Strategy for stereospecific characterization of natural triacylglycerols using multidimensional chromatography and mass spectrometry

Stereoisomeric determination of individual triacylglycerols (TAGs) in natural oils and fats is a challenge due to similar physicochemical properties of TAGs with different fatty acid combinations. In this study, we present a strategy to resolve the enantiomeric composition of nutritionally important TAGs in sea buckthorn (Hippopha & euml; rhamnoides) as an example food matrix. The targeted strategy combines 1) fatty acid profiling with GC, 2) separation of TAGs with RP-HPLC, 3) stereospecific separation with chiral-phase HPLC and 4) structural characterization with MS. Three major asymmetric diacid-and triacid-TAG species were analyzed in sea buckthorn pulp oil. Off-line coupling of RP-HPLC and chiral-phase HPLC allowed separation of several TAG regioisomers and enantiomers, which could not be resolved using one-dimensional techniques. Enantiomeric ratios were determined and specific structural analysis of separated TAGs was performed using direct inlet ammonia negative ion chemical ionization method.Of the TAG 16:0/16:1/16:1 palmitic acid (C16:0) was located predominantly in a primary position and the enantiomeric ratio of TAG sn-16:1-16:1-16:0 to sn-16:0-16:1-16:1 was 70.5/29.5. Among the TAGs 16:0/16:0/18:2 and 16:0/16:0/16:1, only ca 5% had C16:0 in the sn-2 position, thus, ca 95% were symmetric sn-16:0-18:2-16:0 and sn-16:0-16:1-16:0. The enantiomeric ratio of triacid-TAGs containing C16:0 and two unsaturated fatty acids (palmitoleic C16:1, oleic C18:1 or linoleic acids C18:2) could not be resolved due to lack of commercial enantiopure reference compounds. However, it became clear that the targeted strategy presented offer unique and convenient method to study the enantiomeric structure of individual TAGs.(c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/

Effects of Insect Herbivory on Bilberry Production and Removal of Berries by Frugivores

The evolutionary purpose of a fleshy fruit is to attract seed dispersers and get the seeds dispersed by frugivorous animals. For this reason, fruits should be highly rewarding to these mutualists. However, insect herbivory can alter plant reproductive success e.g. by decreasing fruit yield or affecting the attractiveness of the fruits to mutualistic seed dispersers. Under natural conditions, we tested the effects of experimental larval-defoliation on berry ripening and consumption of a non-cultivated dwarf shrub, the bilberry (Vaccinium myrtillus L.), which produces animal-dispersed berries with high sugar and anthocyanin concentration. Bilberry ramets with high fruit yield were most likely to have their berries foraged, indicating that frugivores made foraging choices based on the abundance of berries. Moreover, the probability for berries being foraged was the lowest for non-defoliated ramets that grew adjacent to larval-defoliated ramets, even though larval-defoliation did not affect the biochemical composition (total concentrations of anthocyanins, sugars and organic acids) or the probability of ripening of berries. We hypothesise that the lower probability for berries being foraged in these ramets may be a consequence of rhizome- or volatile-mediated communication between ramets, resulting in a priming effect of the herbivore defence and lower attractiveness of the non-defoliated ramets.Peer reviewe

Direct inlet negative ion chemical ionization tandem mass spectrometric analysis of triacylglycerol regioisomers in human milk and infant formulas

A previously developed direct inlet tandem mass spectrometric method for analysis of triacylglycerol (TAG) regioisomers was updated and validated for operation with current instrumentation with improved sensitivity and throughput. TAG regioisomers in pooled Chinese and Finnish human milk samples, two bovine milk samples and 11 infant formulas were identified and quantified. A total of 241 TAG regioisomers were identified and quantified, consisting of over 60 mol% of all TAGs in the human milk samples. The infant formulas deviated largely from human milk in regioisomeric composition of TAGs. In the Finnish and Chinese human milks, the most abundant ones were 1,3-dioleoyl-2-palmitoylglycerol (OPO; 7.4 and 8.8 mol% of all TAGs) and 1(3)-linoleoyl-2-palmitoyl-3(1)-oleoylglycerol (LPO; 4.7 and 8.3 mol% of all TAGs). In the infant formulas 1,2(2,3)-dioleoyl-3(1)-palmitoylglycerol (OOP) and 1(3)-linoleoyl-2-oleoyl-3(1)-palmitoylglycerol/1(3)-palmitoyl-2-linoleoyl-3(1)-oleoylglycerol (LOP/PLO) were more abundant than OPO and LPO. The differences between human milk and infant formula prompt for further development of current formulas

