Non-aqueous Reversed-phase Research Articles

Quantitative profiling of retinyl esters in milk from different ruminant species by using high performance liquid chromatography-diode array detection-tandem mass spectrometry

An effective high performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) analytical approach was developed for retinoid profiling in raw milk samples (cow, buffalo, ewe, and goat). The analytes were isolated by means of liquid-liquid extraction, including a “lipid freezing” step, with yields exceeding 66%. Since the positive atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) detection is not completely selective, a reliable identification has been accomplished by fully separating the analytes on a tandem C18/C30 column system under non-aqueous reversed phase (NARP) chromatography conditions. After validation, different milk varieties obtained from pasture-fed animals were analysed, providing, for the first time, the retinoid composition of both buffalo’s and ewe’s milk. According to the literature, retinyl palmitate has been found to be the most abundant vitamin A vitamer, but retinyl oleate is the prevalent form in the caprine milk.

Liquid chromatography/mass spectrometry identification of intermediates and vulcanization products by using squalene as vulcanization model compound.

Sulfur-vulcanized rubber is a three-dimensional polymer network, insoluble in all organic solvents. For this reason, vulcanization products are difficult to study and identify by conventional analytical techniques. To simplify this task, low molecular weight olefins have been used as model compounds (MCs) in place of rubber in vulcanization experiments. In this work, the vulcanization process was investigated using squalene (SQ) as MC. By-products, intermediates and products were separated by semipreparative reversed-phase liquid chromatography (RPLC) with UV detection. Each fraction was collected, concentrated and characterized by flow injection analysis (FIA) and non-aqueous reversed-phase (NARP) LC coupled to positive atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Under the latter conditions, an Information-Dependent Acquisition (IDA) was performed on a linear ion trap mass spectrometer to obtain structural information. Several vulcanized compounds containing up to three SQ molecules, cross-linked with chains involving up to 14 sulfur atoms overall, have been identified along with some of their oxidized products (epoxides and hydroperoxides). The FIA-MS spectra showed peak clusters, each of which included two-three subclusters; the interpretation was complicated by the occurrence of more ion species per product, by the unsaturation grade and by the characteristic isotopic distribution of sulfur. The enhanced product ion scan (EPI) spectra, acquired during the IDA experiments, supported the FIA-MS identification allowing one to count the number of sulfur atoms. The sensitivity of the developed analytical strategy was due to the enrichment factor achieved via semipreparative chromatography and the very good response of the APCI detection. Pattern fragmentation and chromatographic behavior simplified the identification of the cured compounds and their oxidized products, whose occurrence was related to the grade of oxidation of SQ used as reagent. Copyright © 2016 John Wiley & Sons, Ltd.

Analysis of diacylglycerols by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry: Double bond location and isomers separation

Diacylglycerols (DAGs) are important lipid intermediates and have been implicated in human diseases. Isomerism complicates their mass spectrometric analysis; in particular, it is difficult to identify fatty acid substituents and locate the double bond positions in unsaturated DAGs. We have developed an analytical strategy using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) in conjunction with dimethyl disulfide (DMDS) derivatization and collision cross-section (CCS) measurement to characterize DAGs in biological samples. The method employs non-aqueous reversed-phase chromatographic separation and profile collision energy (CE) mode for MSE and MS/MS analyses. Three types of fragment ions were produced simultaneously. Hydrocarbon ions (m/z 50–200) obtained at high CE helped to distinguish unsaturated and saturated DAGs rapidly. Neutral loss ions and acylium ions (m/z 300–400) produced at low CE were used to identify fatty acid substituents. Informative methyl thioalkane fragment ions were used to locate the double bonds of unsaturated DAGs. Mono-methylthio derivatives were formed mainly by the reaction of DAGs with DMDS, where methyl thiol underwent addition to the first double bond farthest from the ester terminus of unsaturated fatty acid chains. The addition of CCS values maximized the separation of isomeric DAG species and improved the confidence of DAG identification. Fourteen DAGs were identified in mouse myotube cells based on accurate masses, characteristic fragment ions, DMDS derivatization, and CCS values.

