Non-aqueous Reversed-phase High-performance Liquid Chromatography 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.

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).

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.

Development of an Offline Bidimensional High-Performance Liquid Chromatography Method for Analysis of Stereospecific Triacylglycerols in Cocoa Butter Equivalents

Acyl migration is a serious problem in enzymatic modification of fats and oils, particularly in production of cocoa butter equivalent (CBE) through enzymatic acidolysis reaction, which leads to the formation of non-symmetrical triacylglycerols (TAGs) from symmetrical TAGs. Non-symmetrical TAGs may affect the physical properties of final products and are therefore often undesired. Consequently, an accurate method is needed to determine positional isomer TAGs during the production of CBE. A bidimentional high-performance liquid chromatography (HPLC) method with combination of non-aqueous reversed-phase HPLC and silver ion HPLC joining with an evaporative light scattering detector was successfully developed for the analysis of stereospecific TAGs. The best separation of positional isomer standards was obtained with a heptane/acetone mobile-phase gradient at 25 °C and 1 mL/min. The developed method was then used in multidimensional determination of the TAG positional isomers in fat and oil blends and successfully identified the TAGs and possible isomers in enzymatically acidolyzed CBE.

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.

Exploring the fatty acids of vernix caseosa in form of their methyl esters by off-line coupling of non-aqueous reversed phase high performance liquid chromatography and gas chromatography coupled to mass spectrometry

Vernix caseosa is a greasy biofilm formed on the skin of the human fetus in the last period of pregnancy. This matrix is known to contain a range of uncommon branched chain fatty acids. In this study, we studied the fatty acid composition of vernix caseosa by non-aqueous reversed phase high performance liquid chromatography (RP-HPLC) fractionation followed by gas chromatography–electron ionization mass spectrometry (GC/EI–MS) of the fractions. For this purpose the fatty acids from vernix caseosa were converted into the corresponding methyl esters. These were fractionated by non-aqueous RP-HPLC using three serially connected C 18-columns and pure methanol as the eluent. Aliquots of the HPLC fractions were directly analyzed by GC/EI–MS in the selected ion monitoring mode. Data analysis and visualization were performed by the creation of a two dimensional (2D) contour plot, in which GC retention times were plotted against the HPLC fractions. Inspection of the plot resulted in the detection of 133 different fatty acids but only 16 of them contributed more than 1% to the total fatty acids detected. Identification was based on HPLC and GC retention data, GC/MS–SIM and full scan data, as well as plotting the logarithmic retention times against the longest straight carbon chain. In selected cases, aliquots of the HPLC fractions were hydrogenated or studied by means of the picolinyl esters. Using these techniques, the number of double bonds could be unequivocally assigned to all fatty acids. Moreover, the number of methyl branches, and in many cases the positions of methyl branches could be determined. The enantioselective analysis of chiral anteiso-fatty acids resulted in the dominance of the S-enantiomers. However, high proportions of R-a13:0, R-a15:0, and R-a17:1 were also detected while a17:0 was virtually S-enantiopure.

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.

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.

Ultrasensitive assays of trans- and cis-β-carotenes in vegetable oils by high-performance liquid chromatography–thermal lens detection

The fact that β-carotene might be the protective factor against various cancers, suggests the need for a rapid reliable assay for this potential marker. We have proposed the method for selective, precise and simple profiling of carotenoids as well as for simultaneous ultrasensitive assaying of trans-β-carotene (TBC) and cis-β-carotene(s) (CBC) in five vegetable oils. The oil samples diluted 20 or 100 times were directly injected and analysed by means of the isocratic non-aqueous reversed-phase high-performance liquid chromatography (HPLC) combined with ultrasensitive thermal lens detection (TLS). Elution of TBC was followed by CBC; both were identified and determined in olive, safflower, sesame, wheat germ and linseed oil by standard addition method. The presence of lutein/zeaxanthin, some xanthophyll and/or early eluting carotene may be also presumed in the aforementioned oils. The examined oils showed different and characteristic carotenoid/carotene profiles and characteristic TBC-to-CBC ratios. Both analytes were selectively detected in the presence of palmitic, oleic, linoleic, linolenic and stearic acid, β-sitosterol and dl-α-tocopherol. This extends applicability of the method to other vegetable oils as well. Favourable analytical performances (high sensitivity, low limits of detection (LODs) and wide linearity span) enabled the direct analyses of highly diluted oils. This resulted in several major benefits of the proposed method, among which (i) reduced risk of stationary phase deterioration, (ii) avoiding the risk of carotenoids transformations and (iii) substantial labour and time savings. The TBC and CBC in diluted vegetable oils were reliably measured at ultratrace level (1–26 ng ml −1) with the S/ N ratio ranging from 4 to 140 and precisely determined (imprecision 0.4–8.3%). The concentrations of TBC+CBC estimated in the original oils were as follows: 90.5+51.2 ng ml −1 in sesame oil, 146.0+164 ng ml −1in safflower oil, 464.6+206.1 ng ml −1in linseed oil, 453.7+143.3 ng ml −1 in olive oil and 2.31+2.63 μg ml −1 in wheat germ oil. The characteristic and variable portion of TBC within total β-carotene may serve as a reliable indicator of both, quality and authenticity of the vegetable oil. The HPLC–TLS assay proposed may therefore be successfully applied in nutritional, agricultural and epidemiological studies.

