Molecular Species Of Phosphatidylcholine Research Articles

Hydrolysis and oxidation of lipids in mussel Mytilus edulis during cold storage

Change in quality of mussels (Mytilus edulis) meat when stored at 4 °C was evaluated by determining volatile basic nitrogen (TVB-N), peroxide value (POV), thiobarbituric acid reactive substance (TBARS), total oxidation (TOTOX), lipid class, fatty acid (FA), phosphatidylcholine (PC) and phosphatidylcholine (PE) content, and glycerophospholipid (GP) molecular species. After 4 days of storage, the percentages of triacylglycerol and polar lipid as well as the contents of PC, PE and major GP molecular species significantly decreased (P < 0.05), while the percentages of free FA and monoacylglycerol significantly increased (P < 0.05), indicating the hydrolysis of lipids. The increase in activities of phospholipase and lipase during storage suggests that they may contribute to the hydrolysis of lipids. The cold storage also resulted in significantly increased POV, TBARS and TOTOX as well as lipoxygenase activity (P < 0.05) but slightly decreased percentage of polyunsaturated FA (PUFA), indicating the occurrence of lipid oxidation.

Effects of aging on serum levels of lipid molecular species as determined by lipidomics analysis in Japanese men and women

BackgroundAging is known to be associated with increased risk of lipid disorders related to the development of type 2 diabetes. Recent evidence revealed that change of lipid molecule species in blood is associated with the risk of type 2 diabetes. However, changes in lipid molecular species induced by aging are still unknown.We assessed the effects of age on the serum levels of lipid molecular species as determined by lipidomics analysis.MethodsSerum samples were collected from ten elderly men (71.7 ± 0.5 years old) and women (70.2 ± 1.0 years old), ten young men (23.9 ± 0.4 years old), and women (23.9 ± 0.7 years old). Serum levels of lipid molecular species were determined by liquid chromatography mass spectrometry-based lipidomics analysis.ResultsOur mass spectrometry analysis revealed increases in the levels of multiple triacylglycerol molecular species in the serum of elderly men and women. Moreover, serum levels of total ester-linked phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were increased by aging. In contrast, serum levels of specific ether-linked PC and PE molecular species were lower in elderly individuals than in young individuals.ConclusionsOur finding indicates that specific lipid molecular species, such as ether- and ester- linked phospholipids, may be selectively altered by aging.

Determination of Phosphatidylcholine in Shrimp by High-Resolution Mass Spectrometry

ABSTRACTThe molecular species of phosphatidylcholine from freshwater sources (Macrobranchium nipponense and Macrobranchium rosenbergii) and marine sources (Euphausia superba and Penaeus chinesis) were characterized by high-resolution mass spectrometry. The tandem secondary mass spectrometry (MS/MS) fragmentation allowed for the identification of fatty acyl residues of phosphatidylcholine molecular species. (16:0–18:1)Phosphatidylcholine was the main phosphatidylcholine molecular species determined in all shrimp samples, especially in E. superba. Macrobranchium rosenbergii phosphatidylcholine was particularly rich in (16:0–20:5)phosphatidylcholine and (16:0–22:6)phosphatidylcholine. The proportion of the two molecular species was next to the phosphatidylcholine of E. superba. Therefore, M. rosenbergii appears to be a potential freshwater source for the supplementation of polyunsaturated fatty acids, particularly eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6). This approach may be used as an efficient method for the identification of natural phosphatidylcholine sources from the broad range of plant, animal, and marine origins.

Visualization of local phosphatidylcholine synthesis within hippocampal neurons using a compartmentalized culture system and imaging mass spectrometry

Neurons extend neurites with an increased synthesis of phosphatidylcholine (PC) that is not only a membrane component but also a functional regulator with specific fatty acid composition. To analyze the local synthesis of the PC molecular species within neurons, we combined a compartmentalized culture system with matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). We observed that a newly synthesized PC, which contains exogenously administered palmitic acid-d3, is accumulated at the cell bodies and the tips of the distal neurites. The local accumulation within distal neurites is formed by distinct metabolic activity from cell bodies, suggesting that the local extracellular composition of free fatty acid can be a key to regulate specific functions of each PC molecular species. We expect our simple method to be a starting point for more sophisticated in vitro analytical methods for unveiling detailed lipid metabolisms within neurons.