A Simultaneous Analytical Method to Profile Non-Volatile Components with Low Polarity Elucidating Differences Between Tobacco Leaves Using Atmospheric Pressure Chemical Ionization Mass Spectrometry Detection

Summary A comprehensive analytical method using liquid chromatography atmospheric pressure chemical ionization mass spectrometry detector (LC/APCI-MSD) was developed to determine key non-volatile components with low polarity elucidating holistic difference among tobacco leaves. Nonaqueous reversed-phase chromatography (NARPC) using organic solvent ensured simultaneous separation of various components with low polarity in tobacco resin. Application of full-scan mode to APCI-MSD hyphenated with NARPC enabled simultaneous detection of numerous intense product ions given by APCI interface. Parameters for data processing to filter, feature and align peaks were adjusted in order to strike a balance between comprehensiveness and reproducibility in analysis. 63 types of components such as solanesols, chlorophylls, phytosterols, triacylglycerols, solanachromene and others were determined on total ion chromatograms according to authentic components, wavelength spectrum and mass spectrum. The whole area of identified entities among the ones detected on total ion chromatogram reached to over 60% and major entities among those identified showed favorable linearity of determination coefficient of over 0.99. The developed method and data processing procedure were therefore considered feasible for subsequent multivariate analysis. Data matrix consisting of a number of entities was then subjected to principal component analysis (PCA) and hierarchical clustering analysis. Cultivars of tobacco leaves were distributed far from each cultivar on PCA score plot and each cluster seemed to be characterized by identified non-volatile components with low polarity. While fluecured Virginia (FCV) was loaded by solanachromene, phytosterol esters and triacylglycerols, free phytosterols and chlorophylls loaded Burley (BLY) and Oriental (ORI) respectively. Consequently the whole methodology consisting of comprehensive method and data processing procedure proved useful to determine key-components among cultivars of tobacco leaves, and was expected to additionally expand coverage that metabolomics study has ensured. [Beitr. Tabakforsch. Int. 27 (2016) 60-73]

Synthesis, liquid chromatographic fractionation and partial characterization of polybrominated dibenzofuran congeners

Polybrominated dibenzofurans (PBDFs) are a class of highly toxic environmental contaminants which comprises 135 structurally different congeners. While the gas chromatographic separation and analysis of the most polychlorinated dibenzofurans (PCDFs) are well-documented, comparably little data is currently available in the case of PBDFs. In this study dibenzofuran was brominated to give a mixture of ∼40 PBDFs with one to seven bromine atoms. This synthesis mixture was fractionated by both countercurrent chromatography (CCC) with the solvent system n-hexane/toluene/acetonitrile and non-aqueous reversed-phase high performance liquid chromatography (RP-HPLC) with acetonitrile as the mobile phase. All together 80 consecutive CCC fractions and 40 HPLC fractions were taken and analyzed for PBDFs by gas chromatography coupled to mass spectrometry (GC/MS). CCC and RP-HPLC offered orthogonal separation of the PBDF mixture. As a consequence, selected CCC fractions were further fractionated by RP-HPLC. In this way, eight PBDFs could be isolated and the structures of twelve PBDFs were elucidated by proton magnetic resonance spectroscopy (1H NMR).

Search for Non-Volatile Components with Low Polarity Characterizing Tobacco Leaves Using Liquid Chromatography / Atmospheric Pressure Chemical Ionization Mass Spectrometry Detector

SUMMARY There has been focus on the components with low polarity in tobacco leaf resin due to their probable relation with taste and aroma of tobacco products, the lack of a feasible analytical method and instrument has long been an obstacle to identifying the components with low polarity. The author thereby paid attention to the analysis employing nonaqueous reversed-phase chromatography hyphenated with a photo diode array detector and an atmospheric pressure chemical ionization mass spectrometry detector which has been considered applicable to the separation of significant but unknown non-volatile. The application succeeded in simultaneously separating, detecting and quantifying more than 100 non-volatile components with different low polarity such as solanesols, triacylglycerols, phytosterols, and chlorophylls. However, their compositional differences among various tobacco leaves still remained partial knowledge based on targeted analysis instead of global knowledge based on comprehensive analysis. No investigation searching for key components elucidating different tastes, aromas, species, cultivars, curing processes, and growing districts among tobacco leaves has been carried out so far. For this reason, all the quantification data were consolidated to form complete multidimensional matrix and were statistically processed to observe the categories and the key components of various tobacco leaves by principal component analysis and hierarchical clustering analysis. Tobacco leaves were first classified into three categories consisting of flue-cured Virginia, air-cured leaf, and Oriental. Solanesyl esters, phytosteryl esters and solanachromene contributed to the category of flue-cured Virginia, while air-cured leaf was characterized by free phytosterols. Oriental was featured by chlorophyll in addition to the contributory components to flue-cured Virginia. Non-volatile components with low polarity seemed to be degraded during curing process and to therefore characterize the different curing processes among various tobacco leaves.

