Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument

We describe features of tandem mass spectra of lithiated adducts of triacylglycerol (TAG) species obtained by electrospray ionization mass spectrometry (ms) with low-energy collisionally activated dissociation (CAD) on a triple stage quadrupole instrument. The spectra distinguish isomeric triacylglycerol species and permit assignment of the mass of each fatty acid substituent and positions on the glycerol backbone to which substituents are esterified. Source CAD-MS2 experiments permit assignment of double bond locations in polyunsaturated fatty acid substituents. The ESI/MS/MS spectra contain [M + Li - (RnCO2H)]+, [M + Li - (RnCO2Li)]+, and RnCO+ ions, among others, that permit assignment of the masses of fatty acid substituents. Relative abundances of these ions reflect positions on the glycerol backbone to which substituents are esterified. The tandem spectra also contain ions reflecting combined elimination of two adjacent fatty acid residues, one of which is eliminated as a free fatty acid and the other as an alpha, beta-unsaturated fatty acid. Such combined losses always involve the sn-2 substituent, and this feature provides a robust means to identify that substituent. Fragment ions reflecting combined losses of both sn-1 and sn-3 substituents without loss of the sn-2 substituent are not observed. Schemes are proposed to rationalize formation of major fragment ions in tandem mass spectra of lithiated TAG that are supported by studies with deuterium-labeled TAG and by source CAD-MS2 experiments. These schemes involve initial elimination of a free fatty acid in concert with a hydrogen atom abstracted from the alpha-methylene group of an adjacent fatty acid, followed by formation of a cyclic intermediate that decomposes to yield other characteristic fragment ions. Determination of double bond location in polyunsaturated fatty acid substituents of TAG is achieved by source CAD experiments in which dilithiated adducts of fatty acid substituents are produced in the ion source and subjected to CAD in the collision cell. Product ions are analyzed in the final quadrupole to yield information on double bond location.     

Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using reversed-phase chromatography was developed for the analysis of phospholipids from bacterial extracts of a wild-type strain of Escherichia coli. Product ion mass spectra from [M--H](-) precursor ions allowed an identification of individual phospholipid species that includes both fatty acid composition and fatty acyl location on the glycerol backbone using diagnostic product ions. Thus, complete assignment, including sn-1/sn-2 fatty acyl position, was achieved for this strain of E. coli. In addition, the phospholipids were quantified relative to one another using an internal standard method.     

Structural determination of monoacylglycerols extracted from marine sponge by fast atom bombardment tandem mass spectrometry

Five new monoacylglycerols (MAGs) were isolated from the marine sponge Stelletta sp. by reversed-phase high-performance liquid chromatography and analyzed by positive ion fast atom bombardment mass spectrometry (FAB-MS). FAB mass spectra of these compounds produced abundant sodium-adducted molecules [M+Na]+ from a mixture of 3-nitrobenzyl alcohol and sodium iodide. The structural elucidation of these sponge MAGs was carried out by FAB tandem mass spectrometry (MS/MS). To find diagnostic ions for the characterization of the MAGs, authentic MAGs were initially analyzed by collision-induced dissociation (CID) MS/MS. The CID MS/MS of [M+Na]+ precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge-remote fragmentation (CRF) provided important information for the characterization of acyl chains substituted at the glycerol backbone, and product ions at m/z 84, 97, 113 and 139 were diagnostic for the sodiated glycerol backbone. On the basis of these fragmentation patterns, the structures of five MAGs extracted from marine sponge were elucidated. In addition, high-resolution mass measurement was performed to obtain the elemental compositions of the MAGs.     

Quantification of triacylglycerols in butterfat by gas chromatography-electron impact mass spectrometry using molar correction factors for [M-RCOO]+ ions

A quantitative electron impact GC-MS method using molar correction factors (MCFs) for [M-RCOO]+ ions has been developed for determination of molecular species of triacylglycerols (TAGs). MCFs were determined by linear calibration for 226 ions of 104 TAG species with good reproducibility: on the average, coefficient of determination was 0.975 +/- 0.043 and 0.963 +/- 0.115 for saturated and unsaturated TAGs, respectively. The MCFs of the sn-1(3) regioisomers of short-chain TAGs were lower than those of sn-2 isomers indicating ca. 2-3-fold higher cleavage of butyroyl and caproyl groups from the primary positions than from the secondary position. The method enabled quantification of 139 and 135 individual TAG species of butterfat (BF) and interesterified butterfat, respectively, including several regioisomers of short-chain TAGs. The most abundant molecular species of the even-numbered TAGs in BF were butyroylpalmitoyloleoylglycerol (5.05 mol%), butyroyldipalmitoylglycerol (4.75 mol%), and palmitoyldioleoylglycerol (3.32 mol%). The method provides an alternative for elucidation of nutritional and technological properties of relatively saturated TAG mixtures.     

