AOCS Analytical Division

Serving Lipid Chemists Worldwide

The abstracts and presentations for the

 Mass Spectrometry of Lipids Symposium

that was held on Monday, May 1, 2006 at the

97th American Oil Chemists’ Society

Annual Meeting and Exposition

 

Are Now Available Below!

9.    Richard B. Cole1*, B.M. Ham1, and J.T. Jacob2, 1Department of Chemistry, University of New Orleans, New Orleans, LA; 2Department of Ophthalmology, Louisiana State University Health Sciences Center, New Orleans, LA.

     “MALDI-ToF Mass Spectrometry of Phosphorylated Lipids in Tear Samples”  (3,217 kB)


     The ocular tear fluid is a complex aqueous mixture of salts, mucins, proteins, enzymes, non-polar lipids, and polar phosphorylated lipids that interact with one another to maintain the health and clarity of the cornea and ocular surface. Irregularities in the tear film can cause a number of ocular problems, including ″dry eye″ that afflicts approximately 10 to 14 million people in the United States. The outer lipid layer, secreted by the meibomian glands which are located in the eyelids, serves to retard evaporation of the aqueous layer. Alterations in the tear film composition can result in the development of dry eye syndrome and, ultimately, deleterious effects on vision. Changes in the polar lipid concentration of the outer lipid layer can cause instability of the tear film, and may thus be a potential factor contributing to the development of dry eye syndrome. In this report, we present a MALDI-TOF MS study of the phosphorylated lipids of normal and dry eye tears using an immobilized metal ion affinity chromatography (IMAC) clean-up method coupled with a newly synthesized solid ionic crystal MALDI matrix (comprised of para-nitroaniline and butyric acid) to detect and measure nanogram to picogram quantities of phosphorylated lipids in small volume tear samples. Comparison of the types and relative quantities of the polar phospholipids in normal and dry eye rabbit tears should provide insight into the effect of polar lipid changes on the stability of the tear film.

10.  Fabiola Dionisi* & Elif Buyukpamukcu, Nestlι Research Center, Lausanne, Switzerland.

“Structural Analysis of Phospholipids in Complex Mixtures Using Electrospray Ionization Tandem Mass Spectrometry”  (6,492 kB)


     The term Phospholipids (PLs) defines several classes of molecules differing in polar head groups (e.g. ethanolamine, choline, inositol, serine). Each class is a mixture of different molecules containing fatty acid moities esterified in the sn-1 and sn-2 positions on the glycerol backbone. Due to the presence of these lipophilic (fatty acids) and hydrophilic (polar head) groups, PLs have the ability to form bilayer structures at the interface of two phases, and thus aid in the stabilisation of mainly oil/water emulsions by lowering interfacial surface tension. They have widespread uses in food, cosmetic and pharmaceutical industries. PLs are also essential components of vegetable and animal cell membranes and organelles, and thus provide important structural and functional properties to the cell membranes of all living organisms. PL compositions can affect membrane fluidity, which in turn can alter the activity of membrane-bound proteins.
      To be able to identify and quantify the singular molecular species within the PL classes is important both for nutritional studies and for understanding PL technological functionality. In addition, this knowledge could be useful to define authenticity criteria for important raw materials such as lecithins and other PL rich ingredients. Nevertheless, due to the complexity of their nature, PL analysis is particularly challenging, especially in complex mixtures in food matrices.
      Several methods have been proposed to quantify phospholipids in different matrices. None of them is able to provide an exact quantification of each molecular species in complex mixtures. Therefore, the aim of our study was to develop an Electrospray Ionisation Tandem Mass Spectrometry (ESI-MS/MS) method for the analysis of PLs in mixtures. The developed method allows the identification of the PL polar head group and fatty acid moieties as well as their quantitative analysis. It also may allow the probable attribution of the fatty acids to sn-1 and sn-2 positions. Details regarding the development and the validation of this method will be provided as well as several applications to food matrices.

