A new synthesis of lysophosphatidylcholines and related derivatives. Use of p-toluenesulfonate for hydroxyl group protection

A new stereoselective synthesis of lysophosphatidylcholines is reported. The synthesis is based upon (1) the use of 3-p-toluenesulfonyl-sn-glycerol to provide the stereocenter for construction of the optically active lysophospholipid molecule, (2) tetrahydropyranylation of the secondary alcohol function to achieve orthogonal protection of the sn-2- and sn-3-glycerol positions, and (3) elaboration of the phosphodiester headgroup using a 2-chloro-1,3,2-dioxaphospholane/trimethylamine sequence. In the course of developing the synthesis it has been discovered that methoxyacetate displacement of the sn-3-p-toluenesulfonate yields a reactive methoxyacetyl ester, which in turn can be selectively cleaved with methanol/tert-butylamine, while the ester group at the sn-1-position remains unaffected. The sequence has been shown to be suitable for preparation of spectroscopically labeled lysophosphatidylcholines. One of these compounds was readily converted to a double-labeled mixed-chain phosphatidylcholine applicable for real-time fluorescence resonance energy transfer (FRET) assay of lipolytic enzymes. In addition, the work led to new synthetic strategies based on chemoselective manipulation of the tosyl group in the presence of other base-labile groups such as FMOC derivatives that are often used for the protection of amino and hydroxyl groups in syntheses.     

Synthesis of migration-resistant hydroxyethoxy analogues of lysophosphatidic acid

[reaction: see text] The susceptibility of lysophosphatidic acid (LPA) to intramolecular acyl migration impedes the determination of specific receptor activation by the sn-1 and sn-2 LPA regioisomers. An efficient enantioselective synthesis of hydroxyethoxy (HE)-substituted analogues of sn-1-acyl and 2-acyl LPA derivatives that possess palmitoyl and oleoyl chains is described. While the palmitoyl derivatives fail to activate calcium release in cells transfected with LPA(2) or LPA(3) G-protein-coupled receptors, the LPA(3) receptor is activated by both 1-HE and 2-HE oleoyl LPA analogues with a potency 10-fold lower than that of the parent oleoyl LPA.     

Synthesis, pharmacology, and cell biology of sn-2-aminooxy analogues of lysophosphatidic acid

An efficient enantioselective synthesis of sn-2-aminooxy (AO) analogues of lysophosphatidic acid (LPA) that possess palmitoyl and oleoyl acyl chains is presented. Both sn-2-AO LPA analogues are agonists for the LPA1, LPA2, and LPA4 G-protein-coupled receptors, but antagonists for the LPA3 receptor and inhibitors of autotaxin (ATX). Moreover, both analogues stimulate migration of intestinal epithelial cells in a scratch wound assay.     

Identification of sulfoquinovosyldiacyglycerides from Phaeodactylum tricornutum by matrix-assisted laser desorption/ionization QTrap time-of-flight hybrid mass spectrometry

The pharmaceutical industry is interested in identifying novel target compounds. Due to their versatile pharmacological activities (e.g. antiviral, anti-carcinogen and immunosuppressive) sulfoquinovosyldiacylglycerides (SQDGs) are potential drug candidates. The present publication deals with the purification and structural characterization of SQDGs from three different strains of Phaeodactylum tricornutum. Besides detection of SQDGs (sn-1: C16:1/sn-2: C16:0 and sn-1: C20:5/sn-2: C16:0), two novel 2'-O-acylsulfoquinovosyldiacylglyerides (Ac-SQDGs, sn-1: C16:0/ sn-2: C16:0/2' C20:5 and sn-1: C20:5/sn-2: C16:0/2' C20:5) were identified by using matrix-assisted laser desorption/ionization (MALDI) QTrap time-of-flight (ToF) hybrid mass spectrometry (MS) with multistage MS(n). The analytical method enables the sn-position verification of fatty acids (MS(2)) as well as the confirmation of the regioposition of eicospentanoic acid at the sulfoquinovose (MS(3)).     