Characterization of cardiolipin as the sodiated ions by positive-ion electrospray ionization with multiple stage quadrupole ion-trap mass spectrometry

The application of multiple-stage ion-trap (IT) mass spectrometric methods for the structural characterization of cardiolipin (CL), a 1,3-bisphosphatidyl-sn-glycerol that consists of four fatty acyl chains and three glycerol backbones (designated as A, B, and central glycerol, respectively), as the sodiated adduct ions in the positive-ion mode was evaluated. Following collisionally activated dissociation (CAD), the [M - 2H + 3Na]+ ions of CL yield two prominent fragment ion pairs that consist of the phosphatidyl moieties attached to the 1'- and 3'-position of the central glycerol, respectively, resulting from the differential losses of the diacylglycerol moieties containing A and B glycerol, respectively. The results are consistent with those previously described for the [M - H]- and [M - 2H + Na]- ions in the negative-ion mode, thus permitting assignment of the two phosphatidyl moieties attached to the 1'- or 3'-position of the central glycerol. The identities of the fatty acyl substituents and their positions on the glycerol backbones (glycerol A and B) are deduced from further degradation of the above ion pairs that give the fragment ions reflecting the fatty acid substituents at the sn-1 (or sn-1') and sn-2 (or sn-2') positions. The ions that arise from losses of the fatty acid substituents at sn-1 and sn-1', respectively, are prominent, but the analogous ions from losses of the fatty acid substituents at sn-2 and sn-2', respectively, are of low abundance in the MS2 product-ion spectra. This feature further confirms the assignment of the positions of the fatty acid substituents. The similar IT multiple-stage mass spectrometric approaches including MS2 and MS3 for structural characterization of CL using its [M + Na]+ and the [M - H + 2Na]+ ions are also readily applicable. However, their uses for structural characterization are less desirable because formation of the [M + Na]+ and the [M - H + 2Na]+ ions for CL is not predictable.     

Structural characterization of phosphatidyl-<Emphasis Type="Italic">myo</Emphasis>-inositol mannosides from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> bacillus calmette guérin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. I. PIMs and lyso-PIMs

We described a multiple-stage ion-trap mass spectrometric approach to characterize the structures of phosphatidylinositol and phosphatidyl-myoinositol mannosides (PIMs) in a complex mixture isolated from Mycobacterium bovis Bacillus Calmette Guérin. The positions of the fatty acyl substituents of PIMs at the glycerol backbone can be easily assigned, based on the findings that the ions arising from losses of the fatty acid substituent at sn-2 as molecules of acid and of ketene, respectively (that is, the [M - H - R(2)CO(2)H](-) and [M - H - R(2)CHCO](-) ions), are respectively more abundant than the ions arising from the analogous losses at sn-1 (that is, the [M - H - R(1)CO(2)H](-) and [M - H - R(1)CHCO](-) ions) in the MS(2) product-ion spectra of the [M - H](-) ions desorbed by electrospray ionization (ESI). Further dissociation of the [M - H - R(2)CO(2)H](-) and [M - H - R(1)CO(2)H](-) ions gives rise to a pair of unique ions corresponding to losses of 74 and 56 Da (that is, [M - H - R(x)CO(2)H - 56](-) and [M - H - R(x)CO(2)H - 74](-) ions, x = 1, 2), respectively, probably arising from various losses of the glycerol. The profile of the ion-pair in the MS(3) spectrum of the [M - H - R(2)CO(2)H](-) ion is readily distinguishable from that in the MS(3) spectrum of the [M - H - R(1)CO(2)H](-) ion and thus the assignment of the fatty acid substituents at the glycerol backbone can be confirmed. The product-ion spectra of the [M - H](-) ions from 2-lyso-PIM and from 1-lyso-PIM are discernible and both spectra contain a unique ion that arises from primary loss of the fatty acid substituent at the glycerol backbone, followed by loss of a bicyclic glycerophosphate ester moiety of 136 Da. The combined structural information from the MS(2) and MS(3) product-ion spectra permit the complex structures of PIMs that consist of various isomers to be unveiled in detail.     