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

Publisher's version (útgefin grein)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 % ee).The authors thank Jani Sointusalo at University of Turku for assistance with the recycling system, Dr. Sigridur Jonsdottir at the Science Institute, University of Iceland, for the accurate mass measurements, Novozymes AS in Denmark for the lipase and Olav Thorstad of Pronova BioPharma ASA in Norway for pure DHA. The work was funded by the Academy of Finland (Decision Nos. 310982 and 315274 ), and the Finnish Cultural Foundation . Haraldur G. Gudmundsson acknowledges financial support for a temporary postdoctoral research at University of Turku and Jóhann D. Magnússon financial support from Lýsi hf. in Iceland .Peer Reviewe

A novel UHPLC-ESI-MS/MS method and automatic calculation software for regiospecific analysis of triacylglycerols in natural fats and oils

Regioisomeric analysis of triacylglycerols (TAGs) in natural oils and fats is a highly challenging task in analytical chemistry. Here we present a software (TAG Analyzer) for automatic calculation of regioisomeric composition of TAGs based on the mass spectral data from recently reported ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC−ESI−MS/MS) method for analyzing TAG regioisomers. The software enables fast and accurate processing of complex product ion spectra containing structurally informative diacylglycerol [M+NH4−RCO2H–NH3]+ and fatty acid ketene [RCO]+ fragment ions. Compared to manual processing, the developed software offers higher throughput with faster calculation as well as more accurate interpretation of chromatographically overlapping isobaric TAGs. The software determines results by constructing a synthetic spectrum to match the measured fragment ion spectrum, and by reporting the optimal concentrations of TAGs used to create the synthetic spectrum. This type of calculation is often extremely challenging for manual interpretation of the fragment ion spectra of isobaric TAGs with shared fragments, hence the need for automated data processing. The developed software was validated by analyzing a wide range of mixtures of regiopure TAG reference compounds of known composition and a commercial olive oil sample. Additionally, the method was also applied for regiospecific analysis of TAGs in human milk as an example of natural fats and oils with a highly complex TAG profile. The results indicate that the software is capable of resolving regioisomeric composition of natural TAGs even of the most complex composition. This novel calculation software combined with our existing UHPLC-ESI-MS/MS method form a highly efficient tool for regioisomeric analysis of TAGs in natural fats and oils.</p

Strategies for analyzing the regio- and stereospecific structures of individual triacylglycerols in natural fats and oils