Synthesis of 2-Oxo amide triacylglycerol analogues and study of their inhibition effect on pancreatic and gastric lipases

A general method for the synthesis of chiral 2-oxo amide triacylglycerol analogues, from (R)- or (S)-3-aminopropane-1,2-diol, was developed. These novel inhibitors of digestive lipases are analogues of the triacylglycerol molecule, a natural substrate of lipases, and they were designed to contain the 2-oxo amide functionality in place of the scissile ester bond at the sn-1 or sn-3 position and nonhydrolysable ether bonds instead of ester bonds at the other two remaining positions. The 2-oxo amide derivatives synthesised were tested for their ability to form stable monomolecular films at the air/water interface by recording their surface pressure/molecular area compression isotherms. The inhibition of porcine pancreatic and human gastric lipases by the 2-oxo amides was studied by means of the monolayer technique with mixed films of 1,2-dicaprin and with variable proportions of each inhibitor. The alpha50 values of these triacylglycerol analogues for PPL and HGL varied between 4.4 to 7.0% and 5.6 to 15.9%, respectively. The chirality at the sn-2 position of 2-oxo amide triacylglycerol analogues affected the alpha50 value for HGL, but not for PPL.     

Synthesis of monofluorinated analogues of lysophosphatidic acid

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions both with G-protein coupled seven transmembrane receptors and with the nuclear hormone receptor PPARgamma. Synthesis and biological activities of fluorinated analogues of LPA are still relatively unknown. In an effort to identify receptor-selective LPA analogues and to document in detail the structure-activity relationships of fluorinated LPA isosteres, we describe a series of monofluorinated LPA analogues in which either the sn-1 or the sn-2 hydroxy group was replaced by fluorine, or the bridging oxygen in the monophosphate was replaced by an alpha-monofluoromethylene (-CHF-) moiety. The sn-1 or sn-2 monofluorinated LPA analogues were enantiospecifically prepared from chiral protected glycerol synthons, and the alpha-monofluoromethylene-substituted LPA analogues were prepared from a racemic epoxide with use of a hydrolytic kinetic resolution. The sn-2 and sn-1 fluoro LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. The alpha-monofluoromethylene LPA analogues were unique new nonhydrolyzable ligands with surprising enantiospecific and receptor-specific biological readouts, with one compound showing a 1000-fold higher activity than native LPA for one receptor subtype.     

Original normal-phase high-performance liquid chromatographic separation of monoacylglycerol classes from extra virgin olive oil triacylglycerols for their stereospecific analysis

An innovative procedure to separate the 3 isomeric sn-monoacylglycerols (MAG) classes (sn-1-, sn-2-, sn-3-MAG) is described. MAGs, obtained by chemical deacylation of triacylglycerols (TAGs), have been derivatized with (S)-(+)-1-(1-naphtyl)ethyl-isocyanate, and the resulting urethane derivatives have been separated by normal-phase high-performance liquid chromatography. This procedure allows resolution as diasteroisomers of the 2 enantiomeric classes (sn-1-MAG and sn-3-MAG), without the need of a chiral column, and to separate also the isomeric sn-2-MAG class; moreover, by introducing a chromophoric moiety, this reagent makes possible the ultraviolet detection of the analyte molecules. This procedure has been used to obtain the stereospecific analysis of the TAG fraction of extra virgin olive oil samples. The use of a nondestructive detector permitted the collection of the individual urethane classes; the fatty acid composition of each was determined by high-resolution gas chromatography, obtaining directly from the data the fatty acid distribution within each sn- position of TAGs. To validate this new method, the results have been compared with those obtained by 2 other procedures for TAG stereospecific analysis, and the obtained results were satisfactory since the proposed method gave data very similar to the other procedures.     

Solid-phase synthesis of sn-1,2- and sn-2,3-diacylglycerols via ring-opening of the glycidyl-bound resin

A general method developed for the parallel solid-phase synthesis of sn-1,2- and sn-2,3-diacyglycerol derivatives. The technique relies on the use of carboxylic acid-promoted epoxide ring-opening reactions of the glycidyl-bound resin 3. The polymer-bound monoacylglycerol 5, formed in this manner, is transformed to the respective polymer-bound diacylglycerols 7 and 9 by reaction of the free alcohol moiety with a second carboxylic acid under conditions that lead to retention or inversion of C-2 stereochemistry. The sequence is completed by BCl3-promoted cleavage of 7 and 9 to form the sn-1,2- and sn-2,3-diacylglycerols, respectively.     

