The direct determination of double bond positions in lipid mixtures by liquid chromatography/in-line ozonolysis/mass spectrometry

The direct determination of double bond positions in unsaturated lipids using in-line ozonolysis-mass spectrometry (O₃-MS) is described. In this experiment, ozone penetrates through the semi-permeable Teflon AF-2400 tubing containing a flow of a solution of fatty acid methyl esters (FAME). Unsaturated FAME are thus oxidized by the ozone and cleaved at the double bond positions. The ozonolysis products then flow directly into the atmospheric pressure photoionization (APPI) source of a mass spectrometer for analysis. Aldehyde products retaining the methyl ester group are indicative of the double bond positions in unsaturated FAME. For the first time, O₃-MS is able to couple directly to high performance liquid chromatography (HPLC), making the double bond localization in lipid mixtures possible. The application of LC/O₃-MS has been demonstrated for a fat sample from bovine adipose tissue. A total of 9 unsaturated FAME including 6 positional isomers were identified unambiguously, without comparison to standards. The in-line ozonolysis reaction apparatus is applicable to most mass spectrometers without instrumental modification; it is also directly compatible with various LC columns. The LC/O₃-MS method described here is thus a practical, versatile and easy to use new approach to the direct determination of double bond positions in lipids, even in complex mixtures.     

Ozonolysis of phospholipid double bonds during electrospray ionization: a new tool for structure determination

Ozonolysis of double bonds is observed during the negative ion electrospray ionization of unsaturated phospholipids under conditions that produce a corona discharge. Ionic products of the ozonolysis are detected and characterized by mass spectrometry, and the mass-to-charge ratio of these species can be used to unambiguously assign the double bond position within the unsaturated fatty acid radyls. The reaction products are consistent with the gas-phase ozonolysis of desolvated phospholipid ions in an atmosphere rich in volatilized solvent. Reactions may be carried out in a conventional electrospray ionization mass spectrometer and provide a new method for the structural characterization of phospholipids.     

Synthesis-freindly chiral α-hydroxymethyl ketones from (-)-carvone

Reactions were studied of ozonolysis and periodate cleavage catalyzed by RuCl₃ of a double bond in (1R,5R)-5-(1-methyl-1-chloroethyl)-2-cyclohexen-1-ol and its acetate. The ozonolysis of the unsaturated alcohol led to the formation of cyclic anomeric methoxyspiranes, and its acetate depending on the conditions and oxidant reagent provided the corresponding α, ω-aldehydes, α, ω-ketoacetals, and α, ω-ketoacids.     

Pinpointing Double Bonds in Lipids by Paternò‐Büchi Reactions and Mass Spectrometry

The positions of double bonds in lipids play critical roles in their biochemical and biophysical properties. In this study, by coupling Paternò–Büchi (P‐B) reaction with tandem mass spectrometry, we developed a novel method that can achieve confident, fast, and sensitive determination of double bond locations within various types of lipids. The P‐B reaction is facilitated by UV irradiation of a nanoelectrospray plume entraining lipids and acetone. Tandem mass spectrometry of the on‐line reaction products via collision activation leads to the rupture of oxetane rings and the formation of diagnostic ions specific to the double bond location.     

Distinction among isomeric unsaturated fatty acids as lithiated adducts by electrospray ionization mass spectrometry using low energy collisionally activated dissociation on a triple stage quadrupole instrument

Features of tandem mass spectra of dilithiated adduct ions of unsaturated fatty acids obtained by electrospray ionization mass spectrometry with low-energy collisionally activated dissociation (CAD) on a triple stage quadrupole instrument are described. These spectra distinguish among isomeric unsaturated fatty acids and permit assignment of double-bond location. Informative fragment ions reflect cleavage of bonds remote from the charge site on the dilithiated carboxylate moiety. The spectra contain radical cations reflecting cleavage of bonds between the first and second and between the second and third carbon atoms in the fatty acid chain. These ions are followed by a closed-shell ion series with members separated by 14 m/z units that reflect cleavage of bonds between the third and fourth and then between subsequent adjacent pairs of carbon atoms. This ion series terminates at the member reflecting cleavage of the carbon-carbon single bond vinylic to the first carbon-carbon double bond. Ions reflecting cleavages of bonds distal to the double bond are rarely observed for monounsaturated fatty acids and are not abundant when they occur. For polyunsaturated fatty acids that contain double bonds separated by a single methylene group, ions reflecting cleavage of carbon-carbon single bonds between double bonds are abundant, but ions reflecting cleavages distal to the final double bond are not. Cleavages between double bonds observed in these spectra can be rationalized by a scheme involving a six-membered transition state and subsequent rearrangement of a bis-allylic hydrogen atom to yield a terminally unsaturated charge-carrying fragment and elimination of a neutral alkene. The location of the beta-hydroxy-alkene moiety in ricinoleic acid can be demonstrated by similar methods. These observations offer the opportunity for laboratories that have tandem quadrupole instruments but do not have instruments with high energy CAD capabilities to assign double bond location in unsaturated free fatty acids by mass spectrometric methods without derivatization.     

Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

Imprints of potato sprout (Solanum tuberosum L.), gingko leaves (Gingko biloba L.) and strawberries (Fragaria x ananassa Duch.) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

UV Lamp as a Facile Ozone Source for Structural Analysis of Unsaturated Lipids Via Electrospray Ionization-Mass Spectrometry

Ozonolysis of alkene functional groups is a type of highly specific and effective chemical reaction, which has found increasing applications in structural analysis of unsaturated lipids via coupling with mass spectrometry (MS). In this work, we utilized a low-pressure mercury lamp (6 W) to initiate ozonolysis inside electrospray ionization (ESI) sources. By placing the lamp near a nanoESI emitter that partially transmits 185 nm ultraviolet (UV) emission from the lamp, dissolved dioxygen in the spray solution was converted into ozone, which subsequently cleaved the double bonds within fatty acyls of lipids. Solvent conditions, such as presence of water and acid solution pH, were found to be critical in optimizing ozonolysis yields. Fast (on seconds time scale) and efficient (50%–100% yield) ozonolysis was achieved for model unsaturated phospholipids and fatty acids with UV lamp-induced ozonolysis incorporated on a static and an infusion nanoESI source. The method was able to differentiate double bond location isomers and identify the geometry of the double bond based on yield. The analytical utility of UV lamp-induced ozonolysis was further demonstrated by implementation on a liquid chromatography (LC)-MS platform. Ozonolysis was effected in a flow microreactor that was made from ozone permeable tubing, so that ambient ozone produced by the lamp irradiation could diffuse into the reactor and induce online ozonolysis post-LC separation and before ESI-MS.

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Gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry of cyclic fatty acid monomers isolated from heated fats

The analysis of hydrogenated cyclic fatty acid monomers isolated from heated linseed and sunflower oils is achieved by gas chromatography-tandem mass spectrometry of their pentafluorobenzyl esters. Collisionally activated dissociation of the carboxylate anions produced by electron-capture ionization shows remote charge-site fragmentation that allows location of cyclopentane and cyclohexane rings by examining the resulting mass-analysed ion kinetic energy spectra. Oxidative ozonolysis of the methyl esters of the unsaturated cyclic fatty acid monomers allows location of some double bonds. However, preliminary results obtained with remote charge fragmentation of synthetic unsaturated models make this approach an alternative for double bond location in the cyclic fatty acid monomers isolated from heated fats.     

Hock cleavage of cholesterol 5alpha-hydroperoxide: an ozone-free pathway to the cholesterol ozonolysis products identified in arterial plaque and brain tissue

Cholesterol 5alpha-hydroperoxide, the major product of 1O2-oxidation of cholesterol, readily undergoes acid-catalyzed (Hock) cleavage of the C5-C6 bond to yield the 5,6-secosterol ketoaldehyde and the product of its intramolecular aldolization. These cholesterol oxidation products have long been thought to arise solely from ozonolysis of cholesterol, prompting the recent suggestion that ozone is produced in humans following their identification in arterial and brain tissue extracts.     

基于质谱技术识别脂质双键位置的研究进展

脂质在能量贮存和信号传递方面发挥着巨大作用,同时还是生物膜的主要组成成分。不饱和脂质双键位置不同,生理学意义和生物学功能会有很大差异,因此脂质双键位置的识别至关重要。质谱具有灵敏快速、准确度高等优势,已成为脂质结构研究的重要方法。近年来,不同原理的电离技术与选择性衍生反应迅速发展起来,与质谱相结合已广泛应用于多种脂质双键位置的识别。本文主要对这些新型质谱技术进行总结,并展望了其未来发展趋势。     