Production of Human Milk Fat Replacement Rich of 1,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin,Ethyl Oleate And Mixture of VCO,Soybean Oil And Fish Oil

Human Milk is naturally the only source of food for infant in their early life. It contains 2-6% lipid which provides about 50% of the total energy needed by the infant. Human milk fat (HMF) mainly as TAG with the specific fatty acid composition, palmitic acid (C16:0) (20-25%), which is primary located at sn-2 of glycerol bonds (70%) and oleic acid (C18:1), located at sn-1,3 (35%). HMF also provide fatty acids such as linoleic acid, linolenic acid, EPA, DHA and lauric acid that are very important for infant. The purposes of this research are to synthesize of 1,3-dioleoyl-2-palmitoilglycerol (OPO) and to determine the best composition of OPO, VCO, soybean oil and fish oil for HMFS production for infant formula. Interesterification of tripalmitin and ethyl oleate using immobilized lipase from Rhizomucor miehei (Lipozym RM IM) were used to synthesize of OPO. Interesterification product of mixed VCO, soybean oil and fish oil that are source of lauric acid, linoleic acid, α-linolenic, EPA and DHA, were formulated in mass ratio (58:20:20:2) and (70:18:10:2) for obtaining HMFS which have fatty acids composition similar or close to HMF. Composition of fatty acids from product were analyzed by GCMS. From this research, were obtained HMFS containing palmitic acid as much as 28.89% where 84.49% of that are located at sn-2 while sn-1,3 position are dominated by oleic acid as much as 55.11% from the total 38.7% and 70:18:10:2 w/w is the best composition of interesterification product, VCO, soybean oil and fish oil to obtain HMFS similar to HMF.     

Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: A mechanistic study

Structural characterization of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PI-4P), and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) by collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization is described. In negative ion mode, the major fragmentation pathways under low energy CAD for PI arise from neutral loss of free fatty acid substituents ([M - H - RxCO2H]-) and neutral loss of the corresponding ketenes ([M - H - R'xCH=C=O]-), followed by consecutive loss of the inositol head group. The intensities of the ions arising from neutral loss of the sn-2 substituent as a free fatty acid ([M - H - R2CO2H]-) or as a ketene ([M - H - R'2CH=C=O] ) are greater than those of ions reflecting corresponding losses of the sn-1 substutient. This is consistent with our recent finding that ions reflecting those losses arise from charge-driven processes that occur preferentially at the sn-2 position. These features permit assignment of the position of the fatty acid substituents on the glycerol backbone. Nucleophilic attack of the anionic phosphate onto the C-1 or the C-2 of the glycerol to which the fatty acids attached expels sn-1 (R1CO2-) or sn-2 (R2CO2-) carboxylate anion, respectively. This pathway is sterically more favorable at sn-2 than at sn-1. However, further dissociations of [M - H - RxCO2H - inositol] , [M - H - RxCO2H]-, and [M - H - RxCH=C=O]- precursor ions also yield RxCO2- ions, whose abundance are affected by the collision energy applied. Therefore, relative intensities of the RxCO2- ions in the spectrum do not reflect their positions on the glycerol backbone and determination of their regiospecificities based on their ion intensities is not reliable. The spectra also contain specific ions at m/z 315, 279, 259, 241, and 223, reflecting the inositol head group. The last three ions are also observed in the tandem spectra of the [M - H]- ions of phosphatidylinositol monophosphate (PI-P) and phosphatidylinositol bisphosphate (PI-P2), in addition to the ions at m/z 321 and 303, reflecting the doubly phosphorylated inositol ions. The PI-P2 also contains unique ions at m/z 401 and 383 that reflect the triply phosphorylated inositol ions. The [M - H]- ions of PI-P and PI-P2 undergo fragmentation pathways similar to that of PI upon CAD. However, the doubly charged ([M - 2H]2-) molecular ions undergo fragmentation pathways that are typical of the [M - H]- ions of glycerophosphoethanolamine, which are basic. These results suggest that the further deprotonated gaseous [M - 2H]2 ions of PI-P and PI-P2 are basic precursors.     

