Synthesis and enantiospecific analysis of enantiostructured triacylglycerols containing n-3 polyunsaturated fatty acids

The stereospecific structure of triacylglycerols (TAGs) affects the bioavailability of fatty acids. Lack of enantiopure reference compounds and effective enantiospecific methods have hindered the stereospecific analysis of individual TAGs. Twelve novel enantiostructured AAB-type TAGs were synthesized containing one of the three n-3 polyunsaturated fatty acid: α-linolenic acid (ALA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) in sn-1 or sn-3 position. These compounds formed six enantiomer pairs, which were separated with recycling high-performance liquid chromatography using chiral columns and UV detection. The chromatographic retention behavior of the enantiomers and the stereospecific elution order were studied. The enantiomer with an n-3 PUFA in the sn-1 position eluted faster than the enantiomer with the n-3 PUFA in the sn-3 position, regardless of the carbon chain length and number of double bonds of the PUFA. TAG enantiomers containing DHA exhibited highly different retention on the chiral column and were separated after the first column, whereas recycling was needed to separate the enantiomer pairs containing ALA or EPA. The system using two identical columns and one mobile phase, without sample derivatization, proved to be very effective also for peak purity assessment, confirming the enantiopurity of the synthesized structured TAGs being higher than 98 % (96


Introduction
There is an increasing interest in the health effects of n-3 polyunsaturated fatty acids (PUFAs). α-Linolenic acid (18:3n-3, ALA) is an essential fatty acid and a precursor for synthesis of other long-chain n-3 PUFAs. Due to the low conversion of ALA to eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), also EPA and DHA are often considered as essential components in the diet (Swanson et al., 2012). In addition to fatty acid composition, the positional distribution of the fatty acids in triacylglycerols (TAGs) is an important quality parameter as it affects the bioavailability of fatty acids.
In TAGs, the fatty acyl groups are covalently attached by ester bonds in the glycerol backbone in one of the three stereospecifically distinct locations termed sn-1, sn-2 and sn-3 (IUPAC-IUB Commission on Biochemical Nomenclature, 1977). When the two terminal primary hydroxyl groups of a glycerol are esterified with different fatty acids, the resulting TAG displays chiral asymmetry and thus, has two enantiomers. While enantiomers may differ only slightly in their chemical and physical properties, they are likely to have different biological activities in chiral environments like in the human body due to the three-dimensional structure-activity relationships (Smith, 2009). The stereospecific distribution of fatty acids in TAGs of natural fats and oils is under genetic control, making the positional distribution of fatty acids not random (Brockerhoff, 1971;Vichi et al., 2007).
Chirality of the other major food components like proteins and carbohydrates is well studied (Marchelli et al., 1996;Zawirska-Wojtasiak, 2006), whereas knowledge of the enantiomeric composition of lipids, TAGs in particular, is limited. To understand the health impact of nutritionally important fatty acids in natural TAG molecules in foods, reliable methods and tools are needed to study their enantiospecific location within the TAGs. Synthesis of novel enantiopure reference compounds is crucial for research on structure-activity relationship of TAGs and for development of analytical methods of TAG enantiomers (Kalpio et al., 2015;Kristinsson et al., 2014). Enantioselective HPLC is a promising technique with (Kalpio et al., 2015;Nagai et al., 2011) and without sample recycling systems (Lísa and Holčapek, 2013;Řezanka et al., 2018) to investigate the stereospecific distribution of fatty acids in TAGs of natural sources. Further research related to stereospecific separation of TAGs is still needed, to improve the separation of regioisomers and enantiomers in a single analysis.
