Mass Spectrometric Methodologies for Analysis of Triacylglycerol and Phospholipid Regioisomers in Natural Fats and Oils

Analysis of triacylglycerol (TG) and phospholipid (PL) regioisomers is a challenging task. The liquid chromatographic-mass spectrometric (LC-MS) analysis methods can be divided into two distinct categories: a) direct separation by chromatography or other means such as ion mobility, and b) quantification of regioisomer ratios by structurally informative fragment ions with mass spectrometric methods. Challenges arise from the large number of isobaric lipid species in natural samples, often overlapping chromatographically and sharing structurally informative fragment ions. Due to long retention times and difficult separation, researchers are moving away from direct chromatographic separation of isomers, using mass spectrometry instead. Many established analytical methods are targeting specific isomers of interest, and high-throughput untargeted analysis remains challenging, even though advancements have been made in the recent years. Fragmentation of glycerolipids is influenced by acyl chain lengths and numbers of double bonds of the attached fatty acids, and while certain fragmentation mechanisms offer better sn-specificity than others, the lack of available regiopure standards is still an obstacle for many researchers. In the thesis work, new tandem mass spectrometric (MS2) methods for investigating TG and PL regioisomer compositions in natural samples were developed. Two different instrumental methods and algorithmic calculation models were developed and validated for analysis of TG regioisomers. A direct inlet negative ion chemical ionization method along with an updated version of the MSPECTRA calculation software offers a rapid protocol for investigating TG regioisomers in complex samples such as human milk. While the absolute accuracy of the regioisomer ratio calculations is affected by the nature of the FAs in the TGs, the method is very useful for studying regioisomeric differences between samples. A new fragmentation model for analysis of TG regioisomers utilizing positive electrospray ionization (ESI+) LC-MS2 method was developed in addition to the direct inlet method. The model was created using calibration curves established with a wide range of regiopure TG standards, allowing prediction of fragmentation patterns for TG species without authentic standards. While the triple quadrupole MS instrument used for establishing the model is better suited for targeted approaches, the analysis can later be transferred to an untargeted platform utilizing data-dependent acquisition. Both calculation programs utilize optimization functions, attempting to find concentrations of regioisomers that produce the experimentally observed fragment ion spectra. This approach mitigates the effects of interfering isobaric fragments resulting from different isomeric TG species and the entire fragment spectra of multiple TG molecular species is handled simultaneously instead of each individually. The analysis of PL regioisomers was divided into two parts. In the first part, a hydrophilic interaction liquid chromatography (HILIC) method combined with a data-dependent MS2 acquisition was used to study the fragmentation behavior of different PL classes. Calibration curves were created with PL reference standards, enabling regioisomeric analysis of specific PL molecular species. For second part of the PL regioisomer analysis, the HILIC separation method was replaced by reversed phase chromatography, resulting in more reliable and efficient separation of PL molecular species within classes. An automated data preprocessing algorithm and a fragmentation model for untargeted PC regioisomer identification was created. Together, the developed LC-MS2 methods and calculation software and fragmentation models for TG and PL regioisomer analysis form a strong basis for further development and implementation to lipidomics platforms, enabling more detailed structural analysis in a rapidly growing area of research

Entsymaattisesti puristetun mustaherukkamehun aistinvarainen ja kromatografinen tutkimus