3. David A. Ford, Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO.

“Lipid Mass Spectrometry in the Cardiovascular System” (5,170 kB)


       Alterations in phospholipid metabolism likely are an important contributor to the pathophysiological sequelae of myocardial ischemia and reperfusion. We have used mass spectrometry to show that subcellular phospholipid pools are metabolized during myocardial ischemia and reperfusion. Furthermore, mass spectrometry has been used to demonstrate novel lipid metabolites that are found in ischemic/reperfused myocardium. Utilizing ESI-MS, we have shown that nuclear membrane phospholipids are metabolized in response to myocardial ischemia and reperfusion. In hearts subjected to LAD occlusion followed by release of the occlusion (an in vivo model of myocardial ischemia/reperfusion), plasmalogen oxidation products have been identified. The plasmalogen vinyl ether bond is a target for reactive chlorinating species (RCS) in the ischemic/reperfused heart. These RCS are derived from either infiltrating neutrophils or resident myocardial neutrophils. One of the products from the attack of plasmalogens by RCS in the heart is α-chloro fatty aldehydes. These fatty aldehydes have been measured using GC-MS analyses of their pentafluorobenzyl oxime derivatives. These chlorinated aldehydes accumulate in both infarcted myocardium as well as in ischemic/reperfused myocardium prior to massive infiltration of neutrophils into the myocardium. Recent studies have shown that α-chloro fatty aldehydes produced from RCS attack of plasmalogens form Schiff-base adducts with ethanolamine glycerophospholipids. Taken together, mass spectrometric analyses utilizing GC-MS, direct infusion ESI-MS, as well as LC-ESI-MS have been employed to identify novel mechanisms of lipid metabolism in the ischemic/reperfused heart. It is likely that the loss of key membrane lipids during ischemia/reperfusion, as well as the production of potential lipidic mediators, contribute to cardiac dysfunction from ischemia/reperfusion.

6. Xianlin Han*, K. Wang, J. Yang, H. Cheng, and R. Gross, Department of Medicine, Washington University School of Medicine, St. Louis, MO.

“Shotgun Lipidomics of Cardiolipin Molecular Species”  (835 kB)


       Cardiolipin molecular species are prominent components of mitochondrial inner membranes, contributing to the regulation of multiple discrete mitochondrial functions. In this work, we extend our shotgun lipidomics technology to identify and quantify cardiolipin molecular species directly from lipid extracts of biological samples. Two shotgun lipidomics approaches for analysis of cardiolipin molecular species were developed using either a continuous ion-transmission instrument (i.e., QqQ type) or a high mass resolution hybrid pulsed mass spectrometer (i.e., QqTOF type). Three chemical principles were employed for the development of shotgun lipidomics for cardiolipin molecular species analysis. These include exploiting the marked enrichment of linoleate in cardiolipin to maximize the signal to noise ratio, the specific neutral loss of ketenes from doubly charged cardiolipin molecular ions to yield doubly charged triacyl monolysocardiolipins, and the doubly charged character of two phosphates in each cardiolipin molecular species. Through these techniques, accurate quantitation of multiple low-abundance cardiolipin molecular species identified the specific profiles of cardiolipin molecular species in the lipid extracts of mouse heart, liver, and skeletal muscle. The accuracy (~ 5%) and the low end of the linear dynamic range (10 fmol/mL) for quantitation make these approaches useful for studying alterations in cardiolipin metabolism in multiple disease states using either type of mass spectrometer.

5. Charles L. Hoppel*, E. Lesnefsky, and P. Minkler, Case Western Reserve School of Medicine, Cleveland, OH.

“Rat Heart Mitochondrial Cardiolipin Oxidation During Ischemia in the Aged Rat”  (962 kB)