Structural characterization of unsaturated glycerophospholipids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization

Structural elucidation of glycerophospholipids (GPLs), including the polar head group, the position of double-bond(s) along the fatty acyl substituents, and the positions of acyl groups on the glycerol backbone, using multiple-stage liner ion-trap (LIT) mass spectrometric approach is described in this paper. While the product-ion spectra from MSn (n=2, 3) on the [M+Li]+ or [M-H+2Li]+ ions of GPL are readily applicable for discerning the phospholipid classes and for identifying and locating the fatty acid substituents on the glycerol backbone, the structural information from further dissociation of the dilithiated fatty acid cations produced from MSn (n=3, 4) on the [M-H+2Li]+ ion of GPLs, as well as from further dissociation of the monolithiated fragment ion that bears the unsaturated fatty acid moiety produced from subsequent MSn (n=3,4) on the [M+Li]+ ions of GPLs, affords assignment of the position of double-bond(s) along the fatty acyl groups. The application of the present method in the structural characterization of GPL molecules from the lipid extracts of biological origin, including mixtures of phosphatidylglycerol and of phosphatidylserine without prior chromatographic separation, is also demonstrated. Since lithiated molecular species of GPL are readily formed by ESI, this multiple-stage LIT mass spectrometric approach provides a direct means for the near-complete structural characterization of all the GPLs, including the molecules in the lysophospholipid and plasmalogen subclasses.     

Quantification of distinct molecular species of the 2-lyso metabolite of platelet-activating factor by gas chromatography-negative-ion chemical ionization mass spectrometry.

The biological activity of platelet-activating factor (PAF) is comprised by a few molecular species of phosphatidylcholine which contain a fatty alcohol connected by an ether linkage to the sn-1 position of the glycerol backbone and an acetate ester at the sn-2 position. The various molecular species of PAF differ in chain length and degree of unsaturation in the fatty alcohol residue side-chain. PAF is rapidly hydrolyzed to lyso-PAF by an acetylhydrolase enzyme which is quite active in a number of cells that synthesize PAF. We describe a method for quantitation of lyso-PAF which involves conversion to its propionate derivative in the presence of an internal standard (deuterium-labelled PAF), digestion to the diglyceride with Bacillus cereus phospholipase C, conversion to the pentafluorobenzoate derivative and capillary column gas chromatographic-negative-ion methane chemical ionization mass spectrometric analysis. Distinct molecular species of lyso-PAF can be individually quantitated at levels of 1 ng or less. These methods are applied to the demonstration of lyso-PAF accumulation in renal tissue from transplanted allografts undergoing acute rejection, in renal tissue from kidneys subjected to cold storage and autotransplantation, and in intestinal mucosa subjected to warm ischemia and reperfusion.     

Identification by electrospray tandem mass spectrometry of spin-trapped free radicals from oxidized 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine

GPC radical species formed during oxidation of a glycerophosphocholine (16:0/18:1) under the Fenton reaction conditions were detected using a spin trap, 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO). The stable spin-trapped radical adducts were identified by mass spectrometry (MS) using electrospray (ES) as ionization method and characterized by tandem mass spectrometry (MS/MS). Radical adducts of oxidized free sn-2 fatty acid and of oxidized intact GPC, containing one, two and three additional oxygen atoms, were assigned. DMPO adducts of oxidized intact GPC were observed as singly and doubly charged ions in ES-MS, while adducts of oxidized free fatty acids were observed as singly charged ions. Oxidized free sn-2 fatty acids and intact GPC-DMPO adducts correspond to carbon- and oxygen-centered radicals that were identified by MS/MS as alkyl, hydroxy-alkyl, alkoxyl, hydroxy-alkoxyl, peroxyl and hydroperoxide-alkoxyl spin adducts. The DMPO molecule was attached predominantly at C(9) of the oleic chain. The fragmentation pathway of spin adducts with two DMPO molecules strongly suggests the presence of species that were simultaneously carbon- and oxygen-centered radicals. Several fragments identified are consistent with the presence of isomeric structures contributing to the same ions.     

