Regiospecific characterisation of the triacylglycerols in animal fats using high performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry

High performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI MS) was applied to the characterisation of triacylglycerols (TAGs) in animal fats. The major TAGs in four fats (beef, chicken, lamb and pork) were identified and positional isomers assigned according to their APCI mass spectra. Beef and lamb fat TAGs were confirmed as containing higher proportions of saturated fatty acids compared with those of chicken and pork. HPLC-APCI MS was also shown to be of value in providing regiospecific information for the fatty acids in individual TAG species. For example, beef and lamb fat were shown to contain both cis- and trans-isomers of the 18:1 fatty acid, whilst chicken and pork contained only the cis-isomer. When the position of fatty acid substitution was determined from the APCI spectra, whilst the cis- 18:1 was predominantly found in the 2-position of the TAG, the trans-18:1 showed a preference for the 1/3-position. Similarly, it was confirmed that although the 2-position of beef, chicken and lamb fat TAGs was dominated by unsaturated fatty acids, in pork fat, a characteristically high proportion of palmitic acid was seen in this position. The TAGs identified compared well with those reported previously. The distributions of 2-position fatty acids seen in lamb and pork fat compared favourably with those obtained by the more traditional method of lipase degradation. Although the distributions for chicken and beef showed some discrepancies, these can be attributed to weaknesses in the quantification procedure or the specificity of the lipase. Overall, the technique of HPLC-APCI MS has been shown to be very powerful for the regiospecific analysis of animal fats.     

Examining the collision-induced decomposition spectra of ammoniated triglycerides as a function of fatty acid chain length and degree of unsaturation. II. The PXP/YPY series

A series of positionally pure triglycerides (TAGs) of the form PXP and YPY, where P is the palmitate moiety and X and Y are large arrays of different fatty acid moieties, is synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. The intensities of the collision-induced decomposition (CID) products of ammoniated TAGs were examined as a function of chain length, degree of unsaturation, double-bond position, and cis/trans configuration of X and Y. The major CID products, the diglyceride (DAG) fragment ions and the MH(+) ions, are plotted as functions of chain length for the saturated and monounsaturated series of X and Y. Different trends for each of these series are observed. Trends in the intensities of these fragment ions are also characterized as a function of degree of unsaturation in the TAGs. In general, the fractional intensities of MH(+) increase with increasing degree of unsaturation in the TAGs. MH(+) is absent in the CID spectra of triglycerides containing three saturated fatty acid moieties, suggesting that the presence of double bonds fosters the formation of MH(+). Double bonds positioned close to the carbonyl carbon along the fatty acid chain promote the formation of the DAG fragment ion corresponding to the loss of the fatty acid. The collection of PXP/YPY data produced in this work is used to test the mechanisms of the formation and decomposition of ammoniated TAGs that were previously presented. The YPY data are used to predict the intensities of the fragment ions in the CID spectra of YPX-type TAGs. The limitations of the mathematical approach used in these predictions are discussed in context of a broader plan to develop a software platform for comprehensive analysis of complex TAG mixtures.     

Characterization,thermal properties and phase transitions of amazonian vegetable oils

Thermal profiles of buriti pulp oil (Mauritia flexuosa Mart.), tucumã pulp and kernel oils (Astrocarium vulgare Mart.), rubber seed oil (Hevea brasiliensis), passion fruit oil (Passiflora edulis) and ucuúba butter (Virola surinamensis) were analyzed by thermogravimetry (TG/DTG) and differential scanning calorimetry (DSC). Gas chromatography and calculated iodine values were performed to determine the fatty acid profile and to measure the degree of unsaturation in these oils, respectively. The TG curves showed three steps of mass loss, which can be attributed to the degradation of polyunsaturated, monounsaturated and saturated fatty acids. The DSC crystallization and melting curves are reported and depended on the fatty acid composition. Usually, oil samples with a high degree of saturation showed crystallization and melting profiles at higher temperatures than the oils with a high degree of unsaturation. The data obtained by physicochemical analysis of oil samples were analyzed by principal component analysis and hierarchical cluster analysis to increase understanding of the data set, examining the presence or absence of natural groupings between samples.     