       The aged heart sustains increased damage during ischemia and reperfusion compared to the adult heart, including in the Fischer 344 rat model of aging. Aging decreases the rate of oxidative phosphorylation only in interfibrillar mitochondria (IFM) located among the myofibrils, whereas subsarcolemmal mitochondria (SSM) under the plasma membrane remain unaffected. Twenty five min. of ischemia damages SSM and IFM in both adult and aging hearts. In the aged heart, ischemic damage is superimposed upon defects due to aging. Thus, the combination of aging and ischemic defects increase mitochondrial oxidative damage. Oxidative phosphorylation did not deteriorate further during reperfusion in either the adult or aged heart, supporting the notion that most mitochondrial damage occurred during ischemia. Cardiolipin (CL) is an oxidatively sensitive phospholipid unique to mitochondria. Ischemia did not alter the content of CL in the adult or aging heart. Notably, following ischemia in the aged but not the adult heart, both SSM and IFM contained a new molecular species of CL. The composition of CL was assessed based upon characterization of the individual molecular species of CL using reverse phase chromatography and electrospray mass spectrometry. Unaltered CL consists mainly of a species that contains four linoleic acid residues (C18:2). The new molecular species of CL observed following ischemia in the aged heart showed an increase in molecular weight of 48 amu (M+48), consistent with the addition of three oxygen atoms. Collision induced dissociation of CL localized the addition of 48 amu to an individual acyl-residue. C18:2 is susceptible to lipid peroxidation via the addition of molecular oxygen. Thus, mitochondrial phospholipids sustain oxidative damage following ischemia in the aged heart. Similar oxidative damage was not observed in the adult heart. We next asked if the oxidative damage to CL intensified during reperfusion. Reperfusion did not lead to an increase in content of the M+48 species, nor the formation of additional CL molecular species. Thus, aging-enhanced oxidative damage to cardiac mitochondria occurs during ischemia. The presence of one major oxidized product suggests that oxidative damage is relatively selective, rather occurring than via stochastic lipid peroxidation reactions. Experiments to elucidate the chemical structure of the oxidized CL will provide key insight into the mechanisms of increased oxidative damage to mitochondria during ischemia in the aged heart.
Supported by: NIH POI AG15885 and VA Medical Research Service

8. Fong-Fu Hsu*, Department of Internal Medicine, Washington University, St. Louis, St. Louis, MO.

John Turk, Director, Mass Spectrometry Resource, Washington University, St. Louis, St. Louis, MO.

“Into the Mechanism of Fragment Formation of Lipids: Multiple-Stage Tandem Mass Spectrometric Studies on Complex Lipids”  (1,851 kB)


       Low-energy collisionally activated dissociation (CAD) tandem mass spectrometry of the alkali metal adduct and the deprotonated molecular species of glycerophospholipids desorbed by electrospray ionization give rich structural information that is readily applicable for characterization. To gain further insight into the mechanisms underlying the fragmentation processes, multiple-stage Ion-trap tandem mass spectrometry along with source CAD tandem quadrupole mass spectrometry was used. In positive-ion mode, charge-remote fragmentation (CRF) processes play the major role in the fragmentation, and loss of the fatty acid substituent at sn-1 is a more favorable pathway than the analogous loss of the fatty acid substituent at sn-2. This is due to the fact that the alpha hydrogen of the fatty acid substituent at sn-2, which participates in the loss of the fatty acid at sn-1, is more labile than that at sn-2, which is responsible for the similar loss at sn-1. The differential losses of the fatty acid substituents due to their positions on the glycerol backbone provide simple means for structural determination of glycerophospholipids. In negative-ion mode, charge-driven fragmentations (CDF) are the major processes, and the basicity of the precursor ions is the determinant that leads to the loss of fatty acid substituent as an acid or as a ketene. The distinction of the fatty acid substituents at sn-1 from those at sn-2 is based on the fact that loss of the fatty acid substituent at sn-2 as an acid or as a ketene is more favorable than that at sn-1, because the loss of the former is sterically more favorable.

7. Alan G. Marshall* and R.P. Rodgers, National High Magnetic Field Laboratory ICR Program, Florida State University, Tallahassee, FL.

“Comprehensive Compositional Analysis of Oils, from Crude to Canola”  (7,026 kB)


       Electrospray ionization FT-ICR mass spectrometry resolves and identifies literally thousands of distinct chemical components of oils ranging from petroleum heavy crudes to commercial canola, olive and soybean oils, without extraction or other wet chemical separation pretreatment. For example, compositional fingerprints can reveal both the source and adulteration of vegetable oils. Negative-ion ESI FT-ICR MS distinguishes the acidic components of soybean oil from those of canola and olive oil based on relative abundances of C-18 fatty acids, whereas olive oil differs from canola and soybean oil based on relative abundances of tocopherols. Positive-ion ESI FT-ICR MS distinguishes the three oils according to the relative abundances of di- and triacylglycerols with various numbers of double bonds in the fatty acid chains. This work was supported by the National Science Foundation (CHE-99-09502), Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, FL.

