Resolution of triacylglycerol positional isomers by reversed-phase high-performance liquid chromatography

The ability of reversed-phase high-performance liquid chromatography (RP-HPLC) to separate some positionally isomeric disaturated and monounsaturated triacylglycerols (TAGs) as intact species is demonstrated for the first time. Mobile phases of acetonitrile modified with methanol, ethanol, 2-propanol, 1-propanol, 1-butanol, acetone, or dichloromethane were tested for the separation of POP-PPO, PLP-PPL, PEP-PPE, and PDP-PPD (P-palmitic, O-oleic, L-linoleic, E-eicosapentaenoic, D-docosahexaenoic acid residue) on a single RP-HPLC column. The resolution improved with increasing number of double bonds in the acyl residues. While POP and PPO were only partially resolved, PDP and PPD were fully separated with all tested mobile phases, except those containing methanol. Also separated were the four TAGs having the same equivalent carbon number (ECN = 42), PEP, PPE, PDP, and PPD, on a single RP-HPLC column with mobile phase acetonitrile-2-propanol (70:30, v/v) at 0.8 mL/min. In all cases the isomer with the unsaturated acyl residue in either 1- or 3-position was retained more strongly than the respective 2-isomer.     

Determination of triacylglycerol regioisomers using electrospray ionization-quadrupole ion trap mass spectrometry with a kinetic method

The kinetic method was applied to differentiate and quantify mixtures of regioisomeric triacylglycerols (TAGs) by generating and mass selecting alkali ion bound metal dimeric clusters with a TAG chosen as reference (ref) and examining their competitive dissociations in a quadrupole ion trap mass spectrometer. This methodology readily distinguished pairs of regioisomers (AAB/ABA) such as LLO/LOL, OOP/OPO and SSP/SPS and consequently distinguished sn-1/sn-3, sn-2 substituents on the glycerol backbone. The dimeric complex ions [ref, Li, TAG_{(AAB and/or ABA)}]⁺ generated by electrospray ionization mass spectrometry were subjected to collision induced dissociation causing competitive loss of either the neutral TAG reference (ref) leading to [Li(AAB and/or ABA)]⁺ or the neutral TAG molecule (TAG_{(AAB and/or ABA)}) leading to [ref, Li]⁺. The ratio of the two competitive dissociation rates, defined by the product ion branching ratio (R_iso), was related via the kinetic method to the regioisomeric composition of the investigated TAG mixture. In this work, a linear correlation was established between composition of the mixture of each TAG regioisomer and the logarithm of the branching ratio for competitive fragmentation. Depending on the availability of at least one TAG regioisomer as standard, the kinetic method and the standard additions method led to the quantitative analysis of natural TAG mixtures.     

Elucidation of triacylglycerols in cod liver oil by liquid chromatography electrospray tandem ion-trap mass spectrometry

Though liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS²) has been widely used in the structural elucidation of triacylglycerols (TAG) in vegetable oils, its potentiality for the identification of TAG molecules in omega-3 rich oils remains unexplored till date. Hence, this article investigates the applicability of LC-ESI-MS² for the structural characterization of naturally occurring TAG in cod liver oil without the TAG fractionation during the sample preparation. A computational algorithm was developed to automatically interpret the mass spectra and elucidate the TAG structures respectively. The results were compared against the lipase benchmark method. A principal component analysis study revealed that it is possible to discriminate genuine from adulterated cod liver oil.     

Regioisomeric analysis of triacylglycerols using silver-ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry: comparison of five different mass analyzers

Silver-ion high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) is used for the regioisomeric analysis of triacylglycerols (TGs). Standard mixtures of TG regioisomers are prepared by the randomization reaction from 8 mono-acid TG standards (tripalmitin, tristearin, triarachidin, triolein, trielaidin, trilinolein, trilinolenin and tri-gamma-linolenin). In total, 32 different regioisomeric doublets and 11 triplets are synthesized, separated by silver-ion HPLC using three serial coupled chromatographic columns giving a total length of 75cm. The retention of TGs increases strongly with the double bond (DB) number and slightly for regioisomers having more DBs in sn-1/3 positions. DB positional isomers (linolenic vs. γ-linolenic acids) are also separated and their reverse retention order in two different mobile phases is demonstrated. APCI mass spectra of all separated regioisomers are measured on five different mass spectrometers: single quadrupole LC/MSD (Agilent Technologies), triple quadrupole API 3000 (AB SCIEX), ion trap Esquire 3000 (Bruker Daltonics), quadrupole time-of-flight micrOTOF-Q (Bruker Daltonics) and LTQ Orbitrap XL (Thermo Fisher Scientific). The effect of different types of mass analyzer on the ratio of [M+H-R(i)COOH](+) fragment ions in APCI mass spectra is lower compared to the effect of the number of DBs, their position on the acyl chain and the regiospecific distribution of acyl chains on the glycerol skeleton. Presented data on [M+H-R(i)COOH](+) ratios measured on five different mass analyzers can be used for the direct regioisomeric determination in natural and biological samples.     