3D MALDI Mass Spectrometry Imaging of a Single Cell: Spatial Mapping of Lipids in the Embryonic Development of Zebrafish

The zebrafish (Danio rerio) has been widely used as a model vertebrate system to study lipid metabolism, the roles of lipids in diseases, and lipid dynamics in embryonic development. Here, we applied high-spatial resolution matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) to map and visualize the three-dimensional spatial distribution of phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamines (PE), and phosphatidylinositol (PI), in newly fertilized individual zebrafish embryos. This is the first time MALDI-MSI has been applied for three dimensional chemical imaging of a single cell. PC molecular species are present inside the yolk in addition to the blastodisc, while PE and PI species are mostly absent in the yolk. Two-dimensional MSI was also studied for embryos at different cell stages (1-, 2-, 4-, 8-, and 16-cell stage) to investigate the localization changes of some lipids at various cell developmental stages. Four different normalization approaches were compared to find reliable relative quantification in 2D- and 3D- MALDI MSI data sets.

Hepatic Steatosis Accompanies Pulmonary Alveolar Proteinosis.

Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte-macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.

Spatial and Temporal Mapping of Key Lipid Species in Brassica napus Seeds.

The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape (Brassica napus) is the third most productive vegetable oil crop on the global market; therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionization-mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MALDI-MSI to study the spatial distribution of two major lipid species, triacylglycerols and phosphatidylcholines. A dramatic, heterogenous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various tissue types within the seed. The embryonic axis was found to be particularly enriched in palmitic acid, while the seed coat/aleurone layer accumulated vaccenic, linoleic, and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed from each other, a remarkable discovery given the supposed identical functionality of these two tissues. Triacylglycerol and phosphatidylcholine molecular species distribution was analyzed through a developmental time series covering early seed lipid accumulation to seed maturity. The spatial patterning of lipid molecular species did not vary significantly during seed development. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed imply differential regulation of lipid metabolism between the different tissue types of the seed. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.

Three-dimensional visualization of membrane phospholipid distributions in Arabidopsis thaliana seeds: A spatial perspective of molecular heterogeneity

Arabidopsis thaliana has been widely used as a model plant to study acyl lipid metabolism. Seeds of A. thaliana are quite small (approximately 500×300μm and weigh ~20μg), making lipid compositional analyses of single seeds difficult to achieve. Here we have used matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to map and visualize the three-dimensional spatial distributions of two common membrane phospholipid classes, phosphatidylcholine (PC) and phosphatidylinositol (PI), in single A. thaliana seeds. The 3D images revealed distinct differences in distribution of several molecular species of both phospholipids among different seed tissues. Using data from these 3D reconstructions, the PC and PI mol% lipid profiles were calculated for the embryonic axis, cotyledons, and peripheral endosperm, and these data agreed well with overall quantification of these lipids in bulk seed extracts analyzed by conventional electrospray ionization-mass spectrometry (ESI-MS). In addition, MALDI-MSI was used to profile PC and PI molecular species in seeds of wild type, fad2–1, fad3–2, fad6–1, and fae1–1 acyl lipid mutants. The resulting distributions revealed previously unobserved changes in spatial distribution of several lipid molecular species, and were used to suggest new insights into biochemical heterogeneity of seed lipid metabolism. These studies highlight the value of mass spectrometry imaging to provide unprecedented spatial and chemical resolution of metabolites directly in samples even as small as a single A. thaliana seeds, and allow for expanded imaging of plant metabolites to improve our understanding of plant lipid metabolism from a spatial perspective.

Alteration of Phospholipids during the Mitophagic Process in Lung Cancer Cells.