Determination of the triacylglycerol fraction in fish oil by comprehensive liquid chromatography techniques with the support of gas chromatography and mass spectrometry data.

Fish oil made from menhaden (Brevoortia tyrannus) can be used as a dietary supplement for the presence of high levels of the long-chained omega-3 fatty acids, viz. epentaenoic and docosahexanoic. In this work, for the first time, two different multidimensional approaches were developed and compared, in terms of peak capacity, for triacylglycerol characterization. In particular, silver ion chromatography with a silver-ion column and non-aqueous reverse-phase liquid chromatography with a C18 column were tested in both comprehensive (stop-flow) and off-line modes. The use of mass spectra attained by atmospheric pressure chemical ionization for both LC approaches, and the fatty acids methyl esters profile of menhaden oil obtained by gas chromatography analysis, greatly supported the elucidation of the triacylglycerol content in menhaden oil. The off-line approach afforded a better separation and, thus, higher peak capacity to allow identifying and semiquantifying more than 250 triacylglycerols. Such a huge number has never been reported for a menhaden oil sample.The main disadvantage of such an approach over the stop-flow one was the longer analysis time, mainly attributable to solvent exchange between the two dimensions.

Analysis of 1,2-diol diesters in vernix caseosa by high-performance liquid chromatography – atmospheric pressure chemical ionization mass spectrometry

Fatty acid diesters of long-chain 1,2-diols (1,2-DDE), or type II wax diesters, were analyzed in the vernix caseosa of a newborn girl. 1,2-DDE were isolated from the total lipid extract by the semipreparative TLC using plates coated with silica gel. Chromatographic separation of the 1,2-DDE molecular species was achieved on the non-aqueous reversed-phase HPLC with two Nova-Pak C18 columns connected in series (a total length of 45cm) and using an acetonitrile–ethyl acetate gradient. 1,2-DDE eluted from the column in the order of their equivalent chain number. The analytes were detected as ammonium adducts by an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionization source. Their structures were elucidated using tandem mass spectrometry with MS, MS2 and MS3 steps in a data-dependent mode. More than two thousand molecular species of 1,2-DDE were identified in 141 chromatographic peaks. The most abundant 1,2-DDE were monounsaturated lipids consisting of a C22 diol and a C18:1 fatty acid together with C16:0, C14:0 or C15:0 fatty acids. The positions of double bonds were characterized by the fragmentation of [M+C3H5N]+ formed in the ion source.

Triacylglycerol profile in cocoa liquors using MALDI-TOF and LC-ESI tandem mass spectrometry.

Triacylglycerols are responsible for chocolate's peculiar melting behavior: the type and position of fatty acids on the glycerol molecule strongly affect the melting range of cocoa butter. For this reason, the characterization of triglyceride composition in cocoa products is particularly important. In this work, triacylglycerols extracted from cocoa liquor samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight (TOF) and electrospray ionization tandem mass spectrometry (MS/MS) coupled to liquid chromatography. Extracted samples were initially analyzed by direct injection in MS to obtain information on triglyceride molecular weights; relevant MS parameters were optimized, and the possible formation of the adducts [M + Na](+) and [M + NH(4)](+) was studied. Tandem mass experiments (both with triple quadrupole and TOF/TOF) were performed to study the fragmentation pathways (in particular, the loss of palmitic, stearic and oleic acid) and identify the triacylglycerols in cocoa liquors. Some signals of the spectra obtained with both MS techniques could indicate the presence of diacylglycerols in the cocoa extract, but different experimental evidences demonstrated that they were generated by the in-source fragmentation of triglycerides. A nonaqueous reversed-phase chromatographic separation was also developed and used to support the identification of the analytes; nine triacylglycerols were recognized in the cocoa liquor extracts. The three different batches of Ecuador cocoa liquor did not show significant differences in the triacylglycerol profile.

A Comprehensive Study on Triacylglycerols in Tobacco Leaves Using Liquid Chromatography and Atmospheric- Pressure Chemical-Ionization Mass Spectrometry

Abstract Triacylglycerols in tobacco leaves were investigated to further understand their compositional and metabolic differences among various tobacco leaves and their changes during the curing processes. Non-aqueous reverse-phase chromatography for efficient separation of triacylglycerols and atmospheric-pressure chemical-ionization mass spectrometry for their detection were selected for quantitative analysis. For measuring their absolute amounts, authentic triacylglycerols comprised of the same fatty acids were introduced to generate calibration curves and to quantify the analytes. A validation study using the combination of appropriate separation and authentic triacylglycerols produced very good results in terms of linearity (> 0.999), limit of detection (3-4 ng), limit of quantification (10-15 ng) and recovery ratio (80-120%). The quantification of triacylglycerols in tobacco leaves and a subsequent principal component analysis clarified for the first time their compositional and metabolic differences among various tobacco leaves.

