Electrospray mass spectrometry of human hair wax esters

Wax esters extracted from human hair have been examined by capillary GC-MS and by nano electrospray ionization (ESI) mass spectrometry using a tandem quadrupole mass spectrometer. Initially, the wax esters were examined by capillary GC-MS using conventional means, thus revealing an incomplete chromatographic resolution of the complex array of >200 wax esters ranging from 28 to 40 carbons in length, including saturated/straight-chained, unsaturated/straight-chained, saturated/branched, and unsaturated/branched molecular species. ESI of wax esters produced ammonium adduct ions [M+NH4]+, and collisional activation of these ions formed abundant [RCO2H2]+ product ions. Wax esters containing a double bond in the fatty acyl or fatty alcohol portion of the molecule revealed identical behavior, suggesting little influence of the double bond on the ionization process or subsequent decomposition. The wax ester mixture was analyzed by ESI and tandem mass spectrometry using multiple reaction monitoring and neutral loss scanning. The neutral loss experiment [loss of NH3 and CH2=CH-(CH2)nCH3] was particularly effective at rapidly surveying the complex biological mixture, identifying>160 different wax esters that range from 24 to 42 total carbons.     

Hydroxyl negative chemical ionization mass spectrometry linked with collisionally activated decomposition. A modern analytical tool in inborn errors of metabolism

Two kinds of inborn errors of metabolism, dicarboxylic aciduria and hyperoxaluria, have been studied by means of hydroxyl negative ion chemical ionization [NICI(OH-)], linked with collisionally activated decomposition experiments on the [M-H]- species of the pathognomonic organic acids. This method has led to non-controversial qualitative determinations of C4-C10 dicarboxylic acids and oxalic, glyceric and glyoxylic acids. NICI(OH-) linked with collisionally activated decomposition mass analysed ion kinetic energy spectrometry (CAD MIKES) is proposed herein for diagnostic purposes, as a valid mass spectrometric alternative to standard gas chromatographic/mass spectrometric analysis. The procedure is characterized by simplified sample treatment and by fast execution.     

Structural characterization of saturated branched chain fatty acid methyl esters by collisional dissociation of molecular ions generated by electron ionization

Saturated branched chain fatty acids (BCFA) are present as complex mixtures in numerous biological samples. The traditional method for structure elucidation, electron ionization (EI) mass spectrometry, sometimes does not unambiguously enable assignment of branching in isomeric BCFA. Zirrolli and Murphy (Zirrolli , J. A. , and R. A. Murphy. 1993. Low-energy tandem mass spectrometry of the molecular ion derived from fatty acid methyl esters: a novel method for analysis of branched-chain fatty acids. J. Am. Soc. Mass Spectrom. 4: 223–229.) showed that the molecular ions of four BCFA methyl ester (BCFAME) yield highly characteristic fragments upon collisional dissociation using a triple quadrupole instrument. Here, we confirm and extend these results by analysis using a tabletop 3-D ion trap for activated molecular ion EI-MS/MS to 30 BCFAME. iso-BCFAME produces a prominent ion (30-100% of base peak) for [M-43] (M-C₃H₇), corresponding to the terminal isopropyl moiety in the original iso-BCFAME. Anteiso-FAME yield prominent ions (20-100% of base peak) corresponding to losses on both side of the methyl branch, [M-29] and [M-57], and tend to produce more prominent m/z 115 peaks corresponding to a cyclization product around the ester. Dimethyl and tetramethyl FAME, with branches separated by at least one methylene group, yield fragment on both sides of the sites of methyl branches that are more than 6 C away from the carboxyl carbon. EI-MS/MS yields uniquely specific ions that enable highly confident structural identification and quantification of BCFAME.     

