from sheep and goat as well as in goat subcutaneous tissue. Different columns coated with modified cyclodextrins were tested to resolve racemic 4-alkyl-branched fatty acid methyl ester standards. The best enantiomer resolution was obtained on 25% octakis(2,3,6-tri-O-ethyl)-γ-cyclodextrin (γ-TECD) diluted in OV-1701. For analysis of the food samples, the lipids were extracted and fatty acids in the extracts were converted into fatty acid methyl esters. Non-aqueous reversed-phase high-performance liquid chromatography was used to fractionate the samples in order to gain one solution enriched in 4-Me-8:0 methyl ester and one solution enriched with 4-Et-8:0 methyl ester. Subsequent analysis by enantioselective gas chromatography with mass spectrometry allowed only the detection of one enantiomer of 4-Me-8:0 and 4-Et-8:0 in the samples. By means of a non-racemic standard of 4-Me-8:0, it was found that the predominant enantiomer was (R)-4-Me-8:0.

Separation and identification of an oligomeric light stabilizer Chimassorb 944 by gradient elution chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

A non-aqueous reversed-phase high-performance liquid chromatographic (HPLC) method has been developed to separate a light stabilizer Chimassorb 944 into individual oligomers, which are further identified using pre-column fluorescent derivatization and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Consistent with previous studies, we find that the Chimassorb 944 product is a complex mixture consisting of a homologous series with the amine end groups and the number of repeat units (n) span from 1 to 26. In addition to the dominant linear species, cyclic oligomers are present at relatively high levels in the low-mass range. Their concentration decreases rapidly with the length of the oligomer backbone and becomes undetectable when n>7. Moreover, comparison of the HPLC and MALDI-MS molar mass distributions of Chimassorb 944 shows that the HPLC analysis produces greater molar mass averages and thus offers an effective means for accurate measure of the relative abundances of the oligomers.

A comparative study of the composition of triacylglycerol molecular species in equine and human milks

The composition of triacylglycerols (TAGs) is a very useful parameter to understand several properties of creams and anhydrous milk fat products such as nutritional value, technological applications, and textural properties. In this paper, the molecular species of TAGs of equine milk were identified using a combination of non-aqueous reversed phase high-performance liquid chromatography, electrospray ionization mass spectrometry and high temperature gas chromatography and were then compared to the TAGs composition of human milk. The data showed that equine milk contained a complex mixture of different TAGs molecular species resulting from the widest range of its fatty acids as compared to human milk. In particular, the largest differences in TAGs composition between equine and human milk concerned TAGs containing medium-chain fatty acids; for C8, C10, and C12. In terms of carbon number (CN), TAGs with CN36–CN46 were the most abundant in equine milk with a maximum concentration at CN44 (15.3%), while TAGs with CN44–CN52 were the predominant in human milk with a maximum of CN52 (30%). For both milks studied, the experimentally determined distributions of TAGs molecular species deviated from those predicted by the random hypothesis. TAGs with CN44 corresponding to a fatty acids carbon number distribution of C8/C18/C18, C12/C14/C18, and C10/C16/C18 were apparently preferentially synthesized in equine milk. Whereas, in human milk, mixed TAGs with CN52 containing C16/C18/C18 fatty acids were much higher than would be expected from the random calculation.

Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/ trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position.

Creation and evaluation of a two-dimensional contour plot of fatty acid methyl esters after off-line coupling of reversed-phase HPLC and GC/EI-MS

Fatty acid methyl esters obtained from a fish oil sample were fractionated by non-aqueous reversed-phase high-performance liquid chromatography (RP-HPLC) using three serially connected C(18)-columns and pure methanol as the eluent. The HPLC fractions were analyzed by gas chromatography-electron ionization mass spectrometry in the selected ion monitoring mode. Data analysis and visualization was performed by the creation of a two-dimensional (2D) contour plot, in which GC retention times were plotted against the HPLC fractions. The 2D contour plot resulted in a full resolution of more than 120 fatty acids. The fatty acids were arranged on predictable lines and curves in dependence of the number of carbons and double bonds. The 2D contour plot enabled both the recognition of unknown fatty acids (which were found off the lines and curves) and the prediction of the coordinates of known fatty acids. Finally, selected HPLC fractions were subjected to further experiments (hydrogenation, silver ion fractionation, specific GC/MS measurements) in order to verify the structural assignments predicted from the 2D contour plot. All in all, the structures of over 100 FAs could be assigned to the peaks detected in the 2D contour plots.

Comparison of various types of stationary phases in non-aqueous reversed-phase high-performance liquid chromatography–mass spectrometry of glycerolipids in blackcurrant oil and its enzymatic hydrolysis mixture

The selection of column packing during the development of high-performance liquid chromatography method is a crucial step to achieve sufficient chromatographic resolution of analyzed species in complex mixtures. Various stationary phases are tested in this paper for the analysis of complex mixture of triacylglycerols (TGs) in blackcurrant oil using non-aqueous reversed-phase (NARP) system with acetonitrile–2-propanol mobile phase. Conventional C 18 column in the total length of 45 cm is used for the separation of TGs according to their equivalent carbon number, the number and positions of double bonds and acyl chain lengths. The separation of TGs and their more polar hydrolysis products after the partial enzymatic hydrolysis of blackcurrant oil in one chromatographic run is achieved using conventional C 18 column. Retention times of TGs are reduced almost 10 times without the loss of the chromatographic resolution using ultra high-performance liquid chromatography with 1.7 μm C 18 particles. The separation in NARP system on C 30 column shows an unusual phenomenon, because the retention order of TGs changes depending on the column temperature, which is reported for the first time. The commercial monolithic column modified with C 18 is used for the fast analysis of TGs to increase the sample throughput but at cost of low resolution.