In order to enable the bioavailability studies of n-3 PUFAs located in different sn-positions, various types of structured TAGs possessing EPA and DHA have been synthesized. These TAGs include normal-structured MLM (medium-long-medium) and similar ABA type TAGs possessing pure EPA or DHA located in the sn-2 position along with saturated even carbon number fatty acids ranging from C2 to C18 occupying the sn-1,3-terminal positions of the TAGs (Halldorsson et al., 2003;Haraldsson et al., 2000;Magnusson and Haraldsson, 2010). Furthermore, reversed-structured TAGs of the LML type have also been synthesized, possessing saturated fatty acids (SAFAs) in the sn-2 position and EPA or DHA located in the sn-1,3 positions (Gudmundsdottir et al., 2015;Magnusson and Haraldsson, 2012). These syntheses were all based on the use of a highly regioselective Candida antarctica lipase (CAL) immobilized as CAL-B, which played a key role in the syntheses by offering a full regiocontrol in terms of directing the SAFAs activated as vinyl esters exclusively into the less hindered terminal positions of glycerol. The same lipase offered similar regiocontrol in directing the n-3 PUFAs, activated as oxime esters, into the terminal positions of the glycerol backbone (Magnusson and Haraldsson, 2012). The use of activated fatty acids was essential for ensuring fast action of the lipase under mild temperature to avoid acyl migration side reaction (Compton et al., 2012;Laszlo et al., 2008), which is critical for the regiocontrol of the reactions. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was subsequently used as a coupling agent to chemically incorporate the n-3 PUFAs or SAFAs into the mid-position of the resulting 1,3-DAGs in highly efficient two-step chemoenzymatic processes.
In this study, our aim was to synthesize and chromatographically separate the enantiostructured AAB type TAGs containing one n-3 PUFA (ALA, EPA, or DHA) in sn-1 or sn-3 position. There are various reasons for the interest in enantiostructured TAGs possessing n-3 PUFA esterified to enantiospecific positions of the glycerol molecule. One was to design enantiostructured AAB type TAGs possessing pure ALA, EPA, or DHA in the sn-1, sn-2 or sn-3 position as unique TAG model molecules for investigating the impact of enantiospecific position of EPA and DHA on the bioavailability of these n-3 PUFAs in animals and humans (Linderborg et al., 2019). Symmetric TAGs constituting EPA and DHA in their sn-2 position are already available and fully characterized through the aforementioned synthesis, whereas the synthesis of the corresponding asymmetric TAGs has not been reported before. Secondly, we aim to extend our previous research on synthesis and enantiospecific separation of enantiostructured AAB type TAGs (Kalpio et al., 2015). In that study, the enantiomeric separation was performed on two AAB types of enantiostructured TAGs containing C12-C22 fatty acids with 0-2 double bonds (DBs), namely TAGs possessing two identical saturated fatty acids along with one unsaturated fatty acid, as well as TAGs possessing two identical unsaturated fatty acyl groups and one saturated one. In the current paper, we address a new challenge in the synthesis and analysis of enantiostructured AAB type TAGs possessing n-3 PUFAs with 3-6 DBs (ALA, EPA, or DHA) in one of the enantiomeric sn-1/sn-3 positions along with pure stearic (C18:0) or palmitic acids (C16:0) in the remaining positions. Separation of the resulting TAG enantiomers by the chiral-phase HPLC will produce novel information on elution order of TAG enantiomers containing n-3 PUFAs. Further, this will provide unique data for investigating the influence of carbon chain length and the number of DBs on the elution order of TAG enantiomers.

Materials and methods of synthesis
2.1.1. General 1 H and 13 C nuclear magnetic resonance spectra were recorded on a Bruker Avance 400 spectrometer at 400 and 101 MHz, respectively, and referenced to the residual peak stated (CDCl 3 , δ = 7.26/77.2).
Chemical shifts (δ) are reported in parts per million (ppm) and the coupling constants (J) are reported to the nearest 0.5 Hz. The following abbreviations are used to describe the multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; dd, doublet of doublets; m, multiplet. Infrared spectra were conducted on a Thermo Nicolet FT-IR iS10 Spectrophotometer on a KBr pellet (crystalline material) or as a neat liquid (oils). Melting points were determined on a Büchi 520 melting point apparatus and are uncorrected. The high-resolution mass spectra (HRMS) were acquired on a Bruker micrOTOF-Q mass spectrometer. All data analysis was done on Bruker software. Optical activity measurements were performed on an Autopol R V Automatic Polarimeter from Rudolph Research Analytical using a 40T-2.5-100-0.7 Temp Trol™ polarimetric cell with 2.5 mm inside diameter, 100 mm optical path length and 0.7 mL volume with c referring to g sample/100 mL.