Liquid ionization mass spectrometry of phospholipids

Several phosphatidylcholines (PC) and a phosphatidylethanolamine (PE) were subjected to liquid ionization (LI) mass spectrometry, in which a sample is ionized through energy transfer from metastable argon atoms under atmospheric pressure. Commercially available and synthesized, saturated or unsaturated fatty acid containing phospholipids and their mixtures were studied. A sample either as a concentrated chloroform-methanol solution or with glycerol (matrix) gave characteristic peaks such as MH+ and four fragment ions. One of the fragment ions (e.g., m/z 551 of PC 16:0, 16:0) containing both fatty acid residues has been commonly observed with other ionization methods such as CI, FD, and FAB, but the other fragment ions have not been observed in other mass spectra with one exception on desorption CI. Ions b and d (e.g., m/z 464 and 328, respectively, for PC 16:0, 16:0) contain one fatty acyl residue and the other ion containing the phosphorylcholine moiety appears at m/z 196 for PC. Thus the masses of the MH+ ion and these fragment ions provide useful structural information even in the case of a mixture. The ion b (e.g., m/z 488 of PC 18:0, 18:2) observed during an early period of heating was formed mainly by the loss of one acyl group at sn-1 of the glycerol backbone and thus may be used to differentiate the positional specificities of the constituent fatty acids. The temperature of the sample, however, should be controlled precisely, because it has a significant effect on the mass spectrum. The present method (LI) also provided useful information for a mixture of PC and PE.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

Haemoglobin adducts as biomarkers of exposure to tobacco-related nitrosamines

A sensitive gas chromatography/mass spectrometry (GC/MS) method was developed to measure nitrosamine-haemoglobin adducts (HPB-Hb) (4-hydroxy-3-pyridinyl-1-butanone) at trace levels in red blood cells of smoking and non-smoking mothers and their newborn babies. GC/MS methods with chemical ionization (CI) of methane reagent gas in both positive and negative ion mode as well as electron ionization (EI) were studied to determine differences in sensitivity among the various ionization methods. Detection limits using both positive and negative chemical ionization modes were found to be 30 fmol HPB, whereas detection using electron impact modes yielded a detection limit of 80 fmol HBP. In order to apply the various methods of detection to tobacco-exposed samples from human populations, we characterized adduct levels in maternal as well as paired fetal samples obtained from mothers exposed to tobacco smoke during pregnancy. Maternal samples were characterized using serum cotinine levels and were classified as non-smokers, passively smoke-exposed women, less than one pack per day smokers and greater than one pack per day smokers. Paired maternal and fetal blood samples were obtained at delivery for qualitative and qualitative analysis of nitrosamine adducts. Comparative derivatization of HPB released under alkaline hydrolysis conditions was performed using O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and 2,3,4,5,6-pentafluorobenzoylchloride (PFBC). Both negative CI and positive CI modes of analysis were compared to the more widely accepted EI modes of mass spectrometric analysis. These results suggest that both NICI and PICI modes of detection offer a greater sensitivity of adduct characterization when compared with EI ionization techniques and that either NICI or PICI modes are preferably applicable towards the detection of human biomarker assessment of tobacco-related nitrosamines.     

Soft ionization mass spectral methods for lipid analysis

Chemical ionization (CI), field ionization (FI) and field desorption (FD) are sometimes preferable to electron impact (EI) mass spectrometry as methods for obtaining abundant high-mass ions from lipids. FD often provides mass spectral information which is unobtainable by other methods, and is the best method for obtaining molecular weight information. Fragment ions are observed in the spectra from all the ionization methods, which provide structural information complementing that obtainable from an EI spectrum. Using CI, high-mass ions carrying a large proportion of the total ionization current can be monitored by selected ion monitoring, resulting in enhanced sensitivity for quantitative studies in some cases.     

Steryl glycosides and acyl steryl glycosides from Musa paradisiaca

From peeled fruits of Musa paradisiaca (banana, vegetable variety), two new acyl steryl glycosides, sitoindoside-III and sitoindoside-IV, and two new steryl glycosides, sitosterol gentiobioside and sitosterol myo-inosityl-β-D-glucoside, have been isolated by gradient solvent extraction and extensive chromatography (CC, prep. TLC, GC and HPLC). The compounds have been characterized by comprehensive spectroscopic analyses (IR, ¹H NMR, GC, mass spectra, [α]_D) and crucial chemical transformation. Additionally, seasonal variations of the total sterols, free sterols, steryl esters, steryl glycosides and acyl steryl glycosides in the active samples of banana have been analysed. The results provide a basis for the observed fluctuations in the anti-ulcerogenic activity of the extracts, in different seasons, and the importance of appropriate formulation of the pure principles to optimize the activity.     

