Triacylglycerol Regioisomers Analysis
Triacylglycerols (TAGs) are chemical compounds that are major components of vegetable oils and animal fats. TAG are formed by one molecule of glycerol esterified by fatty acid molecules in all three OH groups. The chemical formula of TAG is RCOO-CH2-CH(-OOCR')CH2OOCR" wherein R, R', and R" are long alkyl chains. The three fatty acids RCOOH, R'COOH and R"COOH may be completely different, or two of them or all of them may be identical. The length of the fatty acid chains in naturally occurring TAG may vary, but the most common are chains having 16, 18, 20, or 22 carbon atoms. Natural fatty acids in plants and animals are typically composed of only even-numbered chain lengths because they are biosynthesized from acetyl-CoA. Bacteria, however, have the ability to synthesize acids with odd chain lengths and branched chains. Therefore, ruminant fat also contains acids with an odd number of carbon atoms, for example 15 or 17, because they are produced by bacteria in the rumen. Most natural fats contain a complex mixture of different TAGs and therefore melt in a wide temperature range. The life functions of the cell have to be retained throughout this temperature range. One of the possibilities how to achieve this is to keep oil bodies in a fluid - non-crystalline state, which could be achieved by transitions between the crystalline forms. Regretfully, data on the melting points of crystalline forms of TAG regioisomers, as well as enantiomers, are available for only a few of them. Despite this, some data have been gathered, e.g. for SLO and SOL, wherein S - stearic, O - oleic, and L - linoleic acid, in which the melting points for individual crystalline α and β forms are -2 to -4 °C and -15 to -17 °C, and -11.5 to -13 °C and -18 to -19 °C, respectively. Although this holds for two regioisomers, i.e. compounds having the same FA and the same molecular weight, it is surprising that a mere change in the positions of two FA on the glycerol backbone shifts the melting point by as much as 10 °C.
A special nomenclature is used for the stereochemistry of TAGs instead of the usual D/L or R/S nomenclature, where the primary hydroxyl groups are often termed α- and α' and the secondary β, or the "stereospecific numbering" (sn) system recommended by the IUPAC-IUB commission.
When the two primary hydroxyl groups are esterified with different fatty acids, the resulting triacylglycerol can be asymmetric and thus can display "optical activity", although this is usually too low to be measured. Thus, e.g., 1-oleoyl-2,3-dipalmitoyl-sn-glycerol showed [α]D20 = 0.00 (c 7.05, CHCl3).
The number of theoretically possible molecular species increases with the growing number of fatty acids forming a mixture of TAGs, see Table 1.
Table 1. The number of possible TAGs.
|Description||Number of Possible TAGs||Number of Possible TAGs for y = 11|
|without isomers||x = (y3 + 3y2 + 2y)/6||286|
|without enantiomers||x = (y2 + y3)/2||726|
|all isomers||x = y3||1331|
where x is the number of TAGs, y is the number of FAs in TAGs
As seen from the Table, for a mere 11 FAs, the number of potentially possible TAGs rises to hundreds of molecular species. Let us point out that 11 FAs is a typical number of FAs obtained in the identification of any natural oil or fat. On the other hand, fish oil contains several dozen FAs and here several hundred molecular species of TAGs (exactly 553) were identified, without counting regioisomers and enantiomers .
Two aims are essentially sought when analyzing TAGs, i.e. the qualitative and, if possible, quantitative representation of individual molecular species. The analysis can thus be divided into several steps, which may or may not follow each other. The first step is to identify the TAG regardless of whether it will be possible to separate, or identify the regioisomers and enantiomers using MS. One of the most suitable methods seems to be shotgun lipidomics of glycerolipids which is mentioned elsewhere on this website. It should be noted that lipidomic analysis or shotgun analysis, in other words MS without separation by HPLC, cannot determine regioisomers and it does not matter whether we use low- or high-resolution MS. Only when a calibration mixture is made from the synthetic standards is it possible to determine the ratio of regioisomers, but not that of enantiomers, whose mass spectra are identical, see Fig. 1a, b, c below.
Fig. 1a. Tandem mass spectrum of natural AEE
Fig. 1b. Tandem mass spectrum of synthetic EEA
Fig. 1c. Tandem mass spectrum of natural EAE
Based on the knowledge of the fatty acids identified by GC-MS of FAME or picolinyl esters (dimethyloxazolines or pyrrolidines), if it is necessary to know and determine the position of double bond(s)), it is at least theoretically possible to determine the number of TAG molecular species. It should be noted, however, that the theoretical minimum number is much higher than the actual number of molecular species of identified TAGs, see an example of one of the most common oils, i.e. olive oil. 15 FA and 37 TAG were identified in the oil , which is less than 5% of the total TAGs, not including regioisomers and enantiomers. This oil essentially comprises only two major FAs, palmitic (P), and oleic acids.