Matrix assisted laser desorption ionization (MALDI)-time of flight/mass spectrometry (TOF/MS) was applied to investigate alterations in phospholipids in mitophagic cancer cells. Several phospholipids, including phosphatidylcholines (PCs), sphingomyelins (SMs), and phosphatidylinositols (PIs), were successfully analyzed in control and mitophagy-induced H460 cells in the positive and negative ion modes. Principal component analysis was applied to differentiate the two groups. The upregulated and downregulated phospholipid species in the mitophagic cells were also represented in a heatmap. In the volcano plot (fold change > 1.3 and p value < 0.01), individual species of seven PCs, two SMs, and three PIs were selected as differentially regulated phospholipids. In particular, almost all the molecular species of PC, SM, and PI were downregulated in the mitophagic cells. Quantification of these lipids indicated that mitophagy induces altered metabolism of phospholipids. Therefore, phospholipid alterations during the mitophagic process of lung cancer cells were well characterized by MALDI-TOF/MS.

Phosphatidylethanolamine and phosphatidylcholine biosynthesis by the Kennedy pathway occurs at different sites in Trypanosoma brucei.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.

Fatty Acid and Lipid Profiles with Emphasis on n-3 Fatty Acids and Phospholipids from Ciona intestinalis.

In order to establish Ciona intestinalis as a new bioresource for n-3 fatty acids-rich marine lipids, the animal was fractionated into tunic and inner body tissues prior to lipid extraction. The lipids obtained were further classified into neutral lipids (NL), glycolipids (GL) and phospholipids (PL) followed by qualitative and quantitative analysis using GC-FID, GC-MS, (1)H NMR, 2D NMR, MALDI-TOF-MS and LC-ESI-MS methods. It was found that the tunic and inner body tissues contained 3.42-4.08% and 15.9-23.4% of lipids respectively. PL was the dominant lipid class (42-60%) irrespective of the anatomic fractions. From all lipid fractions and classes, the major fatty acids were 16:0, 18:1n-9, C20:1n-9, C20:5n-3 (EPA) and C22:6n-3 (DHA). The highest amounts of long chain n-3 fatty acids, mainly EPA and DHA, were located in PL from both body fractions. Cholestanol and cholesterol were the dominant sterols together with noticeable amounts of stellasterol, 22 (Z)-dehydrocholesterol and lathosterol. Several other identified and two yet unidentified sterols were observed for the first time from C. intestinalis. Different molecular species of phosphatidylcholine (34 species), sphingomyelin (2 species), phosphatidylethanolamine (2 species), phosphatidylserine (10 species), phosphatidylglycerol (9 species), ceramide (38 species) and lysophospholipid (5 species) were identified, representing the most systematic PL profiling knowledge so far for the animal. It could be concluded that C. intestinalis lipids should be a good alternative for fish oil with high contents of n-3 fatty acids. The lipids would be more bioavailable due to the presence of the fatty acids being mainly in the form of PL.

Evidence for an N-methyl transfer reaction in phosphatidylcholines with a terminal aldehyde during negative electrospray ionization tandem mass spectrometry.

Lipidomic analysis of the complex mixture of lipids isolated from biological systems can be a challenging process that often involves tandem mass spectrometry and interpretation of both precursor ions and product ions relative to the molecular structure of the lipids. Therefore, detailed understanding of the gas-phase ion chemistry occurring for each class of phospholipids is critically important for an accurate assignment of lipid structure. Some oxidized phosphatidylcholines are known to be biologically active and responsible for pathological events, and are therefore important targets for detection in lipidomic studies. Modification of fatty acyl chains by oxidation may, however, change the behavior of ion formation and decomposition in the mass spectrometer. In this study, we report on the mass-spectrometric behavior of 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, a bioactive product of phosphatidylcholine oxidation. In addition to [M-15](-) and the acetate adduct [M+59](-), three additional adduct ions, including [M-H](-), were present in significant abundance in the negative ion electrospray mass spectrum. It was found that this unexpected [M-H](-) ion was formed by the transfer of a methyl group from the choline residue on the polar head group to the aldehyde functionality of the sn-2 substituent, resulting in a 14-Da increase in the mass of the resulting sn-2 carboxylate anion formed by collisional activation of this ion. These results suggest additional rules for understanding the gas-phase ion chemistry of aldehydic phosphatidylcholine molecular species.