Syntheses 2.1.2.1. 3-O-
Benzyl-sn-glycerol, (R)-1. Sodium hydride (13.4 g, 60 wt % dispersion in mineral oil, 336 mmol, 3.0 equiv.) was treated several times with dry pet. ether using a syringe to remove the mineral oil. To the resulting suspension in THF (280 mL) at 0 °C was added (S)-solketal (14.0 mL, 112 mmol, 1.0 equiv.) dropwise via syringe. The cooling bath was removed, and the resulting suspension was stirred at room temperature for 1 h before being cooled back to 0 °C followed by dropwise addition of benzyl bromide (15.9 mL, 134 mmol, 1.2 equiv.). The resulting mixture was brought to reflux and stirred for 4 h after which it was cooled to 0 °C, diluted carefully with EtOH (172 mL), followed by addition of a 2.0 M aqueous solution of HCl (168 mL). After stirring at 80 °C for 1 h, the resulting mixture was cooled to room temperature and quenched by addition of a saturated aqueous solution of NaHCO 3 (300 mL). The organic layer was separated and the aqueous layer further extracted with EtOAc (3 × 200 mL), the combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo to give the crude product which was purified via column chromatography (petroleum ether / EtOAc (50 %) → petroleum ether / EtOAc (80 %)) to give (R)-1 (19.8 g, 109 mmol, 97 %) as a yellow oil; [ Spectroscopic data was in agreement with that obtained for (S)-1 as previously reported (Kristinsson et al., 2014).

1-O-
Benzyl-sn-glycerol, (S)-1. (S)-1 was prepared in accordance with (R)-1. Spectroscopic data was identical to (R)-1 and in agreement with that as previously reported (Kristinsson et al., 2014). For more details, please see Supplementary material. 2.1.2.3. General procedure a: enzymatic di-acylation procedure. A mixture of benzyl-protected glycerol (1.0 equiv.) and saturated fatty acid (2.1 equiv.) were brought to a melt (80 °C) followed by addition of an immobilized Candida antarctica lipase B (CAL-B) (5.0-10.0 wt % of total mass). Next, vacuum was carefully applied and the resulting suspension stirred at 80 °C at a rate not damaging the lipase carrier material for 16−48 h before being cooled to room temperature. The resulting solid material was dissolved in CH 2 Cl 2 , the lipase separated by filtration and the filtrate concentrated in vacuo to give the crude product which was purified by recrystallization.  Spectroscopic data was in agreement with that as previously reported (Kristinsson et al., 2014).
2.1.2.8. General procedure B: catalytic hydrogenolysis procedure. An oven-dried round-bottom flask, equipped with a stirrer bar, was charged with benzyl-protected glycerol (1.0 equiv.) and Pd/C (10 wt % Pd, 3−5 mol%). The flask was evacuated, filled with nitrogen, the appropriate volume of hexane (10.0 mL / mmol substrate) and THF (1.0 mL / mmol substrate) added, and the flask was purged with hydrogen before a single drop of HClO 3 (70 wt% in water) was added. Hydrogenation (H 2 balloon) was conducted at r.t. with vigorous stirring until completion (monitored by TLC). The reaction mixture was then diluted with THF, filtered through Celite and concentrated in vacuo to give the crude product which was purified by recrystallization.

HPLC enantiomer resolution and enantiopurity analysis 2.2.1. Materials and reagents
The enantiostructured TAGs were analyzed by using chiral-phase LC method to investigate their chromatographic elution behavior in the chiral environment and to confirm the enantiopurity. All samples were diluted to hexane or to a mixture of hexane and isopropanol (3:2, v/v). Both solvents were HPLC grade and purchased from VWR International (Radnor, PA). One enantiomer of each synthesized enantiopair was analyzed as such to get the retention times and to automate the switching between the columns. Enantiopairs were analyzed as a mixture of 75 %:25 % to determine their enantiomeric separation and the elution order and as an enantiomeric mixture of 99 %:1 % to study the enantiopurity.