Food lipids are mainly found in the form of triacylglycerols (TAG) containing three structurally identical or different fatty acids. The positional distribution of fatty acids is under genetic control. In TAG biosynthesis, fatty acids are specifically esterified in glycerol in enzyme-catalyzed reactions resulting in distinctive TAG species that are not randomly built. The chiral nature of other macromolecules in food, such as proteins and carbohydrates, is well established in science but the chirality of lipids and their functions has been a long-standing question. The aim of this work is to contribute to this research area of chirality by giving fresh insights into new methods and chromatographic retention behavior of TAGs, and to study the detailed molecular structure of individual TAGs in natural fats and oils. Novel chromatographic methods (RP- and chiral-phase HPLC) combined with mass spectrometry (MS) were used in the practical work for separation and identification of regioisomers (the positional distribution of fatty acids between the positions sn-2 and sn-1/3) and enantiomers (the positional distribution of fatty acids in the positions sn-1, sn-2, sn-3) of nutritionally important TAGs. ESI-MS/MS and NIAPCI-MS/MS were applied to study the regiospecific composition of TAGs in human milk, and ammonia NICI-MS and MS/MS to confirm the enantiospecific structures of TAGs of sea buckthorn pulp oil as the model food component. The studies included development of a chiral chromatographic method based on sample recycling. The suitability of different MS/MS methods in the analysis of TAG regioisomers was investigated. Overall, the results revealed new information about the methodology and the chiral elution behavior of TAGs. The chiral-phase [2 × cellulose tris-(3,5-dimethylphenylcarbamate)] recycling HPLC method in a polar-organic phase enabled resolution of 17 out of the 21 enantiopairs of TAGs studied. Presence of both saturated and unsaturated fatty acid favored the separation by the chiral columns. A TAG enantiomer with an unsaturated FA in the sn-1 position eluted ahead of its enantiomer despite the length of carbon chains or the number of DBs. Within the TAG regioisomer 16:1-16:1-16:0 in sea buckthorn pulp oil, the enantiomeric ratio of sn-16:1-16:1-16:0 to TAG sn-16:0-16:1-16:1 was 70.5:29.5. Overall, the results clearly showed the non-random positional distribution of natural FAs, and the abundance of unsaturated FAs in the position sn-2, which is common in plant oils. The other most abundant TAG species included mainly TAGs with three different FAs, which were not possible to analyze with the current methodology. The method developed during the PhD project was also successfully applied to an enantiomeric purity assessment of synthesized TAGs. Despite the promising progress in the stereospecific analysis of individual TAGs the elution order of TAG enantiomers is challenging to predict due to the complicated intermolecular forces involved in chiral recognition mechanisms. All new information concerning chiral retention behavior is essential for further development in the field. Chiral phase liquid chromatography combined with mass spectrometric analysis proved to be a reliable method for analysis of lipid stereoisomers in food and other biological systems. These results offer valuable information and a reliable tool to study specific positional distribution of fatty acids in natural samples and can be applied to studies of both food composition and nutritional aspects.Elintarvikkeiden lipidit koostuvat pääosin triasyyliglyseroleista, joissa on kolme samanlaista tai erilaista rasvahappoa. Rasvahappojen paikkajakauma on geneettisesti säädelty. Rasvahapot ovat esteröityneet glyseroliin entsyymikatalysoidussa triasyyliglyserolien biosynteesissä muodostaen kombinaatioita, jotka eivät ole sattumanvaraisia. Muiden ravinnon makromolekyylien kuten proteiinien ja hiilihydraattien kiraalisuus tunnetaan tieteessä jo varsin hyvin, ja myös lipidien kolmiulotteisia rakenteita ja niiden vaikutuksia on pyritty selvittämään jo vuosikymmenten ajan. Tutkimustyön tavoitteena oli kehittää rasvojen kiraalisuuden tutkimusta, soveltaa uusia menetelmiä triasyyliglyserolien kromatografisen retentiokäyttäytymisen selvittämiseen sekä tutkia luonnon rasvojen ja öljyjen yksittäisten triasyyliglyserolien yksityiskohtaista molekyylirakennetta. Nykyaikaisia kromatografisia menetelmiä (käänteisfaasi- ja kiraalifaasinestekromatografia) yhdistettyinä massaspektrometriaan (MS) käytettiin ravitsemuksellisesti tärkeiden triasyyliglyserolien regioisomeerien (rasvahappojen paikkajakauma asemien sn-2 and sn-1/3 välillä) ja enantiomeerien (rasvahappojen paikkajakauma asemien sn-1, sn-2 and sn-3 välillä) erottamiseen ja tunnistamiseen. Regiospesifisessä analyysissä sovellettiin eri massaspektrometrimenetelmiä (ESI-MS/MS ja NIAPCI-MS/MS) äidinmaidon triasyyliglyserolien tutkimiseen. Ammoniakki NICI-MS- and MS/MS -menetelmien avulla varmistettiin mallielintarvikkeena tutkitun tyrnimarjan hedelmälihan öljyn triasyyliglyserolirakenteet enantiospesifisessä analyysissä. Lisäksi kehitettiin näytteen kierrätykseen perustuva kiraalikromatografinen menetelmä. Tutkimuksessa tarkasteltiin eri massaspektrometristen menetelmien soveltuvuutta triasyyliglyserolien regiospesifiseen analyysiin. Kokonaisuudessaan tuloksena saatiin uutta tietoa menetelmistä ja yhdisteiden eluutiokäyttäytymisestä kiraalisessa ympäristössä. Tutkitusta 21:stä triasyyliglyserolista 17:sta saatiin enantiomeerit erotettua toisistaan käyttäen näytteen kierrätykseen perustuvaa nestekromatografiaa, jossa oli kiraalinen stationaarifaasi [2 × selluloosa tris-(3,5-dimetyylifenyylikarbamaatti)] ja eluenttina metanoli. Asymmetristen triasyyliglyserolien tyydyttynyt ja tyydyttymätön rasvahappo edistivät erottumista. Enantiomeeri, jossa tyydyttymätön rasvahappo oli asemassa sn-1, eluoitui toista enantiomeeria nopeammin hiiliketjun pituudesta tai kaksoissidosten lukumäärästä riippumatta. Yhdisteen sn-16:1-16:1-16:0 suhde sen sn-16:0-16:1-16:1 enantiomeeriin oli tyrnimarjan hedelmälihassa 70,5:29,5. Kokonaisuudessaan tulokset osoittivat selvästi luonnon rasvahappojen ei-sattumanvaraisen paikkajakauman ja tyydyttymättömien rasvahappojen runsauden asemassa sn-2, mikä on tyypillistä kasviöljyille. Muut merkittävimmät jakeet koostuivat pääsiassa kolme erilaista rasvahappoa sisältävistä triasyyliglyseroleista, joiden analysoiminen ei ollut mahdollista tässä tutkimuksessa käytetyllä menetelmällä. Väitöskirjatyössä kehitetyn menetelmän osoitettiin olevan tehokas myös synteesituotteiden enantiopuhtauden määrittämisessä. Vaikka yksittäisten triasyyliglyserolien yksityiskohtaisen molekyylirakenteen analysointi on viime vuosikymmenen aikana huomattavasti kehittynyt, on haasteellista ennustaa enantiomeerien eluutiokäyttäytymistä johtuen monimutkaisista molekyylien välisistä kiraalisista vuorovaikutuksista. Yhdisteiden retentiokäyttäytymisen perusteellinen selvittäminen on välttämätöntä jatkokehityksen kannalta. Kiraalinen nestekromatografia yhdistettynä massaspektrometriaan tarjoaa luotettavan menetelmän ravinnon ja muiden biologisten matriisien lipidien isomeerien analysoimiseen. Tuloksena saatiin arvokasta tietoa ja luotettava menetelmä luonnon rasvojen ja öljyjen yksityiskohtaisen paikkajakauman tutkimiseen. Tulevaisuudessa menetelmää voidaan soveltaa niin elintarvike- kuin ravitsemustutkimuksissa