Preferential incorporation of shorter and less unsaturated acyl phospholipids into high density lipoprotein-like particles in the ABCA1- and ABCA7-mediated biogenesis with apoA-I

Molecular species of phosphatidylcholine (PC) and sphingomyelin (SPM) were globally analyzed for lipidomics in the nascent high-density lipoprotein (HDL)-like particles generated with human apolipoprotein A-I (apoA-I) form HEK293 cells where either human ATP binding cassette transporter (ABC) A1 or ABCA7 was transfected and overexpressed. SPM/PC ratio was higher in the ABCA1-mediated HDL than ABCA7-mediated HDL likely being related to their cholesterol content, while it was less than the ratio in the cell membrane in either case. Molecular species composition of hydrocarbon chain moiety in each phospholipid in the HDL largely reflected that in the cells the lipoprotein originated in, without remarkable difference between ABCA1 and ABCA7. Further analysis, however, revealed apparent preference for the molecules with shorter hydrocarbon chain length for both PC and SPM in their relative incorporation into HDL by ABCA1 and ABCA7. Likewise, it was in favor for less-unsaturated hydrocarbon chains of PC while this preference was not apparent for SPM. The results are consistent with the view that assembly of HDL particles with extracellular apoA-I is primarily with the cellular phospholipid molecules being regulated in part by their physicochemical nature.

Age-dependent biochemical dysfunction in skeletal muscle of triple-transgenic mouse model of Alzheimer`s disease.

The emergence of Alzheimer`s disease as a systemic pathology shifted the research paradigm toward a better understanding of the molecular basis of the disease considering the pathophysiological changes in both brain and peripheral tissues. In the present study, we evaluated the impact of disease progression on physiological relevant features of skeletal muscle obtained from 3, 6 and 12 month-old 3xTg-AD mice, a model of Alzheimer`s disease, and respective agematched nonTg mice. Our results showed that skeletal muscle functionality is already affected in 3-month-old 3xTg-AD mice as evidenced by deficient acetylcholinesterase and catalase activities as well as by alterations in fatty acid composition of mitochondrial membranes. Additionally, an age-dependent accumulation of amyloid-β1-40 peptide occurred in skeletal muscle of 3xTg-AD mice, an effect that preceded bioenergetics mitochondrial dysfunction, which was only detected at 12 months of age, characterized by decreased respiratory control ratio and ADP/O index and by an impairment of complex I activity. HPLC-MS/MS analyses revealed significant changes in phospholipid composition of skeletal muscle tissues from 3xTg-AD mice with 12 months of age when compared with age-matched nonTg mice. Increased levels of lyso-phosphatidylcholine associated with a decrease of phosphatidylcholine molecular species containing arachidonic acid were detected in 3xTg-AD mice, indicating an enhancement of phospholipase A2 activity and skeletal muscle inflammation. Additionally, a decrease of phosphatidylethanolamine plasmalogens content and an increase in phosphatidylinositol levels was observed in 3xTg-AD mice when compared with age-matched nonTg mice. Altogether, these observations suggest that the skeletal muscle of 3xTg-AD mice are more prone to oxidative and inflammatory events.

Buffalo milk fat globules and their biological membrane: in situ structural investigations

Milk fat globules and their surrounding biological membrane (the MFGM) are not well understood despite the importance of these milk components in human nutrition and the role of fat globules in determining the properties of dairy products. The objectives of this study were to investigate these unique colloidal assemblies and the microstructure of the MFGM in buffalo milk, which is the second largest global source of dairy products. In-situ structural investigations were performed at room temperature using confocal microscopy with multiple fluorescent probes (Nile Red, Rh-DOPE, the lectin WGA-488). Microscopic observations showed cytoplasmic crescents around fat globules and the heterogeneous distribution of glycosylated molecules and polar lipids with the occurrence of lipid domains. The lipid domains in the buffalo MFGM appear to form by the segregation of lipids with a high phase transition temperature (e.g. sphingomyelin and saturated phosphatidylcholine molecular species) and cholesterol resulting in a gel phase or a Lo phase forming circular domains. The structure of the buffalo MFGM results from a non-random mixing of components, consistent with observations for other species. Structural heterogeneities of the MFGM could affect the processability of buffalo fat globules and the bioavailability of milk lipids.

Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA.

Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds.

Sizing up Surfactant Synthesis

Phosphatidylcholine is generated through de novo synthesis and remodeling involving a lysophospholipid. In this issue of Cell Metabolism, research from the Shimizu lab (Harayama etal., 2014) demonstrates the highly selective enzymatic behavior of lysophospholipid acyltransferases. The authors present an enzymatic model for phosphatidylcholine molecular species diversification that impacts surfactant formation.

Influence of deep-fat frying process on phospholipid molecular species composition of Sardina pilchardus fillet

The effects of deep-fat frying performed using different culinary fats (extra virgin olive oil, conventional sunflower oil and high-oleic sunflower oil) and different frying temperatures (160 and 180 °C) on the composition of the preponderant fish phospholipid classes, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), were investigated on Sardina pilchardus. The total fish lipid fraction was injected into the HPLC system coupled on line with a second order mass spectrometer (MS–MS) by means of electronebulization interface (ESI), without a prior clean-up of the phospholipid fraction.The deep-fat frying process caused significant changes on PE and PC molecular species composition of the fish fillet.In all cases, the deep-fat frying process caused a significant (P < 0.05) increase in the relative proportions of the PE and PC species constituted by the combination of palmitic and docosahexaenoic acids. At the same time, a depletion of the percentage of the PE and PC species formed by two docosahexaenoic acid residues in fried fillets was registered. Anyway, this depletion was statistically significant for PC, but not for PE.Both PE and PC compositions were not influenced by the frying temperature, whereas the nature of the culinary fat had an effect on the PC composition. Particularly, the frying tests with conventional sunflower oil produced a statistically significant increase of PC species containing saturated/polyunsaturated fatty acids and a significant decrease of PC species formed by the combination of two polyunsaturated fatty acids.

Enhanced specificity for phosphatidylcholine analysis by positive ion mode matrix‐assisted laser desorption/ionization imaging mass spectrometry

Visualization of the spatial distribution of phosphatidylcholine (PC) in tissues by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) provides insights into key physiological and pathophysiological processes. In MALDI-IMS analysis, the heterogeneity of adduct ions formed from PC lowers the specificity of detection of PC molecular species and poses a challenge in the identification of these species. To solve this problem, modified matrix solution and desalting with ammonium acetate (NH4 Ac) buffer have been employed. However, the utility of these methods is limited to the analysis of brain sections. The MALDI signal intensities of [PC+H], [PC+Na] and [PC+K] were compared after three different pretreatments (modified matrix solution, desalting with 150 mM ammonium acetate, treatment with 150 mM potassium acetate). Pretreatment of tissue sections with 150 mM potassium acetate resulted in an increase in the signal intensity of [PC+K] ions produced from cryosections of the pancreas, brain, and liver tissues. Pretreatment with potassium acetate can be a simple, improved, and highly useful method for the reliable analysis of PC in tissues.

Elucidation of phosphatidylcholine isomers using two dimensional liquid chromatography coupled in-line with ozonolysis mass spectrometry

In this study, two dimensional (2D) liquid chromatography (LC) was coupled in-line with ozonolysis-mass spectrometry (O3-MS) for the elucidation of phosphatidylcholine (PC) structures in phospholipid extracts. In O3-MS, PC molecules carried by mobile phase pass through a length of gas-permeable tubing surrounded by ozone vapor resulting in oxidative cleavage of double bonds. The characteristic aldehyde products are then directly analyzed by eletrospray ionization (ESI)-MS, allowing assignment of the double bond positions within the PC fatty acyl chains. The on-line 2D-LC method employs hydrophilic interaction chromatography as the first dimension to separate phospholipid classes and reversed phase chromatography in the second dimension to further separate PC molecular species. Thus, by combing the separation power of 2D-LC with in-line O3-MS (2D-LC/O3-MS), PC species in complex mixtures can be identified including the detailed structure of their two fatty acid chains. When the 2D-LC/O3-MS method was applied to a rat liver phospholipid extract, 19 PC molecular species were identified with fatty acid isomers unambiguously assigned.