Examining the collision-induced decomposition spectra of ammoniated triglycerides as a function of fatty acid chain length and degree of unsaturation. I. The OXO/YOY series

A series of positionally pure triglycerides (TAGs) of the form OXO and YOY, where O is the oleate moiety and X and Y are large arrays of different fatty acid moieties, was synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. The intensities of the collision-induced decomposition (CID) products of ammoniated TAGs (ammonium ion adducts) were examined as a function of chain length, degree of unsaturation, double-bond position, and cis/trans configuration of X and Y. The major CID products, the diglyceride fragment ions and the MH+ ion, were plotted as functions of chain length for the saturated and mono-unsaturated series of X and Y. Different trends for each of these series were observed. Trends in the abundances of these fragment ions were also characterized as a function of degree of unsaturation in the TAGs. In general, the fractional abundances of the MH+ ions vary linearly with degree of unsaturation. However, the presence of double bonds positioned close to the carbonyl carbon of the fatty acid chain promotes the formation of the diglyceride fragment ion corresponding to loss of that fatty acid. Mechanisms of the formation and decomposition of ammoniated TAGs are proposed that fit the trends observed in the data. Extensions of this work are described, and a vision of a derived library of CID spectra is discussed as a platform for comprehensive analysis of complex TAG mixtures.     

Silver-phase high-performance liquid chromatography–electrospray mass spectrometry of triacylglycerols

Silver-phase chromatography hyphenated with on-line electrospray mass spectrometry can be applied to characterize triacylglycerols present in vegetable oils with respect to their degree of unsaturation, the position of the most unsaturated fatty acid and the carbon number (CN). The CN information obtained with silver-phase HPLC–ESP–mass spectrometry is complementary to the unsaturation information obtained by silver-phase HPLC-flame ionization detection. Both information is essential to monitor or study modified vegetable oils on the presence of non-natural triacylglycerols. The quantitative results obtained with the method are in agreement with the results obtained in the silver-phase HPLC-flame ionization detection and with theoretical values calculated from the fatty acid distribution of the oil. Silver-phase HPLC–ESP-mass spectrometry gives direct information on fatty acid position and triacylglycerol CN, for each of the triacylglycerols in the sample. This in contrast with non-aqueous reversed-phase HPLC hyphenated with on-line atmospheric pressure chemical ionization-mass spectrometry, which requires a more extensive data processing. The results obtained with silver-phase HPLC–ESP-mass spectrometry can be presented in a three-dimensional overview (relative amount, CN, fatty acid position) serving as a fingerprint for the oil.     

Analysis of triacylglycerols and whole oils by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Since the introduction of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry the majority of research has focused on developing analytical methods for the qualitative determination of water soluble biomolecules such as proteins, peptides, carbohydrates, and oligonucleotides. This paper, however, reports the use of MALDI for the analysis of triacylglycerols and develops a new sample preparation method for nonpolar analytes. MALDI enables the rapid analyses of triacylglycerol (TAG) standards and mixtures of whole oils. The new method provides excellent shot to shot reproducibility, making quantification possible. Detection limits were in the mid femtomole range and the resolution was around 2000 which easily separated TAGs differing by one double bond. Sensitivity decreased with increasing molecular weight, causing biased results when analyzing complex mixtures with a significant range of molecular weight. In all cases only sodiated molecules and prompt losses of a fatty acid sodium salt were observed in the spectra. From this information it was possible to identify the three fatty acids on the glycerol backbone. Collision-induced dissociation was carried out on a triacylglycerol which proved to be useful for additional structural information, including the corroboration of the fatty acid components. With MALDI the percent compositions of TAGs in a standard olive oil was accurately determined. Finally, MALDI was used to examine the differences in lipid components between aged and fresh onion seeds, showing the potential of the technique for observing changes in lipid components in seeds.     

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.     

Reducing fragmentation observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of triacylglycerols in vegetable oils

Edible oils consist primarily of triacylglycerols (TAGs). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of the oils are typically dominated by sodium adducts of these TAGs but also show prominent fragment ions (that do not contain sodium), which can interfere with analytical measurements of other components in oils. The fragments seemingly correspond to the loss of a fatty acid moiety from the sodiated TAGs as a sodium salt: RCOONa. However, a previous study suggested that the fragments actually arise from nearly complete fragmentation of unseen protonated TAGs. These authors suggested that the fragmentation occurs so rapidly and completely that protonated TAGs are not normally observed in the spectra of these oils. In this paper, we present evidence to support their theory and also demonstrate an approach to eliminate these interfering ions from the MALDI-TOF mass spectra via addition of a base to the matrix/sample mixture. The added base does not impede formation of the sodiated TAGs, but does significantly reduce the amount of fragments observed. We propose that this occurs by depleting the H+ ions from the matrix, thus preventing the formation of significant numbers of protonated TAGs in the first place. For measurements by MALDI-TOF, the relative abundances of the fragment ions are related to the strength of the base, and can be almost completely eliminated. However, in longer time-scale experiments such as in post-source decay and Fourier transform mass spectrometry, sodiated and non-sodiated diacylglycerol (DAG)-like fragments are present in spectra, regardless of whether or not a base is added to the sample.     