高效液相色谱法测定猪油甘油三酯中的脂肪酸位置分布

 建立了一种采用高效液相色谱法(HPLC)分析猪油甘油三酯中的脂肪酸组成及其位置分布的方法。利用sn-1,3位专一性脂肪酶对甘油三酯sn-1,3位上的脂肪酸进行水解,形成sn-2位甘油单酯和游离脂肪酸；之后,通过甘油三酯中脂肪酸总含量和sn-1,3位上脂肪酸含量之间的差值计算出sn-2位上的脂肪酸含量。利用2-溴苯乙酮仅同游离脂肪酸作用的特点,将脂肪酸酯化为苯乙酰甲酯,然后进行HPLC分析。分析所用色谱柱为ZORBAX SB C18柱,以十七酸作为内标，甲醇-乙腈-水为流动相,采用梯度洗脱(梯度洗脱程序为甲醇-乙腈-水由80∶10∶10（体积比，下同）在35 min内线性变化到86∶10∶4,然后在5　min内恢复到起始比例，流动相流速为1 mL/min),通过测定苯乙酰甲酯在254 nm处的吸光度值来测定脂肪酸含量。结果表明,猪油甘油三酯中的脂肪酸主要是棕榈酸和油酸(分别占总量的26.61%和43.18%),其中油酸主要分布于sn-1,3位上,而棕榈酸分布于sn-2位上。这些测定结果与传统气相色谱法的测定结果相吻合。该方法简单可行,省去了传统测定中费时的薄层色谱分离步骤,可成为一种有效的实验室分析方法。     

Fast-atom-bombardment and tandem mass spectrometry for determining structures of fatty acids as their picolinyl ester derivatives

Picolinyl ester derivatives of common fatty acids can be readily desorbed by fast atom bombardment (FAB) as positive ions and then collisionally activated. Collisionally activated spectra of the (M + H)⁺ ions of the derivatives reveal that structurally informative remote-charge-site fragmentations occur. The presence of substitutents such as double bond, branch points, cyclopropane rings, hydroxy groups, and epoxy rings interrupts the fragmentation process in such a way that the substituent can be identified and its location on the alkyl chain can be determined. This method is also applicable to the picolinyl esters of short-chain fatty acids and to the analysis of mixtures of fatty acid derivatives. The approach is advantageous becasue the epicolinyl ester derivatives are also amenable to gas chromatography/mass spectrometry (GC/MS). Therefore, the FAB-MS/MS approach developed here is complementary to GC/MS.     

Structural heterogeneity and environmentally regulated remodeling of <Emphasis Type="Italic">Francisella tularensis</Emphasis> subspecies <Emphasis Type="Italic">novicida</Emphasis> lipid a characterized by tandem mass spectrometry

The structural characterization of environmentally-regulated lipid A derived from Francisella tularensis subspecies novicida (Fn) U112 is described using negative electrospray ionization with a linear ion trap Fourier transform ion cyclotron resonance (IT-FT-ICR) hybrid mass spectrometer. The results indicate that a unique profile of lipid A molecular structures are synthesized in response to Fn growth at 25 degrees C versus 37 degrees C. Molecular species were found to be tetra-acylated, sharing a conserved glucosamine disaccharide backbone, a galactosamine-1-phosphate linked to the reducing glucosamine, and multiple O- and N-linked fatty acyl groups. Deprotonated molecules were interrogated by MS(n) scanning techniques at both high and nominal mass resolution and were found to be complex heterogeneous mixtures where structures differed based on the positions and identities of the O- and N-linked fatty acyl substituents. For the dominant ion series, which consisted of five peaks, 30 unique lipid A structures were identified. Estimates for the relative abundance of each structure were derived from MS relative abundance ratios and fragment ion ratios from comparable dissociation pathways from MS(2) through MS(4) experiments. The results suggest a remodeling pathway in which the amide linked fatty acid of the reducing glucosamine favors a 3-hydroxyhexadecanoic acid substituent for growth conditions at 25 degrees C versus a 3-hydroxyoctadecanoic acid substituent for growth conditions at 37 degrees C.     

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 + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.     

Regiospecific analysis of neutral ether lipids by liquid chromatography/electrospray ionization/single quadrupole mass spectrometry: validation with synthetic compounds.