TAGs containing branched FAs occur predominantly in microorganisms, e.g. in bacteria, especially iso- and/or anteiso-fatty acids. TAGs with linear and branched-chain FAs have exactly the same APCI mass spectra and cannot be distinguished from each other. They can be partly separated by RP-HPLC, wherein the branched TAGs are eluted before TAGs with linear FAs. The partitioning is unfortunately inadequate and only TAGs with two or three branched FAs are partially separated from TAGs with linear FAs .
The second step is the performance of regiospecific analysis of TAGs. At the beginning of this century essentially the only available methods were those using enzymes. Pancreatic lipase or extracellular lipase from the mold Rhizopus arrhizus was used for determining the FAs at positions α-(α') and/or β.
Another method was the reaction with Grignard reagent, conversion to phospholipids, and their hydrolysis by phospholipase A2 from snake (or bee) venom. It was also possible to use chiral chromatography, wherein diacylglycerols react with an appropriate isocyanate (e.g. (S)-(+)-1-(1-naphthyl)ethyl isocyanate) to form diastereomers, which are separated on a chiral column. The use of 13C-NMR to determine the FA in the sn-2 position was also described in these pages.
As has been repeatedly mentioned above, if each of the primary hydroxyls (or α'α) is esterified with a different FA, the prochiral secondary hydroxyl of glycerol becomes chiral. If the TAG is composed of two acids, e.g. palmitic and oleic acids, then with one P and two O acids may give rise to the following molecular species: sn-PPO, sn-OPP and sn-POP, where the sn-PPO and sn-OPP are enantiomers and sn-POP and sn-PPO and/or sn-OPP are regioisomers. Because these three isobaric TAGs are three different chemical compounds, they obviously differ in their physical and chemical properties, such as the melting point and [α]D, but also in their mass spectra, in the case of regioisomers, see below.
Only two methods for TAG separation are commonly used and are applicable without problems for the analysis. One of them is RP-HPLC and the other is Ag+-HPLC. Other methods such as TLC on plates with different carriers or supercritical fluid chromatography (SFC)  are only interesting from a theoretical point of view or are used for special purposes.
Separation of regioisomeric TAGs has been described many times, beginning with the pioneering study  and including the review  describing their separations from tens of different oils and fats by non-aqueous reversed-phase liquid chromatography (LC-NARP)  and silver-LC . The separation of regioisomeric TAGs therefore presents no problems for chemists versed in this field. The same holds for identification of regioisomers that has also been solved in principle (see, e.g., the identification of 762 regioisomeric TAG from krill oil , though without any quantification of individual molecular species). However, one should be aware that even this high number of identified TAGs is a mere fraction (about 1.38%) of the theoretical number of TAG which exceeds 54 000 (see Table 1).
Preliminary identification, which may be later confirmed by tandem MS, is based on the elution order of TAG, which best expresses the ECN (equivalent carbon number ECN=ACN-2xDB (ACN = acyl carbon number, DB = number of double bond(s)). Retention time depends mainly on the lengths of the fatty acid chains and the positions and configurations of the double bond(s), and increases with increasing ECN. Retention times within one ECN increase with a decreasing number of double bonds. Regioisomeric TAGs having the same ECN value can be separated on a high-efficiency column such as a combination of two or three columns in series, which provides a minimal efficiency of about 50,000 theoretical plates. So, for example, separation of regioisomers (OPP and POP, or OPO and POO) can be achieved . With this method, large differences were found between the representation of regioisomers in animal fat (wild boar) and in vegetable oil (sunflower), wherein the ratio of POP/OPP is 10/90 and 100/0 respectively.
Silver ion (Ag+-HPLC) is another technique for the separation of TAGs. The method is based on the principle of interaction of Π electrons of the double bond(s) with d electrons of silver. Weak reversible complexes are produced, wherein the silver ions are bound to the stationary phase, preferably on an ion-exchange column. With an increasing number of double bonds, the retention times of eluting TAGs also increase; however, it depends also on the steric availability of the double bond. It is thus possible to separate positional isomers of the FAs, for instance TAGs containing linolenic acids (αLn and γLn), e.g. αLnαLnαLn and/or αLnαLnγLn, etc. The best separation was, however, achieved using two-dimensional HPLC, either off-line or on-line (a combination of Ag+-HPLC and RP-HPLC). An advantage of the off-line 2D separation is the possibility to optimize the conditions in both dimensions; the disadvantage is that the method is labor intensive and time consuming. On-line 2D-HPLC is fully automated, but the efficiency of separation in the second dimension is reduced due to the short period of time (usually 1 minute) spent on sampling from the first column .