HPLC instrumentation
To improve the chromatographic separation by increasing the column length, chiral recycling HPLC (R-HPLC) method described previously (Kalpio et al., 2015) was applied, using two polysaccharidebased chiral stationary phases, CHIRALCEL OD-RH (150 × 4.6 mm, 5 μm, Chiral Technologies Europe, Illkirch, France), in polar-organic phase mode. A precolumn (10 × 4.0 mm, 5 μm) was connected outside to equalize the cycles. Methanol (Sigma-Aldrich Corporation, St. Louis, MO) was used as a mobile phase and isopropanol as the washing solution. The column temperature was 25 °C, flow rate 0.5 mL/min, and injection volume 10 μL. Before injections, samples were diluted to isopropanol to a concentration of 1 mg/mL. The samples were detected using a UV detector at 205 nm. The sample recycling system with a tenposition valve allowed analytes to alternate between two similar chiral columns enabling enantioseparation. Once timed separately for each enantiopair by used input, the switching between two columns was automated. Before enantiomeric resolution, good chromatographic performance, including peak shape and width, was ensured. The flow rate of the mobile phase and sample size were optimized to narrow down the peak width. With the R-HPLC configuration containing two columns, the influence of flow rate on column pressure was followed carefully.

Data treatment
The peak areas were integrated using a LabSolutions 5.57 software (Shimadzu, Kyoto, Japan). The following data analysis parameters were used: width 200 s, slope 150-200 μV/min, drift 0 (automatic) and minimum area 1000 counts. Due to the different enantiomeric resolution, the value for slope had to be adjusted. However, the similar data analysis parameters for each enantiopair were applied, and consequently, the results were comparable. Within each enantiopair, the peak area ratio was regarded as the ratio of concentrations between the two enantiomers separated. For calculations, the average value of 2-4 last peak pairs was used depending on the efficiency of enantiomeric separation (Table 3).
Enantiomeric resolution (R s ) was calculated by the instrument software according to Eq. (1). (1) where t R1 = retention time, t R2 = retention time of previous peak, W = peak width, W p = peak width of previous peak The peak width (W) is the time between two intersection points on the baseline by the tangent lines drawn from the left and right inflection of a peak.
Enantiopurity. There are different calculation procedures for enantiopurity due to the different methods used for determination. IUPAC defines enantiopurity as enantiomer excess (Moss, 2009) M. Kalpio, et al. Chemistry and Physics of Lipids 231 (2020) 104937 3. Results and discussion