In situ quantitative 1H NMR spectroscopy discriminates between raw and steam cooked potato strips based on their metabolites

A direct quantitative proton nuclear magnetic resonance spectroscopy method was developed for the measurement of saccharides, organic acids and amino acids in potato (Solanum tuberosum L.) tuber filaments, a complex gel-like food matrix. The method requires minimal sample preparation. It is thus a faster alternative compared to liquid sample matrices, as well as an extension to methods analyzing only selected metabolites in the sample. The samples in this study were either raw or steamed potato strips that were either measured as D2O extracts or directly without extraction or derivatization steps (in situ technique). A total of 22 compounds were identified in extracts and 18 in potato strips. Of these, 20 compounds were quantifiable in potato extracts and 13 compounds in potato strips. The effect of thermal processing was reflected in the profile of analyzed compounds. One example was fumaric acid, which was completely lost in steamed samples in both measurement techniques. Additionally, the content of γ-aminobutyric acid in steamed potato strips was lower. In potato extracts, the contents of additional 7 compounds were statistically different. The raw and steamed samples separated into two groups with multivariate models both in extracts and potato strips, and these groups were linked to changes in aforementioned compounds. These results demonstrated that the in situ quantitative 1H NMR technique is a useful tool to analyze potato metabolites. This technique could be further applied to any gel-like complex matrix, meaning that lengthy sample pretreatment could be skipped

Triacylglycerol biosynthesis in developing Ribes nigrum and Ribes rubrum seeds from gene expression to oil composition

201