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.

Analysis of triacylglycerol and phospholipid sn-positional isomers by liquid chromatographic and mass spectrometric methodologies.

Analysis of triacylglycerol (TG) and phospholipid sn-positional isomers can be divided into two main categories: (a) direct separation by chromatography or other means such as ion mobility mass spectrometry and (b) quantification of regioisomer ratios by structurally informative fragment ions with mass spectrometric methods. Due to long retention times and limited performance, researchers are moving away from direct chromatographic separation of isomers, using mass spectrometry instead. Many established analytical methods are targeting specific isomers of interest instead of untargeted analysis of comprehensive profiles of regioisomers. Challenges remain arising from the large number of isobaric and isomeric lipid species in natural samples, often overlapping chromatographically and sharing structurally informative fragment ions. Further, fragmentation of glycerolipids is influenced by the nature of the attached fatty acids, and the lack of available regiopure standards is still an obstacle for establishing calibration curves required for accurate quantification of regioisomers. Additionally, throughput of many methods is still quite limited. Optimization algorithms and fragmentation models are useful especially for analysis of TG regioisomers, as identification using calibration curves alone without proper separation is difficult with complex samples.

Enzyme-assisted aqueous extraction of fish oil from Baltic herring (Clupea harengus membras) with special reference to emulsion-formation, extraction efficiency, and composition of crude oil.

Enzyme-assisted aqueous extraction (EAAE) is a green, and scalable method to produce oil and protein hydrolysates from fish. This study investigated the role of different parameters on emulsion formation, oil recovery, and the composition of crude oil during EAAE of Baltic herring (Clupea harengus membras). Fatty acid compositions, lipid classes, tocopherols, and oxidation status of the EAAE crude oils were studied. Compared to solvent-extracted oil, EAAE resulted in a lower content of phospholipids accompanied by a 57% decrease in docosahexaenoic acid. Changing fish to water ratio from 1:1 to 2:1 (w/w) with ethanol addition led to the greatest reduction (72%) of emulsion, which resulted in an increase in oil recovery by 11%. The addition of ethanol alone, or reduction of enzyme concentration from 0.4% to 0.1% also reduced emulsion-formation significantly. Overall, emulsion reduction resulted in higher content of triacylglycerols and n - 3 polyunsaturated fatty acids in the crude oil extracted.

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.

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 (R2  >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 (Hippophaë 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.

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.

Determination of vitamin K composition of fermented food.

A rapid ultra-high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometric (UHPLC-APCI-MS/MS) method was developed for the analysis of vitamin K compounds: phylloquinone (PK) and menaquinones (MK-n). Non-chlorinated mobile phase composition was optimized for separation of eight vitamin K compounds on a reversed phase column in 10 min. 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 method was used to screen and quantitate vitamin K from 17 fermented food products. The highest amount of PK was detected in kimchi (42 µg/100 g), whereas the highest MK-7 content was detected in natto (902 µg/100 g). Some MK-9 was present in kefir (5 µg/100 g). Two Chinese fermented soybean pastes contained significant amount of MK-6 (5-36 µg/100 g), MK-7 (12-86 µg/100 g), and MK-8 (22-44 µg/100 g).