Synthesis
The synthetic route illustrated in Scheme 1 was followed for the synthesis of the structured TAGs (shown for the R-enantiomers) commencing from (S)-solketal. This is a very similar approach to the one that was followed in our previously described synthesis of enantiopure AAB type TAGs possessing two identical saturated fatty acyl groups (palmitic or stearic acids) along with one unsaturated fatty acid, either as oleic acid or linoleic acid (Kristinsson et al., 2014). In that case no enzyme was involved and the EDCI coupling agent was used to introduce the saturated fatty acyl groups chemically into the sn-3 and sn-2 positions of the benzylated glycerol adduct (S)-1, whereas in the current approach this was executed enzymatically by use of immobilized Candida antarctica lipase (CAL). In the final step, the pure n-3 PUFAs (ALA, EPA and DHA) were introduced to the sn-1 terminal position by Steglich esterification by use of the EDCI coupling agent after removal of the benzyl protective moiety by catalytic hydrogenolysis over Pd/C.
Referring to Scheme 1 the benzyl-protected glycerol derivatives (R)-1 and the corresponding (S)-1 were accomplished by a previously described two-step procedure (Kristinsson et al., 2014), starting from (S)and (R)-solketals, respectively, and obtained in excellent yields (94 and 95 % over the two steps, respectively). Palmitic acid or stearic acid was introduced to both hydroxyl groups of (R)-1 by use of CAL-B to accomplish benzyl protected (S)-2a (palmitic acid) and (S)-2b (stearic acid) in excellent yields, 97 and 92 %, respectively. In our previous report these derivatives were prepared by aid of EDCI coupling agent and were not purified prior to the debenzylation. The enzymatic reaction was performed without solvent under vacuum at 80 °C. It took 24 h for the reaction to proceed to completion and in this reaction, we have firm reasons to believe that the fatty acids were incorporated into the 2position by acyl-migration, promoted by the high temperature and the presence of protons from the fatty acids (Haraldsson et al., 1995). The diacylated adducts (S)-2a and (S)-2b were isolated and purified by crystallization from ethanol. Similarly, the corresponding antipodal adducts (R)-2a and (R)-2b were obtained in 95 and 80 % yields, respectively.
As before, deprotection of the benzyl moiety was executed by catalytic hydrogenolysis over Pd/C by aid of catalytic amounts of perchloric acid (Hartung and Simonoff, 1953;Rylander, 1990). The deprotection took place very cleanly with no sign of acyl-migration as was firmly established by 400 MHz 1 H and 13 C NMR spectroscopy analysis. That technique is of great use to monitor a full regiocontrol of reactions involving 1,3-and 1,2-DAGs as well as 1-and 2-MAGs as has been described in details in previous reports (Halldorsson et al., 2003;Haraldsson, 2012, 2010). There were no indications of any 1,3-DAGs present in the products from the hydrogenolysis reaction. The resulting enantiostructured DAGs (S)-3a and (S)-3b were accomplished in 98 and 80 % yields, respectively, after recrystallization from hexane. Similarly, the antipodal (R)-derivatives were obtained in 97 and 87 % yields, respectively, for (R)-3a and (R)-3b.

Table 1
The identities of the enantiostructured TAG products (S)-4a,b -6a,b and (R)-4a,b -6a,b, constituting pure ALA (4), EPA (5) and DHA (6) together with palmitic (a) and stearic (b) acids, along with their obtained yields and specific optical rotations.  Kalpio, et al. Chemistry and Physics of Lipids 231 (2020) 104937 mass spectrometry analyses (HRMS) offered satisfactory results. The specific optical rotation values warrant a comment in remaining extremely low. This was to be expected as a result of the cryptoactivity behavior that is characteristic of chiral TAGs that was also observed in our previous studies involving the AAB type TAGs. The cryptoactivity term refers to compounds displaying specific rotation remaining close to zero and hardly measureable (Foss et al., 2005;Schlenk, 1965). Baer and Fischer were the first to report such behavior for TAGs in the late 1930s (Baer and Fischer, 1939;Fischer and Baer, 1941). As can be noticed from the results in Table 1 the specific optical rotation absolute values ranged between 0.02 to 0.18, lowest for the ALA adducts, however, the signs remained consistent in terms of negative values for all the (S)-and positive for the (R)-TAGs. By the methodology described we have also succeeded in synthesizing tens of grams of enantiostructured TAGs possessing pure DHA along with stearic acid (25 g of each of the TAG enantiomers (R)-and (S)-6b) and palmitic acid (50 g each of the (R)-and (S)-6a enantiomers). The former TAGs with DHA and stearic acid have already been submitted to animal bioavailability studies on rats along with 25 g of the corresponding symmetrically ABA structured TAG possessing DHA in the 2-position and stearic acid in the remaining 1,3-positions accomplished by previously described procedures (Linderborg et al., 2019). The corresponding TAGs possessing DHA and palmitic acid syntheses have also been completed (50 g of each TAG type) that will be used in further animal studies. For these large-scale syntheses DHA of purity levels ≥ 95 % was used, whereas in the material intended for the chiral separation studies the purity levels of all n-3 PUFAs including DHA were 99 %.