A reversed-phase high-performance liquid chromatography (HPLC) method with on-line electrospray ionization/collision-induced dissociation/mass spectrometry (ESI/CID/MS) is presented for the regiospecific analysis of synthetic reference compounds of neutral ether lipids. The reference compounds were characterized by chromatographic retention times, full mass spectra, and fragmentation patterns as an aid to clarify the regiospecificity of ether lipids from natural sources. The results clearly show that single quadrupole mass spectroscopic analysis may elucidate the regiospecific structure of neutral ether lipids. Ether lipid reference compounds were characterized by five to six major ions in the positive ion mode. The 1-O-alkyl-sn-glycerols were analyzed as the diacetoyl derivative, and showed the [M - acetoyl](+) ion as an important diagnostic ion. The diagnostic ions of directly analyzed 1-O-alkyl-2-acyl-sn-glycerols and 1-O-alkyl-3-acyl-sn-glycerols were the [M - alkyl](+), [M + H - H(2)O](+) and [M + H](+) ions. Regiospecific characterization of the fatty acid position was evident from the relative ion intensities, as the sn-2 species had relatively high [M + H](+) ion intensities compared with [M + H - H(2)O](+), whereas the reverse situation characterized the sn-3 species. Furthermore, corresponding sn-2 and sn-3 species were separated by the chromatographic system. However, loss of water was promoted as fatty acid unsaturation was raised, which may complicate interpretation of the mass spectra. The diagnostic ions of directly analyzed 1-O-alkyl-2,3-diacyl-sn-glycerols were the [M - alkyl](+), [M - sn-2-acyl](+) and [M - sn-3-acyl](+) ions. Regiospecific characterization of the fatty acid identity and position was evident from the relative ion intensities, as fragmentation of the sn-2 fatty acids was preferred to the sn-3 fatty acids; however, loss of fatty acids was also promoted by higher degrees of unsaturation. Therefore, both structural and positional effects of the fatty acids affect the spectra of the neutral ether lipids. Fragmentation patterns and optimal capillary exit voltages are suggested for each neutral ether lipid class. The present study demonstrates that reversed-phase HPLC and positive ion ESI/CID/MS provide direct and unambiguous information about the configuration and identity of molecular species in neutral 1-O-alkyl-sn-glycerol classes.     

Regiospecific characterisation of the triacylglycerols in animal fats using high performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry

High performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI MS) was applied to the characterisation of triacylglycerols (TAGs) in animal fats. The major TAGs in four fats (beef, chicken, lamb and pork) were identified and positional isomers assigned according to their APCI mass spectra. Beef and lamb fat TAGs were confirmed as containing higher proportions of saturated fatty acids compared with those of chicken and pork. HPLC-APCI MS was also shown to be of value in providing regiospecific information for the fatty acids in individual TAG species. For example, beef and lamb fat were shown to contain both cis- and trans-isomers of the 18:1 fatty acid, whilst chicken and pork contained only the cis-isomer. When the position of fatty acid substitution was determined from the APCI spectra, whilst the cis- 18:1 was predominantly found in the 2-position of the TAG, the trans-18:1 showed a preference for the 1/3-position. Similarly, it was confirmed that although the 2-position of beef, chicken and lamb fat TAGs was dominated by unsaturated fatty acids, in pork fat, a characteristically high proportion of palmitic acid was seen in this position. The TAGs identified compared well with those reported previously. The distributions of 2-position fatty acids seen in lamb and pork fat compared favourably with those obtained by the more traditional method of lipase degradation. Although the distributions for chicken and beef showed some discrepancies, these can be attributed to weaknesses in the quantification procedure or the specificity of the lipase. Overall, the technique of HPLC-APCI MS has been shown to be very powerful for the regiospecific analysis of animal fats.     

Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry

Electrospray ionization (ESI) tandem mass spectrometry (MS) has simplified analysis of phospholipid mixtures, and, in negative ion mode, permits structural identification of picomole amounts of phospholipid species. Collisionally activated dissociation (CAD) of phospholipid anions yields negative ion tandem mass spectra that contain fragment ions representing the fatty acid substituents as carboxylate anions. Glycerophosphocholine (GPC) lipids contain a quaternary nitrogen moiety and more readily form cationic adducts than anionic species, and positive ion tandem mass spectra of protonated GPC species contain no abundant ions that identify fatty acid substituents. We report here that lithiated adducts of GPC species are readily formed by adding lithium hydroxide to the solution in which phospholipid mixtures are infused into the ESI source. CAD of [MLi⁺] ions of GPC species yields tandem mass spectra that contain prominent ions representing losses of the fatty acid substituents. These ions and their relative abundances can be used to assign the identities and positions of the fatty acid substituents of GPC species. Tandem mass spectrometric scans monitoring neutral losses of the head-group or of fatty acid substituents from lithiated adducts can be used to identify GPC species in tissue phospholipid mixtures. Similar scans monitoring parents of specific product ions can also be used to identify the fatty acid substituents of GPC species, and this facilitates identification of distinct isobaric contributors to ions observed in the ESI/MS total ion current.     