Simple complementary liquid chromatography and mass spectrometry approaches for the characterization of triacylglycerols in Pinus koraiensis seed oil

A new simple strategy to identify triacylglycerols (TAGs) in oils and fats was performed using on line coupling of non aqueous reversed phase chromatography-electrospray ionization-mass spectrometry (NARP-LC-ESI-MS(2)) with silver nitrate (AgNO(3)) as post-column additive, and chromatographic data (partition number information and both the graphs of log k vs. number of double bond (DBN) and carbon number (CN)). NARP liquid chromatography permitted to separate TAGs composed of Δ5 and Δ9 but not from Δ11 double bond location on alkyl chain of fatty acid residues. Silver cationization improved the sensitivity by a factor one hundred. MS(2) information gave unambiguously the nature of three fatty acid residues bonded to glyceryl backbone of TAGs while log k against DBN and CN curves discriminated between the same molecular mass TAG isomers (whose constitutive fatty acid residues are double bond position and configuration isomers). Combination of structural information given by MS with chromatographic retention laws led to the development of a general methodology for determination of the structure of TAGs in lipids. This methodology was applied to Pinus koraiensis seed oil for which some uncommon TAGs are present. It permitted the identification of 58 TAGs in this oil. The experimental proof of 29 uncommon TAGs as component of this oil is demonstrated. Among them 26 were minor constituents.     

Examining the collision-induced decomposition spectra of ammoniated triglycerides. III. The linoleate and arachidonate series

A series of positionally pure triglycerides (TAGs) of the form LXL, YLY, AXA, and YAY was synthesized and analyzed by reversed-phase high-performance liquid chromatography/tandem mass spectrometry. L and A represent the linoleate and arachidate moieties, respectively, and X and Y represent large arrays of fatty acid moieties of various chain lengths, degree of unsaturations, double-bond positions, and cis/trans configurations. The abundances of the collision-induced decomposition (CID) products of ammoniated TAGs were examined as a function of these parameters. The major CID products, the diglyceride (DAG) product ions and the MH(+) ions, are plotted as functions of chain length for the saturated and monounsaturated series of X and Y. The following trends are observed in the data. TAGs with higher degrees of unsaturation tend to show greater relative abundances of MH(+) in the CID spectra of their ammoniated precursor ions. The position of the fatty acid constituents along the glycerol backbone also seems to influence the abundances of the MH(+) ion in the CID spectra of the ammoniated precursor ions. A fatty acid constituent with double bonds along the fatty acid chain positioned close to the carbonyl promotes the formation of the DAG product ion that corresponds to its loss upon CID of the ammoniated precursor ion. Linoleic acid substituents also seem to promote the formation of DAG product ions, but to a lesser extent. Data for the YAY TAGs are used to predict the abundances of the product ions in the CID spectra of ammoniated YAX TAGs. These data are discussed in context of a broader project to develop and validate software algorithims to support a platform for comprehensive analysis of complex mixtures of TAGs.     

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.     

Serum albumins as differential receptors for the discrimination of fatty acids and oils

Using fluorescent indicators and several serum albumins, a sensing system for fatty acids was developed. Fatty acids were able to be differentiated based on their carbon chain length and degree of unsaturation. A sensing ensemble was then applied to study complex mixtures of triglycerides, namely, edible oils. The oils, with different fatty acid compositions, were successfully differentiated using principal component analysis.     

植物油的质谱分析：1.蓖麻油

运用一种植物油的系统分析方法测定蓖麻油的精细组分，这包括皂化，ＢＦ３－ＭｅＯＨ甲基化，ＧＣ－ＣＩＭＳ法测定脂肪酸碳链长度和不饱和度，ＡＭＰ衍生化，ＧＣ－ＥＩＭＳ测定不饱和脂肪酸的双链位置，分析了蓖麻油的十一种脂肪酸组分，有五个为未知组分，并测定了其中三个组分的双链位置。     

Olive oil or lard?: Distinguishing plant oils from animal fats in the archeological record of the eastern Mediterranean using gas chromatography/combustion/isotope ratio mass spectrometry