2. M. Cameron Sullards, Bioanalytical Mass Spectrometry Facility, Georgia Institute of Technology, Atlanta, GA.

“Identification and Structure Determination of Higher Order Glycosphingolipids via LC-MS/MS”  (4,367 kB)


       Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the sphingolipids are bioactive and are often closely related in both structure and metabolism, to determine the role(s) of sphingolipids in a given biological context one must conduct a sphingolipidomic analysis--i.e., a structure-specific and quantitative measurement of all of these compounds, or at least all members of a critical subset. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is currently the only technology with the requisite structural specificity, sensitivity, quantitative precision, and relatively high-throughput capabilities for such analyses in small samples (~ 10e6 cells). Here we will present methods that have been developed for the relatively rapid analysis of some higher order glycosphingolipids (including all the compounds that are presently regarded as sphingolipid second messengers) using normal- and reverse-phase HPLC in combination with triple quadrupole (for MS/MS) and hybrid quadrupole-ion trap (for enhanced resolution, product ion analysis, and MS/MS/MS) mass spectrometry.

4. Jack Syage* & Sheng-Suan (Victor) Cai, Syagen Technologies, Tustin, CA.

“Comparison of APPI, APCI, and ESI Mass Spectrometry for Analysis of Lipids and Related Compounds”  (1,698 kB)


       Lipid analysis has been traditionally difficult by GC, GC/MS and HPLC. GC or GC/MS requires tedious sample pretreatment and derivatization. HPLC with UV or ELSD avoids derivatization, but lacks sensitivity and specificity. LC/MS allows simple sample preparation without derivatization and meanwhile offers higher sensitivity and specificity. Currently, the most commonly used LC/MS technique for lipid analysis is electrospray ionization (ESI). However, nonpolar lipids may be difficult to analyze by ESI. In this work we compare the quantitative accuracy and sensitivity of analyzing lipids by atmospheric pressure photoionization (APPI), chemical ionization (APCI), and ESI. These results demonstrate the benefits of using LC/APPI-MS for lipid analysis.

               Analysis was performed on a Waters ZQ LC/MS. Normal phase solvent systems were used due to the low solubility of these compounds in aqueous reversed phase solvent systems. By comparison, APPI offers the most stable signals, lower background noises, lower detection limits, generally highest signal intensities and therefore the highest S/N ratio. APPI is 2-4 times more sensitive than APCI and much more sensitive than ESI without mobile phase modifiers. APPI and APCI offer comparable linear range (i.e., 4-5 decades). ESI sensitivity is dramatically enhanced by use of mobile phase modifiers (i.e., ammonium formate or sodium acetate). However these adduct signals are less stable, and are either nonlinear or have dramatically reduced linear ranges. Analysis of fish oils by APPI shows significantly enhanced target analyte intensities in comparison with by APCI and ESI. Furthermore, APPI allows analysis of thermo labile compounds at a lower probe temperature without compromising sensitivity.

1. Wm. Craig Byrdwell, USDA, ARS, BHNRC, Food Composition Laboratory, Beltsville, Maryland

“Dual Parallel Mass Spectrometers with 31P NMR for Sphingolipid Analysis”  (3,895 kB)


     Two complementary atmospheric pressure ionization (API) mass spectrometry (MS) techniques were used simultaneously, in parallel, for detection of commercially available sphingolipids separated using normal-phase chromatography on amine columns. Atmospheric pressure chemical ionization (APCI) MS was used in parallel with electrospray ionization (ESI) MS for analysis of bovine brain sphingolipids, bovine milk sphingolipids and chicken egg sphingolipids. APCI-MS mass spectra exhibited mostly ceramide-like fragment ions, [Cer-H2O+H]+ and [Cer-2H2O+H]+, from sphingolipids, such as those listed at www.sphingomyelin.com. ESI-MS produced mostly intact protonated molecules, [M+H]+. Separate runs employing APCI-MS/MS and MS3 were used to differentiate between isobaric sphingolipids. APCI-MS/MS mass spectra exhibited long-chain base related fragments, [LCB]+ and [LCB-H2O]+, that allowed the sphinganine backbone to be differentiated from the sphingenine backbone. Fragments formed from the fatty amide chain, [FA(long)]+ and [FA(short)]+ allowed the fatty acid chains in isobaric species to be identified, and also allowed an overall fatty acid composition to be determined. A substantial number of dihydrosphingomyelin (DHS) molecular species were identified that have previously gone unreported using LC/API-MS techniques. Identification of DHS species and SPM species was confirmed using 31P NMR spectroscopy. This provided an independent method for confirmation of the presence of substantial abundances of dihydrosphingomyelin species in bovine brain and in bovine milk.