Chromatographic results
The adjusted retention times (t 'R =t R -t M ) after the first column and equivalent carbon numbers of the TAGs [ECN, defined as the number of carbon atoms in the fatty acid residues (ACN) minus twice the number of DBs] are presented at Table 2.
Both the length of the acyl chain and the number of DBs of the fatty acids have a crucial impact on retention of TAGs. In our previous study, the retention of TAGs decreased with increasing unsaturation and with decreasing acyl chain length when polar mobile phase was used. In the current study, TAGs with C16-C20 fatty acids were eluted in the order of ECN, in agreement with our previous findings (Kalpio et al., 2015). Additionally, the current study showed that presence of an n-3 PUFA with 0-5 DBs in the TAG did not change elution order based on the ECN. However, when the number of DBs increased from 5 (EPA) to 6 (DHA), the elution order of TAGs did not follow the ECN values. TAGs containing DHA with six DBs eluted later than TAGs containing EPA with five DBs, even though the ECN were the same. The main reason is likely the spatial configuration of very long carbon chains of the n-3 PUFAs in the TAG molecules (Řezanka et al., 2018). Řezanka and coworkers also noticed certain abnormalities in the retention with even up to 20 min difference in the retention times of TAGs with the same ECN, when TAGs containing at least one very long chain fatty acid were chromatographed on two chiral (3,5-dimethylphenylcarbamate modified β-cyclodextrin) columns using gradient of hexane and hexane/2propanol. Despite the different chromatographic conditions applied, similar mechanisms probably explain the elution behavior of DHA-TAGs: the interactions of the DHA enantiomers with the mobile phase and the stationary phase. Cis-unsaturated fatty acid tails of DHA-TAGs bent to a greater extent than the less unsaturated PUFAs, due to the higher number of cis-double bonds present, each causing a permanent kick and bending in the hydrocarbon chain.
Based on the findings of previous research and the current study, ECN values should be used to predict the order of elution in chiral environment only for TAGs containing long chain PUFAs with 1-5 DBs under similar chromatographic conditions. Even though some systematic behavior can be noticed, it is a challenging task to find any common rule about the elution order of chiral TAGs. This is due to the fact that the chiral recognition mechanisms at molecular level are still not fully understood in the chromatography of lipids. In addition to the traditional comprehensive lipid profiling, one trend of lipidomic studies is to aim for a deeper understanding of a specific lipid class (Yang et al., 2016). Thus, all new information on the chiral retention behavior at the molecular level is essential for further development in the field.

Enantiomer resolution and elution order
The applied R-HPLC method confirmed the presence of two enantiomers in each of the pairs synthesized. The method was demonstrated to enable the resolution of TAG enantiomers with at least up to six DBs. Resolution of the chiral separation of enantiopairs examined after the last column calculated by the instrument software varied between 1.0 and 3.8 (Fig. 1). Only TAGs containing DHA were separated with one column without recycling, but not to the baseline. The peak shape and area remained constant and the peak broadening did not limit the analysis. Consequently, recycling was continued until the desired resolution was achieved. All enantiopairs were clearly separated after four columns. Previously, less peak broadening and better separation was offered with two identical columns compared to singlecolumn (Liu et al., 2014).
The non-destructive UV-vis detection enabled elucidation of the whole separation. As TAG enantiomers have identical physicochemical properties in an isotropic environment (Sánchez et al., 2012), the detector response is the same for both enantiomers. Usual disadvantages in lipid analysis, namely differences in sensitivity between saturated and unsaturated TAGs and insufficient sensitivity for all saturated TAGs (Holčapek et al., 2005), did not hinder the analysis in this study.
Elution order for SSU/USS type enantiopairs with two saturated and one unsaturated fatty acids was in accordance with earlier findings (Kalpio et al., 2015). The enantiomer with an n-3 PUFA in the sn-1 position eluted faster than the enantiomer with the unsaturated fatty acid in the sn-3 position. Despite the different retention behavior of DHA-TAGs compared to other TAGs, there was no difference in elution order between DHA-TAG enantiomers. The enantiomeric elution order has been opposite when non-polar solvents like hexane-based mobile phases were used (Lísa and Holčapek, 2013;Řezanka et al., 2018).