Distinguishing animal fats from plant oils in archaeological residues is not straightforward. Characteristic plant sterols, such as β-sitosterol, are often missing in archaeological samples and specific biomarkers do not exist for most plant fats. Identification is usually based on a range of characteristics such as fatty acid ratios, all of which indicate that a plant oil may be present, none of which uniquely distinguish plant oils from other fats. Degradation and dissolution during burial alter fatty acid ratios and remove short-chain fatty acids, resulting in degraded plant oils with similar fatty acid profiles to other degraded fats. Compound-specific stable isotope analysis of δ(13)C(18:0) and δ(13)C(16:0), carried out by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), has provided a means of distinguishing fish oils, dairy fats, ruminant and non-ruminant adipose fats, but plant oils are rarely included in these analyses. For modern plant oils where C(18:1) is abundant, δ(13)C(18:1) and δ(13)C(16:0) are usually measured. These results cannot be compared with archaeological data or data from other modern reference fats where δ(13)C(18:0) and δ(13)C(16:0) are measured, as C(18:0) and C(18:1) are formed by different processes resulting in different isotopic values. Eight samples of six modern plant oils were saponified, releasing sufficient C(18:0) to measure the isotopic values, which were plotted against δ(13)C(16:0). The isotopic values for these oils, with one exception, formed a tight cluster between ruminant and non-ruminant animal fats. This result complicates the interpretation of mixed fatty residues in geographical areas where both animal fats and plant oils were in use.     

食用植物油中甘油三酯色谱分析方法研究进展

甘油三酯 (TAGs) 是植物油的主要成分(占95%~98%),对植物油的性质有着重要的影响。对其结构组成的分析有助于监控食用植物油质量,保障植物油食用安全。由于甘油骨架上可以结合的脂肪酸很多,导致甘油三酯的种类十分庞大,且一般天然油脂中的甘油三酯不仅物理化学性质非常接近,同时还存在大量的同分异构体和位置异构体。因此甘油三酯的分析是一项非常有挑战性的工作。该文对各类色谱分析技术在食用油中甘油三酯分析方面的应用进行了综述,评述了各方法的优缺点,并对指纹图谱技术和化学计量学方法在植物油鉴定中的应用进行了介绍。     

Comprehensive two-dimensional liquid chromatography x gas chromatography: evaluation of the applicability for the analysis of edible oils and fats

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LCXGC for detailed oil and fat analysis is evaluated. Comprehensive LCXGC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.     

Comprehensive two-dimensional liquid chromatography×gas chromatography: evaluation of the applicability for the analysis of edible oils and fats

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LC×GC for detailed oil and fat analysis is evaluated. Comprehensive LC×GC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.     

Determination of triacylglycerols of manketti oil by reversed-phase HPLC

Using reversed-phase HPLC with refractometric detection, we determined the composition of triacylglycerols (TAGs) of manketti oil and calculated its fatty acid composition. It was shown that the oil consists of octadecatrienoic (34.6 ± 0.8 mol %), linoleic (35.4 ± 0.8 mol %), oleic (14.6 ± 0.4 mol %), palmitic (7.2 ± 0.3 mol %), and stearic acids. Using spectrophotometric detection, for the oils of centrathus and jacaranda seeds, it was demonstrated that octadecatrienoic acid is presented mainly by α-eleostearic acid and small impurities of β-eleostearic (2.8 mol %) and jacarandic (0.3 mol %) acids. The results of the quantitative determination of TAGs, obtained by refractometric detection (with corrections of the peak areas to the calculated values of the refractive indices of TAGs), are consistent with the data obtained using spectrophotometric detection.     

Characterization and quantification of triacylglycerols in peanut oil by off‐line comprehensive two‐dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

The complexity of natural triacylglycerols (TAGs) in various edible oils is prodigious due to the hundreds of set is of TAG compositions, which makes the identification of TAGs quite difficult. In this investigation, the off‐line 2D system coupling of nonaqueous RP and silver‐ion HPLC with atmospheric pressure chemical ionization MS detection has been applied to the identification and quantification of TAGs in peanut oil. The method was successful in the separation of a high number of TAG solutes, and the TAG structures were evaluated by analyzing their atmospheric pressure chemical ionization mass spectra information. HPLC and MS conditions have been optimized and the fragmentation mechanisms of isomers have been validated. In addition, an internal standard approach has been developed for TAG quantification. Then this system was applied in peanut oil samples and there was a total of 48 TAGs including regioisomers that have been determined and quantified.     

Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag⁺ after their separation by non‐aqueous reversed‐phase liquid chromatography (NARP‐LC) before MS to improve MS sensitivity. Electrospray ionization–MS (ESI‐MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.