Conjugated Linoleic Acid-Rich Soy Oil Triacylglycerol Identification

Conjugated linoleic acid (CLA)-rich soy oil has been produced by soy oil linoleic acid (LA) photoisomerization, but CLA-rich oil triacylglycerol (TAG) characterization was not described. Therefore, the objectives were to identify and quantify new TAG fractions in CLA-rich oil by nonaqueous reversed-phase high-performance liquid chromatography (NARP-HPLC). Analytical NARP-HPLC with an acetonitrile/dichloromethane (ACN/DCM) gradient and an evaporating light scattering detector/ultraviolet (ELSD/UV) detector was used. New TAG peaks from LA-containing TAGs were observed. The LnLL, LLL, LLO, and LLP (Ln, linolenic; L, linoleic; O, oleic; and P, palmitic) peaks reduced after isomerization with an increase in adjacent peaks that coeluted with LnLnO, LnLO, LnOO, and LnPP. The newly formed peaks were wider than those of the original oil and absorbed at 233 nm, suggesting the possibility of various CLA containing TAGs. The HPLC profile showed five fractions of mixed TAGs, and fatty acid analysis showed that CLA isomers were found predominately in fractions 2 and 3, which originally contained most LA. The CLA isomers were 70-80% trans,trans and 20-30% cis,trans and trans,cis.

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.

Continuous counter current extraction, isolation and determination of solanesol in Nicotiana tobacum L. by non-aqueous reversed phase high performance liquid chromatography

A method of continuous counter current extraction in a large-scale production of solanesol from tobacco leaves was developed. The crude extract containing 15–20% solanesol was subjected to a series of steps, viz., saponification, solvent recrystallization and column chromatography. The pure material was characterized by FT-IR, ESI-MS, 1H NMR and 13C NMR spectrometry. The analysis was carried out by a simple and rapid non-aqueous reversed-phase high performance liquid chromatographic method on a Hypersil BDS C 18 column (250 mm × 4.6 mm, particle size 5 μm) with isopropyl alcohol–methanol (60:40, v/v) as mobile phase and detection at 215 nm. The product purity was between 95 and 98% (w/w) as determined by HPLC.

Investigation of the pharmacokinetics and determination of cholesteryl carbonate zidovudine in rat plasma by non-aqueous reversed-phase high performance liquid chromatography with UV detection

A simple reversed phase high-performance liquid chromatographic method was established for the separation and quantification of a novel prodrug of zidovudine in rat plasma. Zidovudine was one of the original drugs used to treat HIV infection. Appropriate aliquots of rat plasma were spiked with cholesteryl carbonate zidovudine (AZTC) and treated with acetonitrile to precipitate plasma proteins. The supernatant after supercentrifugation was collected and an aliquot of 20μL was injected directly into an HPLC system consisting of a Diamonsil™ C18 column and a diode array detector. The mobile phase consisted of methanol (85%, v/v) and diethyl ether (15%, v/v) at a flow rate of 1.0mL/min. The extraction recoveries of AZTC at the three concentrations examined were all higher than 80%. The HPLC assay was linear over the concentration range 0.5–80μg/mL. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration–time profile for AZTC after administration via the tail vein. The mean elimination half-life (t1/2) was 292.4min. This RP-HPLC method will be useful for the evaluation of the pharmacokinetics of AZTC in rats.

High-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry and gas chromatography–flame ionization detection characterization of Δ5-polyenoic fatty acids in triacylglycerols from conifer seed oils

Edible conifer seeds can serve as a source of triacylglycerols (TGs) with unusual Δ5 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 Δ5-UPIFAs on the retention and fragmentation behavior is described and used for the successful identification of TGs in each oil. TGs containing Δ5-UPIFAs have a higher retention in comparison with common TGs found in plant oils with single methylene interrupted Δ6(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 Δ5-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.

Off-line coupling of non-aqueous reversed-phase and silver ion high-performance liquid chromatography–mass spectrometry for the characterization of rice oil triacylglycerol positional isomers

The determination of the triacylglycerol (TAG) profile in real world matrices is rather difficult as these compounds present a complex composition and are characterized by similar physico-chemical properties. This investigation is based on the high-performance liquid chromatography (HPLC) multidimensional determination of the TAG profile in terms of TAG species and positional isomers in a rice oil sample. The off-line bi-dimensional system was attained through the coupling of non-aqueous reversed-phase HPLC and silver ion (Ag)-HPLC. The primary column eluate was fractionated and the fractions of interest were then injected onto the secondary column, allowing the separation of several TAG positional isomers, unresolved in the first dimension. Peak assignment was carried out by combining retention data with atmospheric pressure chemical ionisation (APCI) MS spectra information. The fatty acid distribution along the glycerol backbone, determined by Ag-HPLC, was confirmed through diglyceride ion ratios derived from APCI-MS analysis. Method validation, where both precision and accuracy were measured, was carried out in preliminary applications on standard compounds. The analytical results obtained show that rice oil TAGs follow a distribution which can be considered typical for vegetable oils.