Analysis of medium-chain acyl-coenzyme A esters in mouse tissues by liquid chromatography-electrospray ionization mass spectrometry

Medium-chain acyl-coenzyme A (CoA) esters are key metabolites in lipid metabolism. Liquid chromatography-electrospray ionization mass spectrometry analysis of medium-chain acyl-CoA esters is described. Eight medium-chain acyl-CoA esters were well separated on a C(8)-MS reversed-phase column using a linear gradient of ammonium acetate buffer (pH 5.3)-acetonitrile. The positive-ion mass spectra of all the saturated and unsaturated medium-chain acyl-CoA esters gave dominant [M+H](+) ions, whereas their negative-ion mass spectra showed abundant [M-H](-) and [M-2H](2-) ions. The positive-ion mode of operation was slightly less sensitive than the negative-ion detection mode. Five medium-chain acyl-CoA esters of C(6:0), C(8:0), C(8:1), C(10:0), and C(10:1) in liver, heart, kidney, and brain from the mouse were identified. The predominant acyl-CoA peaks were C(6:0), C(8:0), and C(10:0). Small amounts of medium-chain acyl-CoAs of C(8:1) and C(10:1) were detected only in heart and kidney. The analytical method is very useful for the analysis of medium-chain acyl-CoA esters in the tissues.     

Analysis of cell membrane aminophospholipids as isotope-tagged derivatives

Glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) lipids were reacted with a multiplexed set of differentially isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The resulting derivatized aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions (m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectrometric response of N-methylpiperazine amide-tagged aminophospholipids was probed using glycerophosphoethanolamine and glycerophosphoserine lipid standards. The [M+H]+ of each tagged aminophospholipid shifted 144 Da, and during collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting aminophospholipids was useful for an unbiased analysis of plasmalogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the [M-H]- ions. The isotope-tagged reagents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu2+/H2O2 to illustrate the ability of these reagents to aid in the mass spectrometric identification of aminophospholipid changes that occur during biological stimuli.     

Synthesis and characterization of long-chain 1,2-dioxo compounds

A series of long-chain methyl esters with vicinal oxo groups (1,2-diones; 1,2-diketones) were synthesized by potassium permanganate-based oxidation of methyl esters of mono-unsaturated fatty acids. The presence of two additional carbonyl groups may facilitate the synthesis of other derivatives. The starting materials were selected in such a fashion to give the 1,2-dioxo moiety in consecutive positions from the methyl ester group. The compounds were characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. In mass spectrometry, both electron and chemical ionization (methane as reagent gas) were investigated. The position of the dioxo moiety can be determined in both ionization modes, however, in electron ionization mode the corresponding fragment ions are considerably stronger. In electron ionization mode, a fragmentation mechanism depending on the position of the 1,2-dioxo moiety occurs while the spectra derived from chemical ionization mode are mainly characterized by peaks around the molecular ion with both ionization modes appearing suitable.     

Fast atom bombardment,electron impact and chemical ionization mass spectrometry of the neutral 18-electron species M(CO)2(CNR)2(PR′3)2 (MMo or W)

The molybdenum(0) and tungsten(O) complexes of the type M(CO)₂(CNR)₂(PR′₃)₂ have been studied using a variety of mass spectral techniques, viz. fast atom bombardment (FAB), electron impact (EI), and both positive- and negative-ion chemical ionization (PICI and NICI) mass spectrometry. The FABMS technique gave the most structurally informative spectra with the observation of the molecular ions M⁺ (100% relative abundance in the case of MW) and in some instances the pseudomolecular ion (M + H)⁺. Fragmentation ions arising from competitive ligand loss (CO versus RNC versus PR′₃) were observed, as well as those formed by loss of H from fragment ions and dealkylation of RNC ligands. The EI and PICI spectra were not especially useful due to the relatively low thermal stability of these complexes, while the NICI spectra gave an abundance of ions that resulted from ligand redistribution reactions. Of special note were anions that contained M(CO)₄ and M(CO)₃ fragments. Dealkylation of the RNC ligands to give cyanometallate anions was also prevalent.     