Atmospheric pressure chemical ionization (APCI) as the ionization technique is most suitable for MS analysis of TAG since it gives sufficiently abundant ions in the range of the molecular ion, i.e. ions of the type [M+H]+, [M+Li]+, [M+Na]+, [M+NH4]+, etc., and produces substantial diacylglycerol-like fragment ions, i.e. [DAG]+, which are ions corresponding to the loss of fatty acyl groups esterified to the glycerol backbone, [M+H-RiCOOH]+. Weak [M+H]+ ions are observed from saturated TAGs, but polyunsaturated TAGs produce an [M+H]+ as the base peak. In the case of the fully saturated TAGs like tripalmitin, the use of ammonium can yield [M+NH4]+, which has a greater abundance than the [M+H]+ and also important and abundant ions, often base peaks, of the type [M+H-RiCOOH]+ (DAG+ type ions), see also below. Tandem mass spectrometry (MS/MS) has yielded further structural information on the regioisomers, i.e. those TAGs in which the acyl chains are at different positions (i.e. AAB, ABA), or the double bond(s) in one or more of the acyl chains.
Already in 1996  APCI mass spectra of TAG regioisomers were found to contain variously abundant ions of the type [M+H-RiCOOH]+. The ratio of fragment ions, i.e. [M+H-R1(3)COOH]+ versus [M+H-R2COOH]+ has since been used for identification of regioisomers, since the FA esterified at the sn-2 position (β-OH, secondary hydroxyl of glycerol) is poorly displaceable in the mass spectrometer and thus a fragment [M+H-R2COOH]+ corresponding to loss of the FA from this position is less abundant than it should theoretically be, see Figs. 2a, b. It should be noted that similar rules generally apply for all lipids having fatty acids at the sn-1 and sn-2 positions [12,13].
Fig. 2a. Semipreparative RP-HPLC of regioisomers of triacylglycerol rac-PPE in the diatom Phaeodactylum tricornutum
Fig. 2b. Mass spectrum of synthetic PPE
Fig. 2c. Mass spectrum of synthetic PEP
In other words, the ratios of ions of type 1,3-[DAG]+/(1,2-[DAG]+ + 2,3-[DAG]+) are below unity so that e.g. the formation of the 1,2-isomer of the [R1(2)R2(3)]+ ion requires less energy than that involved in generating the analogous 1,3-ion from the TAG . Consequently, the two regioisomers show two different mass spectra.
The cleavage of the trifunctional TAGs, i.e. TAGs composed of 3 different FAs, gives rise to 18 isomeric TAG and thus the mass spectrum features 9 [DAG]+ ions including three pairs of enantiomers (AB, BA, AC, CA BC, CB). When enantiomers were also analyzed, e.g. in a model mixture, all 27 molecular species were identified. This rich mixture cannot be successfully analyzed using only MS without separation by HPLC, see e.g. papers by the Holcapek group [15,16]. It should be noted that the analysis of a natural sample is always more complex than the above-mentioned model mixture and, in particular, that in the model mixture resulting from randomization all the TAG are statistically represented, which does not hold for natural mixtures.
The result of the above rule is shown in the specific case of the triacylglycerol PoPoP (Po - palmitoleic acid) and its regioisomers and enantiomers. Positive ion APCI mass spectra of TAGs typically exhibited an [M+H]+ ion and fragment ions of the type [M+H-RCOOH]+ ([DAG]+) resulting from the loss of an acyl chain from the TAG. A monoacylglycerol-like fragment ([MAG]+) is equivalent to an [RCO+74]+ ion (see structures). The positive ion APCI mass spectrum of PoPPo shows an [M+H]+ ion at m/z 803 and fragment ions [M+H-RCOOH]+ at m/z 549 [PPo]+ and at m/z 547 [PoPo]+, [RCO+74]+ at m/z 313 for [P+74]+, or at m/z 311 for [Po+74]+, and [RCO]+ at m/z 239 [palmitoyl] and at m/z 237 [palmitoleoyl]. These ions were observed with sufficient intensity to analyze the mass spectrum. Another example is shown in Fig. 3, where the APCI mass spectrum of the TAG shows that this is a StLnSt (St-stearidonic, Ln-linolenic acid), as indicated by the following ions: [M+H]+ at m/z 869, [M+H-RCOOH]+ at m/z 591 (StSt) and m/z 593 (StLn), [RCO+74]+ at m/z 333 [St]+ and m/z 335 [Ln]+, RCO+ at m/z 259 (St) and m/z 261 (Ln).
Fig. 3a. Chiral LC of natural mixture of TAG, i.e., fraction having 11 double bonds after Ag-LC obtained from snow alga.
Fig. 3b. Mass spectrum of natural StLnSt
At this point it should be noted, however, that an accurate quantitative determination of two regioisomers requires the measurement of a calibration curve. That involves mixing pure regioisomers in at least three different ratios, which is often very difficult because the commercial availability of regioisomers is very low; the section below concerns their synthesis.