Työn tarkoituksena oli tutkia viidestä eri mustaherukkalajikkeesta puristettua mehua kemiallisesti käyttäen kaasukromatografia sekä aistinvaraisen arvioinnin avulla. Kromatografisen analyysin avulla mehuista tutkittiin sokeri- ja happopitoisuudet. Aistinvaraisessa arvioinnissa keskityttiin mehun makuominaisuuksien arviointiin. Työ oli osa laajempaa CurrantFood -projektia, jonka tarkoituksena oli mm. kehittää uusia käyttökohteita mustaherukalle. Ennen mehun puristusta marjoille tehtiin entsymaattinen käsittely ja puristus suoritettiin käyttämällä pientä laboratoriomittakaavan levypuristinta. Aistinvarainen arviointi suoritettiin koulutetun raadin avulla käyttäen kuvailevia menetelmiä. Tulosten tilastollisessa tarkastelussa havaittiin lajikkeiden välillä useita eroavaisuuksia, jotka olivat tilastollisesti merkittäviä, mutta eivät erityisen suuria. Selvin korrelaatio havaittiin mehujen sisältämien sokerien ja aistitun makeuden välillä. Lisäksi maun kokonaisvoimakkuus korreloi jonkin verran happamuuden ja karvauden kanssa. Tiettyjen ominaisuuksien osalta, kuten esimerkiksi astringoivuus, täyteläisyys ja marjaisuus, ei havaittu ollenkaan tilastollisesti merkittäviä lajikkeiden välisiä eroja.The purpose of the thesis was to study juice pressed from five different cultivars of black currant. The analyses were conducted using a gas chromatograph to study the amount of sugars and acids in the juice. The taste properties of the juice were studied by sensory evaluation. The thesis was part of a larger project called CurrantFood. The purpose of the CurrantFood project was to develop new applications for black currant. The berries were treated enzymatically before juice pressing. The juice pressing was conducted using a laboratory scale plate extractor. The sensory evaluation was carried out using descriptive methods. A statistical analysis of the results found a number of differences between cultivars that were statistically significant. The scope of the differences, however, was relatively small. A clear correlation was detected between the amount of sugars in the juice and the perceived sweetness. Also, the total intensity of the juice somewhat correlated with the perceived sourness and bitterness. Some of the studied properties such as astringency, roundness and berryness did not show any statistical variance between cultivars

Direct infusion and ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis of phospholipid regioisomers

Rationale Phospholipids are important components of cell membranes that are linked to several beneficial health effects such as increasing plasma HDL cholesterol levels, improving cognitive abilities and inhibiting growth of colon cancer. The role of phospholipid (PL) regioisomers in all these health effects is, however, largely not studied due to lack of analytical methods. Methods Electrospray ionization mass spectrometry in negative mode produces structurally informative fragment ions resulting from differential dissociation of fatty acids (FAs) from the sn-1 and sn-2 positions, primarily high-abundance [RCOO](-) ions. The fragment ion ratios obtained with different ratios of regiopure phospholipid reference compounds were used to construct calibration curves, which allow determination of regioisomeric ratios of an unknown sample. The method was developed using both direct infusion mass spectrometry (MS) and ultra-high-performance liquid chromatography and hydrophilic interaction liquid chromatography mass spectrometry (UHPLC-HILIC-MS). Results The produced calibration curves have high coefficients of determination (R-2 >0.98) and the fragment ion ratios in replicate analyses were very consistent. A test mixture containing 60/40% ratios of all available regioisomer pairs was analyzed to test and validate the functionality of the calibration curves. The results were accurate and reproducible. However, regioisomeric quantification of certain chromatographically overlapping compounds is restricted by the relatively wide window in precursor ion selection of the MS instrument used. Conclusions This method establishes a framework for analysis of phospholipid regioisomers. Specific regioisomers can be quantified using the existing data, and method development will continue with improving chromatographic separation and exploring the fragmentation patterns and efficiencies of different PL classes and FA combinations, ultimately to refine this method for routine analysis of natural fats and oils