Development of an Offline Bidimensional High-Performance Liquid Chromatography Method for Analysis of Stereospecific Triacylglycerols in Cocoa Butter Equivalents

Acyl migration is a serious problem in enzymatic modification of fats and oils, particularly in production of cocoa butter equivalent (CBE) through enzymatic acidolysis reaction, which leads to the formation of non-symmetrical triacylglycerols (TAGs) from symmetrical TAGs. Non-symmetrical TAGs may affect the physical properties of final products and are therefore often undesired. Consequently, an accurate method is needed to determine positional isomer TAGs during the production of CBE. A bidimentional high-performance liquid chromatography (HPLC) method with combination of non-aqueous reversed-phase HPLC and silver ion HPLC joining with an evaporative light scattering detector was successfully developed for the analysis of stereospecific TAGs. The best separation of positional isomer standards was obtained with a heptane/acetone mobile-phase gradient at 25 °C and 1 mL/min. The developed method was then used in multidimensional determination of the TAG positional isomers in fat and oil blends and successfully identified the TAGs and possible isomers in enzymatically acidolyzed CBE.

Comprehensive Profiling of Carotenoids and Fat-Soluble Vitamins in Milk from Different Animal Species by LC-DAD-MS/MS Hyphenation

This paper describes a novel and efficient analytical method to define the profile of fat-soluble micronutrients in milk from different animal species. Overnight cold saponification was optimized as a simultaneous extraction procedure. Analytes were separated by nonaqueous reversed-phase (NARP) chromatography: carotenoids on a C(30) column and fat-soluble vitamins on a tandem C(18) column system. Besides 12 target analytes for which standards are available (all-trans-lutein, all-trans-zeaxanthin, all-trans-β-cryptoxanthin, all-trans-β-carotene, all-trans-retinol, α-tocopherol, γ-tocopherol, δ-tocopherol, ergocalciferol, cholecalciferol, phylloquinone, and menaquinone-4), the DAD-MS combined detection allowed the provisional identification of other carotenoids on the basis of the expected retention times, the absorbance spectra, and the mass spectrometric data. Retinol and α-tocopherol were the most abundant fat-soluble micronutrients and the only ones found in donkey's milk along with γ-tocopherol. Ewe's milk also proved to be a good source of vitamin K vitamers. Bovine milk showed a large variety of carotenoids that were absent in milk samples from other species with the only exception of all-trans-lutein and all-trans-zeaxanthin.

Simultaneous Quantification of Oleins (triolein, diolein and monoolein) in Mouse Feces using Liquid Chromatography-Electrospray Ionization/Mass Spectrometry

Fat malabsorption is an important cause of poor growth in infancy and childhood. Steatorrhea tests have been developedusing various methods. Traditional measurements of stool fat, however, require large samples and it often takes as a week tocomplete the analysis. In this paper, a liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) methodwas developed for simultaneous quantitative analysis of triacylglycerols, triolein, diolein and monoolein, in mouse feces. Moreover,the procedure was rapid, simple as well as compatible with LC-ESI/MS. Chloroform-isopropyl alcohol solution was usedfor fat-soluble sample extraction. After centrifugation and filtration, an analytical solution was prepared. Triolein, diolein andmonoolein were separated using non-aqueous reversed-phase column with the mobile phase consisting of A (methanol) and B(acetone-isopropyl alcohol). The precision (% CV) and accuracy (% bias) of the assay were 3.8-14.7% and 85.2-114.9%, respectively. This method has been successfully applied to simultaneous determination of triolein, diolein and monoolein in feces from30 mice. This method can therefore be applied to measure triacylglycerols in mouse feces accurately and precisely by LC-ESI/MS, thereby helping to predictive biomarker in fat malabsorption and diagnostic research.