Studies on phosphatidylglycerol with triple quadrupole tandem mass spectrometry with electrospray ionization: Fragmentation processes and structural characterization

Negative-ion low-energy collisionally activated dissociation (CAD) tandem mass spectrometry of electrospray-produced ions permits structural characterization of phosphatidylglycerol (PG). The major ions that identify the structures arise from neutral loss of free fatty acid substituents ([M − H − R _x CO₂H]⁻) and neutral loss of the fatty acids as ketenes ([M − H − R′ _x CH = C = O]⁻), followed by consecutive loss of the glycerol head group. The abundances of the ions arising from neutral loss of the sn-2 substutient as a free fatty acid ([M − H − R₂CO₂H]⁻) or as a ketene ([M − H − R′₂CH = C = O]⁻) are greater than those of the product ions from the analogous losses at sn-1. Nucleophilic attack of the anionic phosphate site on the C-1 or the C-2 of the glycerol to which the carboxylates attached expels the sn-1 (R₁CO₂⁻) or the sn-2 (R₂CO₂⁻) carboxylate anion, resulting in a greater abundance of R₂COO⁻ than R₁COO⁻. These features permit assignments of fatty acid substituents and their position in the glycerol backbone. The results are also consistent with our earlier findings that pathways leading to those losses at sn-2 are sterically more favorable than those at sn-1. Fragment ions at m/z 227, 209 and 171 reflect the glycerol polar head group and identify the various PG molecules. Both charge-remote fragmentation (CRF) and charge-drive fragmentation (CDF) processes are the major pathways for the formation of [M − H − R _x COOH]⁻ ions. The CRF process involves participation of the hydrogen atoms on the glycerol backbone, whereas the CDF process involves participation of the exchangeable hydrogen atoms of the glycerol head group. The proposed fragmentation pathways are supported by CAD tandem mass spectrometry of the analogous precursor ions arising from the H-D exchange experiment, and further confirmed by source CAD in combination with tandem mass spectrometry.     

Collision-induced dissociation of glycero phospholipids using electrospray ion-trap mass spectrometry.

Characterisation of phospholipids was achieved using collision-induced dissociation (CID) with an ion-trap mass spectrometer. The product ions were compared with those obtained with a triple quadrupole mass spectrometer. In the negative ion mode the product ions were mainly sn-1 and sn-2 lyso-phospholipids with neutral loss of ketene in combination with neutral loss of the polar head group. Less abundant product ions were sn-1 and sn-2 carboxylate anions. CID using a triple quadrupole mass spectrometer, however, gave primarily the sn-1 and sn-2 carboxylate anions together with lyso-phosphatidic acid with neutral loss of water. For the ion trap a charge-remote-type mechanism is proposed for formation of the lyso-phospholipid product ions by loss of alpha-hydrogen on the fatty acid moiety, electron rearrangement and neutral loss of ketene. A second mechanism involves nucleophilic attack of the phosphate oxygen on the sn-1 and sn-2 glycerol backbone to form carboxylate anions with neutral loss of cyclo lyso-phospholipids. CID (MS(3) and MS(4)) of the lyso-phospholipids using the ion-trap gave the same carboxylate anions as those obtained with a triple quadrupole instrument where multiple collisions in the collision cell are expected to occur. The data demonstrate that phospholipid species determination can be performed by using LC/MS(n) with an ion-trap mass spectrometer with detection of the lyso-phospholipid anions. The ion-trap showed no loss in sensitivity in full scan MS(n) compared to multiple reaction monitoring data acquisition. In combination with on-line liquid chromatography this feature makes the ion-trap useful in the scanning modes for rapid screening of low concentrations of phospholipid species in biological samples as recently described (Uran S, Larsen A, Jacobsen PB, Skotland T. J. Chromatogr. B 2001; 758: 265).