Negative APCI and also ESI of TAGs have also been described . The regioisomeric composition of TAGs in various vegetable oils was determined by direct inlet of ammonia to a negative APCI tandem MS system. The method is based on the preferential formation of [M-H-RCOOH-100]- ions (for structure see ) during collision-induced dissociation (CID) by loss of the sn-1/3 fatty acids from [M-H]- ions. For accurate determination of the ratio of regioisomers, the calibration curve was prepared from pure regioisomers in proportions 25:75, 50:50, and 75:25. The ratio of product ion [M-H-RCOOH-100]- intensities was determined and it has been found that e.g. the ratio of intensities of two regioisomers, (sn-LLO + sn-OLL) to LOL, is more than one. The product ions in the mass spectra corresponding to the m/z 497.4 and m/z 499.4 are [M-H-O-100]- (LL) and [M-H-L-100]- (OL or LB), with the ratios of the abundances of the ions being approximately 60:100 (sn-LLO+sn-OLL) or 10:100 (LO) .
Characterization and quantification of TAGs that have no electrostatic charge in solution can be carried out with electrospray ionization (ESI), but it requires the addition of an electrolyte such as ammonium or metal ions (e.g. Li or Na) to produce adduct ions [M+NH4 or Li or Na, etc]+.
Fragments of the type [M+Alk-RiCOOH]+ are also present but, unfortunately, they have lower abundances than in APCI. This is because the adducts [M+Na]+ or [M+K]+ are more stable and fragmentation in tandem ESI is more difficult than in APCI. The interpretation is also complicated by the presence of both protonated and sodiated fragment ions. It is therefore appropriate to add lithium salt, usually 1 mM, into the mobile phase . Naturally, as described previously , dual ionization is one of the most appropriate combinations.
At this point it is necessary to point out the difficulties caused by stable isotopes. Natural TAGs always contain certain and predetermined amounts of heavier isotopes, particularly 2H, 13C, 18O, etc. Their presence is manifested in two unpleasant effects. Firstly, e.g. the [M+H+2]+ (PPPo) TAG at m/z 807.7352 is a pseudomolecular ion with an abundance of about 15%, whereas the [M+H]+ from PPP is 807.7442 Da. The difference is so small that a mass spectrometer with low resolution cannot distinguish between these ions and thus, e.g. in shotgun lipidomics, the ion abundances of [M+H+2]+ (PPPo) are added to the abundances of [M+H]+ (PPP) , etc.
Another problem arises when the TAGs contain acids having more than 22 carbon atoms. For instance, for trinervonin (TAG containing three nervonic acids, i.e. 15(Z)-tetracosenoic acid), the [M+H]+ has a value of 1138.0728 Da and the rounded value of this ion is thus even and the nitrogen rule thus seemingly does not apply. Therefore, the use of nominal masses (accurate mass minus the mass defect) is recommended and preferred.
Preparation of standards
Two methods were used for the synthesis of standards. The first one consisted in using phospholipase C to obtain, from commercially available phosphatidylcholines, one of the enantiomers of diacylglycerol. This was then esterified to yield a corresponding TAG. This enabled us to obtain both regioisomers and enantiomers of TAGs having one or two PUFAs, e.g. eicosapentaenoic or arachidonic acids and palmitic acid .
The other method has already been successfully used and described. The principle of the method consists of reacting the free acid with 2,3-isopropylidene-sn-glycerol, and then deprotecting and esterifying the remaining two free hydroxyls (at the sn-2 and sn-3 positions) with the corresponding FA .
Although it is not the task of this website to deal with separation and identification of enantiomers, we at least partially did so, as this area is developing rapidly, as evidenced for example by the survey of literature data on the separation of TAGs on chiral columns written by Rezanka , which is accessible to readers without paid access to the articles in the journal Lipids.
Finally, it should be noted that the separation and identification of regioisomers and enantiomers is rapidly developing in parallel with the rapid development of instrumental techniques. Based on many dozens of published articles we unfortunately have to state that previously published molecular theories have nowadays essentially no informative value. This can be documented, for instance, on the example of marine fish and mammals that feed on the fish . Thus e.g. the sn-EEP/EPE/sn-PEE TAG ratios in harp seal and sardine are 0:77.8:22.2 and 9.8:17.8:72.4, respectively, which cannot be explained by the dependence on the food chain, but rather varying intensity biosynthetic pathways.
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Reviews or Chapters in Books
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- Byrdwell, WC. Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids. DOI: 10.1007/s11745-001-0725-5).
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- Mottram, HR. Regiospecific analysis of triacylglycerols using high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. In: Byrdwell WC. (eds) Modern Methods for Lipid Analysis by Liquid Chromatography/Mass Spectrometry and Related Techniques. AOCS Press, Champaign, IL, 276–297 (2005).
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