Electron transfer dissociation of doubly sodiated glycerophosphocholine lipids

The ability to generate gaseous doubly charged cations of glycerophosphocholine (GPC) lipids via electrospray ionization has made possible the evaluation of electron-transfer dissociation (ETD) for their structural characterization. Doubly sodiated GPC cations have been reacted with azobenzene radical anions in a linear ion trap mass spectrometer. The ion/ion reactions proceed through sodium transfer, electron-transfer, and complex formation. Electron-transfer reactions are shown to give rise to cleavage at each ester linkage with the subsequent loss of a neutral quaternary nitrogen moiety. Electron-transfer without dissociation produces [M + 2Na](+.) radical cations, which undergo collision-induced dissociation (CID) to give products that arise from bond cleavage of each fatty acid chain. The CID of the complex ions yields products similar to those produced directly from the electron-transfer reactions of doubly sodiated GPC, although with different relative abundances. These findings indicate that the analysis of GPC lipids by ETD in conjunction with CID can provide some structural information, such as the number of carbons, degree of unsaturation for each fatty acid substituent, and the positions of the fatty acid substituents; some information about the location of the double bonds may be present in low intensity CID product ions.     

Regioselective Synthesis of β‐Aryl‐ and β‐Amino‐Substituted Aliphatic Esters by Rhodium‐Catalyzed Tandem Double‐Bond Migration/Conjugate Addition

Rhodium–phosphite catalysts were found to effectively mediate double‐bond migrations within unsaturated esters. Once the double‐bond is in conjugation with the carboxylate group, they also catalyze the Michael addition of carbon and nitrogen nucleophiles. In the presence of these catalysts, unsaturated carboxylates enter a dynamic equilibrium of positional and geometrical double‐bond isomers. The conjugated species are continuously removed through 1,4‐additions with formation of β‐amino esters or β‐arylated products, depending on the nucleophile employed. The applicability of both protocols to a range of substrates, such as fatty esters of different chain lengths and double‐bond positions, and several nucleophiles including arylborates and primary and secondary amines, is demonstrated.     

Secondary organic aerosol formation from limonene ozonolysis: homogeneous and heterogeneous influences as a function of NO(x)

Limonene has a high emission rate both from biogenic sources and from household solvents. Here we examine the limonene + ozone reaction as a source for secondary organic aerosol (SOA). Our data show that limonene has very high potential to form SOA and that NO(x) levels, O(3) levels, and UV radiation all influence SOA formation. High SOA formation is observed under conditions where both double bonds in limonene are oxidized, but those conditions depend strongly on NO(x). At low NO(x), heterogeneous oxidation of the terminal double bond follows the initial limonene ozonolysis (at the endocyclic double bond) almost immediately, making the initial reaction rate limiting. This requires a high uptake coefficient between ozone and the first-generation, unsaturated organic particles. However, at high NO(x), this heterogeneous processing is inhibited and gas-phase oxidation of the terminal double bond dominates. Although this chemistry is slower, it also yields products with low volatility. UV light suppresses production of the lowest volatility products, as we have shown in earlier studies of the alpha-pinene + ozone reaction.     

Secondary organic aerosol from limona ketone: insights into terpene ozonolysis via synthesis of key intermediates

Limona ketone was synthesized to explore the secondary organic aerosol (SOA) formation mechanism from limonene ozonolysis and also to test group-additivity concepts describing the volatility distribution of ozonolysis products from similar precursors. Limona ketone SOA production is indistinguishable from alpha-pinene, confirming the expected similarity. However, limona ketone SOA production is significantly less intense than limonene SOA production. The very low vapor pressure of limonene ozonolysis products is consistent with full oxidation of both double bonds in limonene and furthermore with production of products other than ketones after oxidation of the exo double bond in limonene. Mass-balance constraints confirm that ketone products from exo double-bond ozonolysis have a minimal contribution to the ultimate product yield. These results serve as the foundation for an emerging framework to describe the effect on volatility of successive generations of organic compounds in the atmosphere.     

HPTLC/DESI-MS imaging of tryptic protein digests separated in two dimensions

Desorption electrospray ionization mass spectrometry (DESI-MS) was demonstrated as a method to detect and identify peptides from two-dimensional separations of cytochrome c and myoglobin tryptic digests on ProteoChrom HPTLC Cellulose sheets. Data-dependent tandem mass spectra were acquired during lane scans across the TLC plates. Peptides and the corresponding proteins were identified using a protein database search software. Two-dimensional distributions of identified peptides were mapped for each separated protein digest. Sequence coverages for cytochrome c and myoglobin were 81 and 74%, respectively. These compared well with those determined using the more standard HPLC/ESI-MS/MS approach (89 and 84%, respectively). Preliminary results show that use of more sensitive instrumentation has the potential for improved detection of peptides with low R(f) values and improvement in sequence coverage. However, less multiple charging and more sodiation were seen in HPTLC/DESI-MS spectra relative to HPLC/ESI-MS spectra, which can affect peptide identification by MS/MS. Methods to increase multiple charging and reduce the extent of sodiation are currently under investigation.     