Enantiopurity
With all enantiomers examined the ee (%) was more than 96 % (Table 3). No impurities of the opposite enantiomer were detected from sn-ALA-16:0-16:0. The small shoulder of the opposite enantiomer i.e. the peak area of impurity was less than 2 % of the peak area of the actual target compound. Performance of the system to detect small enantiomeric impurities was confirmed using a sample of each enantiomer spiked with 1 % of the opposite enantiomer. When comparing a chromatogram of enantiostructured TAG with the one spiked with 1 % of the less abundant enantiomer, the opposite enantiomer was clearly visible in the latter (Fig. 2).   2017). Thus, enantiopurity is essential in many applications. In pharmaceutical applications, the methods that determine the specific optical rotation are commonly used for the characterization of the optical purity (Gergely, 2000). The chiroptical techniques like the measurement of optical rotatory dispersion (ORD) and circular dichroism (CD) have also been applied to study saturated glycerides (Gronowitz and Herslof, 1975). Because the optical activity of TAGs is very low, the chiral chromatographic methods have a crucial role in studying enantiopurity. Recently, enantioselective chromatography has been increasingly used not only as a tool for chiral analyses but also as a technique to determine enantiomeric impurities. The R-HPLC can be used to enhance separation efficiency and for example to be applied to study enantiopurity or other impurities in the sample (Liu et al., 2014;Trone et al., 2006). Enantioresolution is the main requirement of the reliable method to study enantiopurity due to the inability of UV detector to detect the co-eluting impurities.

Conclusions
Twelve enantiostructured TAGs were successfully synthesized in a five-step chemoenzymatic approach by use of a lipase. Six pairs of TAG enantiomers were formed, all of the AAB type containing one n-3 PUFA (ALA, EPA or DHA) attached to the terminal sn-1 or sn-3 position of the glycerol backbone along with two palmitic or stearic acids located in the remaining positions. They were all obtained in high chemical purity within very high to excellent yields. These compounds are valuable material for bioavailability studies and as enantiopure reference compounds for chromatographic separation and analysis.
The chiral-phase recycling HPLC (R-HPLC) method enabled resolution of the synthesized TAG enantiomers, and their elution order and chromatographic behavior were firmly established. Elution order of the enantiomers was found to be in agreement with our previous findings determined in our previous study, with the enantiomer with an n-3 PUFA in the sn-1 position eluting faster than its enantiomer with the n-3 PUFA in the sn-3 position. However, the retention behavior of the TAG enantiomers containing very long chain fatty acid with six DBs (DHA) was different compared to the TAG enantiomers containing C12-C22 fatty acids with 0-5 DBs. Retention of DHA-TAGs was higher than TAGs containing ALA or EPA and they were enantioseparated without recycling. The elution order was based on the ECN for TAGs containing C16-C20 fatty acids with 0-5 DBs. However, DHA-containing TAGs did not follow this elution order. All the DHA-TAGs eluted later than the EPA-TAGs with the same ECN values. Based on the results, we can conclude that no common rules can be applied to predict the elution behavior of all chiral TAGs, which highlights the importance of further studies in the field.
The compounds analyzed with the chiral R-HPLC-UV were all of excellent enantiomeric excess (>96 %). Among the immense number of molecular species of natural TAGs, only a limited number of TAG reference compounds, and practically no enantiopure TAGs, is commercially available. Thus, reliable and workable synthesis is an obligatory route in order to study the bioactivities of TAG enantiomers and to study the chromatographic behavior of these compounds.
Synthesis of enantiostructured TAGs together with the chiral R-HPLC method enables studies on the role of chirality in food and other biological systems. The knowledge whether a compound is naturally present as a pure enantiomer or with a specific enantiomeric ratio can lead to speculations on the importance of the enantiospecificity for a certain function. The stereospecific information on TAGs offers a new platform for investigations into lipid synthesis and metabolism.

Author contributions
All authors have given approval to the final version of the manuscript.

Declaration of Competing Interest
The authors declare no competing financial interest.