Use of triacylglycerol profiles established by high performance liquid chromatography with ultraviolet-visible detection to predict the botanical origin of vegetable oils

A method for the determination of triacylglycerols (TAGs) in vegetable oils from different botanical origins by HPLC with UV-vis detection has been developed. Using a core-shell particle packed column (C18, 2.6 μm), TAG separation was optimized in terms of mobile phase composition and column temperature. Using isocratic elution with acetonitrile/n-pentanol at 10 °C, excellent efficiency with good resolution between most of the TAG peak pairs, within a total analysis time of 15 min, was achieved. Using mass spectrometry detection, a total of 15 peaks, which were common to oils of six different botanical origins (corn, extra virgin olive, grapeseed, hazelnut, peanut and soybean) were identified. These peaks were used to construct linear discriminant analysis (LDA) models for botanical origin prediction. Ratios of the peak areas selected by pairs were used as predictors. All the oils were correctly classified with assignment probabilities higher than 95%.     

Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined. The choice of modifier depended on the nature of the stationary phase. The more polar solvents were better suited for stationary phases with higher percentage of free silanol groups. Isomeric species were eluted according to chain length, number of double bonds, and the position of the unsaturated acyl chain in the glycerol molecule. Retention increases in the order 20:5 < 22:6 < 18:3 < 20:4 < 18:2. Within each isomeric pair, the species with unsaturated acyl chain occupying either the sn-1- or the 3-position were retained preferentially. Complete simultaneous regioselective separation of 10 isomeric triacylglycerols in a single isocratic run on a single ODS column was demonstrated.     

Identification of triacylglycerol using automated annotation of high resolution multistage mass spectral trees

High complexity of identification for non-target triacylglycerols (TAGs) is a major challenge in lipidomics analysis. To identify non-target TAGs, a powerful tool named accurate MSⁿ spectrometry generating so-called ion trees is used. In this paper, we presented a technique for efficient structural elucidation of TAGs on MSⁿ spectral trees produced by LTQ Orbitrap MSⁿ, which was implemented as an open source software package, or TIT. The TIT software was used to support automatic annotation of non-target TAGs on MSⁿ ion trees from a self-built fragment ion database. This database includes 19108 simulate TAG molecules from a random combination of fatty acids and corresponding 500582 self-built multistage fragment ions (MS ≤ 3). Our software can identify TAGs using a “stage-by-stage elimination” strategy. By utilizing the MS¹ accurate mass and referenced RKMD, the TIT software can discriminate unique elemental composition candidates. The regiospecific isomers of fatty acyl chains will be distinguished using MS² and MS³ fragment spectra. We applied the algorithm to the selection of 45 TAG standards and demonstrated that the molecular ions could be 100% correctly assigned. Therefore, the TIT software could be applied to TAG identification in complex biological samples such as mouse plasma extracts.     

A laser desorption ionization time-of-flight mass spectrometry investigation into triacylglycerols oxidation during thermal stressing of edible oils

Laser desorption ionization time-of-flight mass spectrometry (LDI–TOF MS) was used to characterize olive and sunflower oils before and after thermally assisted oxidation in order to develop a rapid fingerprinting method for oil that contains unchanged and oxidized components. No matrix was used to assist laser desorption, and simplified mass spectra were obtained in the mass range of interest (m/z 500–1000), where triacyl- and diacylglycerol ions were observed. Sample preparation was reduced to dissolving oil in chloroform saturated with NaCl. Sodiated triacylglycerols (TAGs), their epoxy/hydroxy and hydroperoxy derivatives, as well as TAGs with shortened chain fatty acids (β-scission products) were clearly observed in the spectra. LDI–TOF MS rapidly provides semiquantitative information about the oxidation level of edible oil, and thus represents a very useful quality control tool. Dedicated to Professor Pier Giorgio Zambonin on the occasion of his 72nd birthday.     

Separation of triacylglycerols and free fatty acids in microalgal lipids by solid-phase extraction for separate fatty acid profiling analysis by gas chromatography

Microalgal lipids were separated into two fractions, triacylglycerols (TAGs) and free fatty acids (FFAs), by solid-phase extraction employing sodium carbonate as the sorbent and dichloromethane (20% by volume) in n-hexane as the extracting solvent. The TAG fraction was then saponified, followed by acidification, extraction and tert-butyldimethylsilyl esterification. The FFA fraction was directly acidified, extracted and derivatized. From the lipid extracts of eight microalgal species examined, a total of 13 fatty acids were detected in the TAG fractions and nine were found in the FFA fractions, with at much higher total TAG content in all microalgae. Oleic acid was the most prominent fatty acid in three species, α-linolenic acid was more abundant in two others, and palmitic acid was present in highest concentration in the remaining three species.     

Application of selected ion monitoring to the analysis of triacylglycerols in olive oil by high temperature-gas chromatography/mass spectrometry

The analysis of the triacylglycerol (TAG) composition of oils is a very challenging task, since the TAGs have very similar physico-chemical properties. In this work, a high temperature-gas chromatographic method coupled to electron ionization-mass spectrometry (HT-GC/EI-MS), in the Selected Ion Monitoring (SIM) mode, method was developed for the analysis of TAGs in the olive oil; this is a method suitable for routine analysis. This method was developed using commercially available standard TAGs. The TAGs studied were separated according to their equivalent carbon number and degree of unsaturation. The peak assignment was carried out by locating the characteristic fragment ions having the same retention time on the SIM profile such as [RCO+74]⁺ and [RCO+128]⁺ ions, due to the fatty acyl residues on sn-1, sn-2 and sn-3 positions of the TAG molecule and the [M−OCOR]⁺ ions corresponding to the acyl ions. The developed method was very useful to eliminate the interferences that appeared in the mass spectrum since electron ionization can prevent satisfactory interpretation of spectra.