Electron‐induced dissociation (EID) for structure characterization of glycerophosphatidylcholine: determination of double‐bond positions and localization of acyl chains

Glycerophospholipids are a highly abundant and diverse collection of biologically relevant lipids, and distinction between isomeric and isobaric species is a fundamental aspect for confident identification. The ability to confidently assign a unique structure to a glycerophospholipid of interest is dependent on determining the number and location of the points of unsaturation and assignment of acyl chain position. The use of high‐energy electrons (>20 eV) to induce gas‐phase dissociation of intact precursor ions results in diagnostic product ions for localizing double‐bond positions and determining acyl chain assignment. We describe a high‐resolution, tandem mass spectrometry method for structure characterization of glycerophospholipids using electron‐induced dissociation (EID). Furthermore, the inclusion of nomenclature to systematically assign bond cleavage sites with acyl chain position and double‐bond location enables a uniform platform for lipid identification. The EID methodology detailed here combines novel application of an electron‐based dissociation technique with high‐resolution mass spectrometry that facilitates a new experimental approach for lipid biomarker discovery and validation. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis of sidisterone, a phytoecdysteroid from Silene dioica (L.) Clairv.

The first synthesis of the natural phytoecdysteroid sidisterone is realized. It is based on the isoxazoline ring reductive cleavage of 20-hydroxy-20-isoxazolynylecdysterone, acid-catalyzed cyclization of the resultant ketodiol into a dihydrofuran derivative, and ozonolysis of the exocyclic double bond of the latter.     

Charging and Charge Switching of Unsaturated Lipids and Apolar Compounds Using Paternò-Büchi Reactions

The ability to control the charge state and ionization efficiency of lipids and hydrocarbons by means of in-source Paternò-Büchi functionalization in nano-electrospray ionization mass spectrometry experiments is investigated. Ultraviolet light irradiation of acetylpyridine filled nano-electrospray emitter tips, containing unsaturated analytes, generates protonated lipid and hydrocarbon ions. Comparison of reaction yields and fragment ion abundances of functionalized unsaturated fatty acids indicate that acetylpyridine Paternò-Büchi functionalization allows to readily detect fatty acids and determine double bond positions, but fragmentation efficiency and reactivity depend on double bond position and varies between different acetylpyridine isomers. Results for methyl oleate and olefins suggest that fragment ion abundances of unsaturated compounds depend on interactions between acetylpyridine and nearby functional groups. Paternò-Büchi functionalization with acetylpyridine was used to detect and assign double bond positions of mono- and polyunsaturated fatty acid, cholesterol ester, triglyceride, and hydrocarbon standards with ion abundances that are up to 631 times higher than abundances of the same compounds prior Paternò-Büchi reaction. To demonstrate the scope and analytical robustness of the newly developed method, free fatty acids in mouse brain as well as male Schistosoma mansoni extracts and hydrocarbons in an olefin mixture are investigated. For this complex set of analytes, charging and charge switching using acetylpyridine Paternò-Büchi functionalization enable double bond position assignment and relative quantification in positive ion mode.

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Transformations of peroxide olefin ozonolysis products in methanol in the presence of water

Transformations of peroxide products of ozonolysis of various olefins with different degrees of substitution at the double bond by the action of hydroxylamine and semicarbazide hydrochloride in methanol in the presence of water as co-solvent were studied.     

Localization of cyclopropyl groups and alkenes within glycerophospholipids using gas-phase ion/ion chemistry

Shotgun lipid analysis using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a common approach for the identification and characterization of glycerophohspholipids GPs. ESI-MS/MS, with the aid of collision-induced dissociation (CID), enables the characterization of GP species at the headgroup and fatty acyl sum compositional levels. However, important structural features that are often present, such as carbon–carbon double bond(s) and cyclopropane ring(s), can be difficult to determine. Here, we report the use of gas-phase charge inversion reactions that, in combination with CID, allow for more detailed structural elucidation of GPs. CID of a singly deprotonated GP, [GP − H]⁻, generates FA anions, [FA − H]⁻. The fatty acid anions can then react with doubly charged cationic magnesium tris-phenanthroline complex, [Mg(Phen)₃]²⁺, to form charge inverted complex cations of the form [FA − H + MgPhen₂]⁺. CID of the complex generates product ion spectral patterns that allow for the identification of carbon–carbon double bond position(s) as well as the sites of cyclopropyl position(s) in unsaturated lipids. This approach to determining both double bond and cyclopropane positions is demonstrated with GPs for the first time using standards and is applied to lipids extracted from Escherichia coli.