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/

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

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

Selvitys akkuklusterin tutkimusinfrastruktuureista

Kansallisessa akkustrategiassa 2025 todetaan, että alan julkiset ja yksityiset tutkimus­infrastruktuurit tulisi verkottaa ja tunnistaa näissä olevat puutteet. Kehittämistyön pohjaksi työ- ja elinkeinoministeriö on tilannut teknologian tutkimuskeskus VTT:ltä selvityksen nykyisistä akkuklusterin julkisista ja yksityisistä tutkimusinfrastruktuureista. Selvityksessä kartoitettiin akkuklusterin nykyiset tutkimusinfrastruktuurit. Infrastruktuurit on koottu raportin liitteeksi sisältäen yleisluontoiset ja teknisemmät kuvaukset. Raportti sisältää kuvauksen nykytilasta. Lisäksi tunnistettiin aukkoja ja kehityskohteita. Selvityksen pohjalta esitetään useita toimenpiteitä, joiden avulla varmistetaan hyvät tutkimuksen perusedellytykset akkuklusterilla. Toimenpiteet sisältävät ehdotuksia laitteistoihin, kuten akkujen turvallisuustestaukseen, akkujen mekaaniseen kierrätykseen ja materiaalitutkimuksen analytiikkaan. Lisäksi toimenpiteinä ehdotetaan datan hyödyntämisen parantamista laatimalla pelisäännöt ja toteuttamalla akkuklusterin data-alusta. Lisäksi erilaiset testiympäristöt, kuten laajat sähköajoneuvojen latauksen testiympäristöt tulisi toteuttaa. Kansallisesti on tärkeää yhteistyön lisääminen ja osaamisen kehittäminen. Tutkimusinfrastruktuureja on mahdollista hyödyntää houkuttelemaan kansainvälisiä hankkeita ja invest-in -toimintaa, siksi ehdotetaan akkuklusterin infrastruktuurien verkottamista ja esittelyä kansainvälisille toimijoille

Determination of vitamin K from fermented foods

A quick and sensitive analysis method was developed for the analysis of vitamin K compounds, including phylloquinone (PK) and menaquinones (MK-n). The method utilizes ultra-high performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization (UHPLC-APCI-MS/MS). Non-chlorinated mobile phase composition was optimized for separation of eight vitamin K compounds on a reversed phase column in 10 minutes. Sample treatment with liquid and solid phase extractions and by the use of MK-4 as an internal standard enabled the quantitation of microgram level of vitamin K compounds in food. The UHPLC-MS/MS analysis is one of the fastest described methods for vitamin K analysis and consumes low amounts of solvents per sample. Sensitivity of the developed method is comparable to the other previously described vitamin K analysis methods using similar equipment. The method was tested with various different food samples and it was determined to be sufficient for most food matrices. Only one limiting factor was detected: high fat content of food. Food samples with less than 8% fat were quantified properly with high signal intensity. With higher fat content the sensitivity for each vitamin K compound would have to be checked before quantification. Due to restrictions in schedule the method was not optimized further for high-fat food samples. The method was used to screen and quantify vitamin K from 17 fermented food products. The highest amount of PK was detected in kimchi (42.4 ± 2.5 µg/100 g fresh weight), whereas the highest MK-7 content was detected in natto (901.7 ± 29.2). MK-9 was detected only in kefir (4.5 ± 2.4). Two Chinese fermented soybean pastes contained significant amount of MK-6 (4.5 ± 0.1 and 35.8 ± 15.6), MK-7 (86.3 ± 0.2, 11.9 ± 2.9), and MK-8 (44.0 ± 1.4 and, 22.3 ± 3.3)

Advanced Tandem Mass Spectrometric Analysis of Complex Mixtures of Triacylglycerol Regioisomers: A Case Study of Bovine Milk Fat

Comprehensive analysis of triacylglycerol (TAG) regioisomers is extremely challenging, with many variables that can influence the results. Previously, we reported a novel algorithmic method for resolving regioisomers of complex mixtures of TAGs. In the current study, the TAG Analyzer software and its mass spectrometric fragmentation model were further developed and validated for a much wider range of TAGs. To demonstrate the method, we performed for the first time a comprehensive analysis of TAG regioisomers of bovine milk fat, a very important and one of the most complex TAG mixtures in nature containing FAs ranging from short to long carbon chains. This analysis method forms a solid basis for further investigation of TAG regioisomer profiles in various natural fats and oils, potentially aiding in the development of new and healthier foods and nutraceuticals with targeted lipid structures