Characterization of acylphosphatidylglycerols from <Emphasis Type="Italic">salmonella typhimurium</Emphasis> by tandem mass spectrometry with electrospray ionization

Acylphosphatidylglycerol (Acyl-PG), a polar lipid class containing three fatty acyl groups, was isolated from Salmonella bacteria and characterized by tandem quadrupole and quadrupole ion-trap mass spectrometric methods with electrospray ionization. The structural characterization of the acyl-PG with various acyl groups (A-B/C-PG, where A not equal B not equal C) is based on the findings that the carboxylate anions (R(x)CO(2)(-)) arising from sn-2 (R(2)CO(2)(-)) is more abundant than that arising from sn-3' (R(3')CO(2)(-)), which is much more abundant than that arising from sn-1 (R(1)CO(2)(-)). This information provides a simple method for determination of the fatty acyl moieties and their positions in the molecule. The structural identification of the molecule can also be achieved by the findings that the fragment ion reflecting the ketene loss at sn-2 is more prominent than that reflecting the acid loss (i.e., [M - H - R'(2)CH=CO](-) > [M - H - R(2)CO(2)H](-)), while the ion arising from acid loss at sn-1 or sn-3' is, respectively, more abundant than the corresponding ketene loss (i.e., [M - H - R(1)CO(2)H](-) > [M - H - R'(1)CH=CO](-); [M - H - R(3')CO(2)H](-) > [M - H -R'(3')CH=CO](-)). The identity of the acyl moiety at sn-3' can be confirmed by an acyl-glycerophosphate anion observed in the product-ion spectrum obtained with a triple-stage quadrupole (TSQ) instrument, but not in that obtained with an ion-trap mass spectrometer (ITMS). However, the MS(2)-spectrum obtained with an ITMS is featured by the ion series that abundances of [M - H - R'(2)CH=CO - R(3)CO(2)H - 74](-) > [M - H - R'(2)CH=CO - R(1)CO(2)H - 74](-) z.Gt; [M - H - R'(1(or 3'))CH=CO - R(3'(or 1))CO(2)H - 74](-). This information also facilitates structural elucidation of the acyl-PG subclass that contains various acyl substituents. Structural identifications of molecular species having two identical fatty acyl substituents at sn-1, sn-2, or sn-3' or consisting of more than one isomeric structures are also demonstrated. The identities of the minor isomeric species in the molecules can be revealed by the aforementioned structural information arising from the various ion series combined.     

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.     

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).     

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。     

Synthesis of alpha-fluorinated phosphonates from alpha-fluorovinylphosphonates: a new route to analogues of lysophosphatidic acid

A versatile, efficient method for the preparation of alpha-monofluoromethylene (-CHF-) phosphonates from alpha-fluorovinylphosphonate provides access to a class of lysophosphatidic acid (LPA) receptor-subtype agonists. In addition, sn-2 O-methylation of alpha-monofluoromethylene phosphonates using trimethylsilyldiazomethane generated sn-1-acyl, 2-O-methyl alpha-monofluoromethylene derivatives. Finally, a novel method for the selective etherification of 1,2-diols was developed and a new class of sn-1 O-methyl, 2-acyl alpha-monofluoromethylene LPA analogues was prepared. [reaction: see text]     

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.     

Molecular organization of cholesterol in unsaturated phosphatidylethanolamines: X-ray diffraction and solid state 2H NMR reveal differences with phosphatidylcholines

The major mammalian plasma membrane lipids are phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and cholesterol. Whereas PC-cholesterol interactions are well studied, far less is known about those between PE and cholesterol. Here, we investigated the molecular organization of cholesterol in PEs that vary in their degree of acyl chain unsaturation. For heteroacid sn-1 saturated (palmitoyl), sn-2 unsaturated (various acyl chain) PEs, cholesterol solubility determined by X-ray diffraction was essentially identical with 1 (oleoyl, 51 +/- 3 mol %) and 2 (linoleoyl, 49 +/- 2 mol %) double bonds before decreasing progressively with 4 (arachidonyl, 41 +/- 3 mol %) and 6 (docosahexaenoyl, 31 +/- 3 mol %) double bonds. With 6 double bonds in each chain, cholesterol solubility was further reduced to 8.5 +/- 1 mol %. However, (2)H NMR experiments established that the orientation of cholesterol in the same heteroacid PE membranes was unaffected by the degree of acyl chain unsaturation. A tilt angle of 15 +/- 1 degrees was measured when equimolar [3alpha-(2)H(1)]cholesterol was added, regardless of the number of double bonds in the sn-2 chain. The finding that solubility of cholesterol in sn-1 saturated PEs depends on the amount of polyunsaturation in the sn-2 chain of PE differs from the equivalent PCs that universally incorporate approximately 50 mol % sterol. Unlike PCs, a differential in affinity for cholesterol and tendency to drive lateral segregation is inferred between polyunsaturated PEs. This distinction may have biological implications reflected by the health benefits of dietary polyunsaturated fatty acids that are often taken up into PE > PC.     