Separation and identification of perchlorinated polycyclic aromatic hydrocarbons and fullerenes (C 60, C 70) by coupling high-performance liquid chromatography with ultraviolet absorption spectroscopy and atmospheric pressure chemical ionization mass spectrometry

Simultaneous separation and identification of perchlorinated polycyclic aromatic hydrocarbons (PCPAHs) and fullerenes is of practical interest due to the growth mechanism of fullerenes involved with PCPAHs. Non-aqueous reversed-phase high-performance liquid chromatography (HPLC), with an ODS column and a gradient mobile phase of methanol–ethanol–cyclohexane mixtures, was combined with both rapid-scan ultraviolet spectrometry (UV) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) for the separation and identification of over 80 PCPAHs as well as fullerenes C 60 and C 70, that were synthesized in the discharge reaction of chloroform. PCPAH retention was found to depend on the number of aromatic rings and the degree of non-planarity of PCPAH structure. Based on the isotopic pattern of molecular ion or/and quasi-molecular ion peaks in corresponding mass spectra, molecular compositions of the PCPAH products were unambiguously determined. The results obtained from the HPLC–UV–MS analysis not only are helpful for the understanding of the fullerenes formation mechanism, but also contribute to the analytical technique capable of separating and identifying the complicated mixture of PCPAHs and fullerenes.

Prediction of isocratic nonaqueous reversed-phase high-performance liquid chromatography retention parameters and response factors of triacylglycerols detected by an ultraviolet-diode array-evaporative light-scattering on-line system.

Nonaqueous reversed-phase high-performance liquid chromatography of 10 homogeneous triacylglycerol molecular species (TAG), both saturated and unsaturated, is carried out. The eluate from the column is detected by an ultraviolet diode array detector (DAD) on-line with an evaporative light-scattering detector (ELSD). The retention parameters (as selectivities, alpha) for 220 TAGs are determined, and the obtained values are related to the following structural parameters: total carbon number; mono-, di-, and triunsaturated fatty acid residues number/molecule; and monounsaturated fatty acid carbon number. Multiple regression analysis is carried out to obtain a relationship for the prediction of alpha values of any TAG when the same experimental conditions are used. In regard to the quantitative analysis of the separated TAG species, the dependence of response of the two on-line detectors on the aforementioned structural parameters is studied. Three different wavelengths (205, 210, and 215 nm) are considered for TAG detection by DAD; in each case, the obtained multiple regression model shows a good correlation between the dependent variable and predictive values of the TAG species (response factors and considered structural parameters, respectively). The ELSD gives responses exponentially related to injected amounts. Also, in this case, an attempt to relate the response factors of each considered detector to some structural parameters of TAG species is carried out. The results of this study are used to analyze the TAG fraction from an olive oil.

Prediction of relative retention times of triacylglycerols in non-aqueous reversed-phase high-performance liquid chromatography

Accurate and simple methods for prediction of relative retention times of triacylglycerols in non-aqueous reversed-phase HPLC are presented here. The prediction is based on a simple calculation using the experimental relative retention time (min) of the triacylglycerol with the closest corresponding structure and taking into account the contributions (min) of functional groups present in the triacylglycerol for which the standard is not available to predict the relative retention time. The contribution of functional groups on the fatty acyl in the s n-2 position were greater than those at the s n-1(3) position, especially for polar functional groups such as the hydroxy group of ricinoleate. Another method of prediction which is based on the experimental relative retention time of tristearin and disregards the difference of s n-1(3) and s n-2 is also presented here.