A study of the ozonolysis of model lipids by electrospray ionization mass spectrometry

The ozonolysis of vegetable oils has been proposed as step to functionalise unsaturated lipids for the production of bio-based chemicals and materials. The observation of ozonolysis products by mass spectrometry will facilitate mechanistic studies that are essential for the further development of industrial processes based on lipid ozonolysis. Ozonolysis of the model lipids methyl oleate and triolein was performed and samples were taken over a range of reaction times. Ozonlysis products were separated by normal and non-aqueous reversed-phase (NARP) liquid chromatography (LC) coupled to electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole-time of flight instrument. Post-column addition of ammonium acetate solutions aided ionization. Volatile reaction products were observed using gas chromatography/electron impact mass spectrometry (GC/MS). Secondary ozonides, reaction intermediates and previously unreported high molecular weight product dimers were observed as intact molecular ammonium ion adducts under positive ESI. The main fragment ions obtained by MS/MS were from cleavage of the trioxolane group, loss of fatty acyl chain and fragmentation between n-8 and n-9 on the fatty acyl chain. The MS/MS spectra and exact mass measurements explain most of the ozonolysis reaction products. LC/MS, LC/MS/MS and GC/MS results demonstrate that the products from the ozonolysis of the model lipids methyl oleate and triolein are consistent with known ozonolysis reaction pathways. LC/MS techniques using non-aqueous chromatography have permitted the direct observation of mono-, di- and tri-1,2,4-triloxanes which are the secondary ozonides formed by the ozonolysis of triolein. It was also shown that intermediates formed during the ozonolysis of triolein can combine to form high molecular weight ozonide dimers.

Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/ trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position.

Comprehensive two‐dimensional liquid chromatography with evaporative light‐scattering detection for the analysis of triacylglycerols in Borago officinalis

An optimized 2-D liquid chromatography (LC×LC) set-up, based on the different selectivities of a silver ion (Ag) and a non-aqueous reversed phase (NARP), employed in the first (D1) and the second dimension (D2), respectively, in combination with evaporative light-scattering detection (ELSD), has been developed for the analysis of the triacylglycerol (TAG) fraction in a Borago officinalis oil. The 2-D set-up, thanks to the complementary separation selectivity provided by the two columns, allowed to distribute 78 TAGs throughout the 2-D LC retention plane otherwise unachievable by 1-D LC.

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape(TM) from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.

Analysis of wax ester molecular species by high performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry

High chromatographic resolution of wax esters (WEs) was achieved by non-aqueous reversed-phase liquid chromatography on a Nova-Pak C18 column by optimising the acetonitrile/ethyl acetate mobile phase gradient. The retention behaviour of WEs was studied in this chromatographic system. The WEs eluted according to their equivalent carbon number (ECN) values; within the group of WEs with the identical ECN, the most unsaturated species tended to elute first. The isobaric WEs with different positions of the ester moiety were separated from each other whenever the lengths of the chains were sufficiently different. The methyl-branched esters eluted at shorter retention times than the straight-chained analogues, and the resolution among methyl-branched WEs depended on the position of the branching. The analytes were detected by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) using data-dependent scanning. WEs provided simple full-scan spectra with abundant protonated molecules and low-intensity fragments. Collision-induced dissociation (CID) promoted identification of the WE molecular species. The responses of WEs were found to be dependent on the number of double bonds and on the alkyl-chain length; the limits of the detection ranged from 20 μmol/L to 200 nmol/L. The HPLC/APCI-MS was applied for the analysis of the WEs isolated from honeycomb beeswax, jojoba oil and human hair. Good agreement between reported results and the literature data was achieved, with several novel polyunsaturated WEs also being found.

Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag(+) after their separation by non-aqueous reversed-phase liquid chromatography (NARP-LC) before MS to improve MS sensitivity. Electrospray ionization-MS (ESI-MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO(3)](+) ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS(5) spectra. The observation of ions produced by LC-MS(5) of on-line Ag(+)-cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS(5) experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils.

Contribution of two approaches using electrospray ionization with multi‐stage mass spectrometry for the characterization of linseed oil

A detailed characterization of triacylglycerols (TAGs) present in linseed oil samples from a local producer was performed using electrospray ionization and two mass spectrometric approaches; direct infusion multi-stage mass spectrometry (MS(n)) experiments and liquid chromatography/tandem mass spectrometry (LC/MS/MS) using non-aqueous reversed-phase chromatographic conditions. The combination of both approaches permitted the identification of 26 TAGs. Comparison of the two analytical approaches showed that discrimination of regioisomers was achieved from MS3 data while other isobaric species were separated and identified by LC/MS/MS analysis. The results we obtained were also compared with those previously reported. The TAG composition of the studied linseed oil is qualitatively identical to that of linseed oils from various sources in Europe, Canada, Argentina or India. However, a few differences were observed with regard to the proportions of some TAGs; these can be explained by variations in the culture conditions, climate, and variety of the seeds.