Structural determination of hexadecanoic lysophosphatidylcholine regioisomers by fast atom bombardment tandem mass spectrometry

The structural determination of sn-1 and sn-2 hexadecanoic lysophosphatidylcholine (LPC) regioisomers was carried out using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The collision-induced dissociation (CID) of protonated and sodiated molecules produced diverse product ions due mainly to charge remote fragmentations. Based on the information obtained from the CID spectra of protonated and sodiated molecules, sn-1 and sn-2 hexadecanoic LPC isomers could be discriminated. Especially, the abundance ratio of the diagnostic ion pair [m/z 224/226] in the CID spectra of [M + H](+) ions was shown to be greatly different. Moreover, the CID-MS/MS spectra of sodium-adducted molecules for hexadecanoic LPC isomers showed characteristic product ions such as [M + Na - 103](+), [M + Na - 85](+), and [M + Na - 59](+), by which their regio-specificity can be differentiated.     

Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine derivatives: potential fluorinated probes for the study of membrane topology

The synthesis of three monofluorinated dimyristoylphosphatidylcholine derivatives (F-DMPC), with the fluorine atom located on the acyl chain in position 2 of the glycerol (sn-2) is described. The synthetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) and three different fluorinated fatty acids. The latter were obtained from two different and complementary synthetic routes. Preliminary FTIR studies suggest that the presence of the fluorine atom does not significantly perturb the lipid conformational order and phase transition temperature and that these monofluorinated PC derivatives could be used as probes for the study of membrane topology, i.e. the location of drugs, peptides or proteins in membranes.     

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.     

Direct analysis by electrospray ionization tandem mass spectrometry of mixtures of phosphatidyldiacylglycerols from Lactobacillus

Electrospray ionization followed by collision-induced dissociation in a quadrupole ion trap mass spectrometer of mixtures of deprotonated phosphatidyldiacylglycerols afforded a group of three diagnostic ions of convenient abundance for each phosphatidyldiacylglycerol (PG) present in the mixture. Thus, it was possible to determine unmistakably the identity and substitution positions (sn-1 or sn-2) for both acyl groups of each PG present in the mixture. The method also allows the study of isomeric mixtures of PG and mixtures containing minor amounts of some PG from crude extracts of Lactobacillus acidophillus. The present results improve those of previous studies using fast atom bombardment and electrospray ionization tanden mass spectrometry, in which it was reported that it was possible to differentiate the identity and position of the sn-2 acyl substituent only by the presence of one ion, with variable abundance.     

Analysis of triacylglycerols with non-aqueous reversed-phase liquid chromatography and positive ion electrospray tandem mass spectrometry

A non-aqueous reversed-phase liquid chromatographic method coupled to electrospray ionisation (ESI) tandem mass spectrometry was developed for the analysis of triacylglycerols (TGs). The synthetic TGs studied were separated according to their equivalent carbon number with a gradient of methanol (containing 0.01% (v/v) formate adjusted to pH 5.3 with ammonia) and chloroform. ESI mass spectra of TGs yielded positive ion current signals for [M + NH(4)](+) and [M + NH(4)-RCOONH(4)](+). The mass spectra also showed signals believed to arise from [M + K](+). Collision-induced dissociation (CID) of the [M + NH(4)](+) precursor ion yielded [M + NH(4) - RCOONH(4)](+), [RCO + 74](+) and [RCO](+) product ions as aids for the structural elucidation of the TGs. In addition, [RCO - 18](+) and small amounts of [RCO - 2](+) product ions were also found. The latter ions were observed only for TGs containing unsaturated fatty acids. CID of ammoniated 1-stearoyl-2-oleoyl-3-linoleoyl-glycerol (18:0/18:1/18:2) indicated that neutral loss of the sn-2 fatty acid was energetically less favourable than loss of the fatty acid from the sn-1 or sn-3 position.