Acylation of steryl glucosides by phospholipids. Solubilization and properties of the acyl transferase.

Particulate enzyme preparations of cotton fibers catalyze the acylation of exogenous steryl glucoside to form acylated steryl glucoside. The acyl transferase involved in this reaction was solubilized by treatment of the membrane fractions with Triton X-100 and was partially purified by chromatography on DEAE-cellulose and gel filtration. This solubilized enzyme had an absolute requirement for Triton X-100 and phospholipid in order to catalyze the acylation of the steryl glucoside. The best phospholipid substrate was phosphatidylethanolamine but egg and soybean phosphatidylcholine were also active. The phospholipid was shown to function as an acyl donor by demonstrating that [¹⁴C]fatty acid from ¹⁴C-labeled phospholipid could be transferred to steryl-[³H]glucoside to form [¹⁴C,³H]acylated steryl glucoside. Saponification of this compound yielded [¹⁴C]fatty acid and steryl-[⁷H]glucoside.     

Partial characterization of steryl ester biosynthesis in spinach leaves

Acetone powders of a 20,000g pellet fraction from spinach leaves (Spinacia oleracea L.) synthesized [4-(14)C]cholesteryl esters when incubated with [4-(14)C]cholesterol. The reaction was inhibited by digitonin. There was a reciprocal relationship between the decline of label in cholesterol and its incorporation into cholesteryl ester, indicating that free cholesterol was the direct precursor for cholesteryl ester biosynthesis. The hydrolysis of cholesteryl [1-(14)C]palmitate into free cholesterol and [1-(14)C]palmitate was not detected in these acetone powder preparations. Exogenous cholesteryl palmitate had no effect on the esterification of [4-(14)C]cholesterol. The data indicate that an esterase-type mechanism was not involved in the biosynthesis of these steryl esters. Label from [1-(14)C]palmitoyl-CoA was incorporated into steryl esters when incubated with spinach leaf acetone powder preparations. The optimal buffer for steryl ester biosynthesis was 2-(N-morpholino)ethanesulfonate and the optimal pH was 6. Iodoacetamide, N-ethylmaleimide, and dithiothreitol had no effect on the esterification reaction. Ethylenediaminetetraacetate, MgCl(2), CaCl(2), MnCl(2), and ZnSO(4) inhibited at concentrations of 10 to 30 mm.     

Steryl esters and their relationship to normal and diseased human central nervous system

The composition and distribution of steryl esters in human diseased or developing brain tissue has been studied. The abnormal brain conditions included sudanophilic leukodystrophy, multiple sclerosis plaque, subacute sclerosing panencephalitis, and an old cerebral infarction and two types of brain-derived tumors. In addition to the above abnormal tissue, steryl esters were also examined in developing and normal adult human brain. It was found upon subcellular fractionation that the steryl ester was localized mainly in the soluble nonparticulate material. A cholesteryl ester-rich fraction, floating on top of distilled water after centrifugation, was recovered only in the developing brain or in instances where there was myelin damage. The sterol portion of the steryl ester was largely cholesterol. The fatty acid moiety was mainly composed of C(16), C(18), and C(20) fatty acids. The dominant fatty acid was oleic acid, and the proportion of this fatty acid increased in demyelination. Although there were great differences in the quantities of steryl ester found, the fatty acid profiles of normal developing and adult brain were quite similar. As has been noted by others, the fatty acid composition of brain steryl esters most closely resembles that of brain phosphatidylcholine.     

Analysis of acidic carbohydrates as their quaternary ammonium or phosphonium salts by matrix-assisted laser desorption/ionization mass spectrometry

New two-component systems using quaternary ammonium or phosphonium salts as a co-matrix have been developed for the analysis of acidic carbohydrates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In the analysis of the sodium salt of heparin disaccharide I-S, the combination of 2-amino-5-nitropyridine with tetraphenylphosphonium bromide gave the best result. In the analysis of gangliosides containing the sialic acid moiety, the combination of 2,4,6-trihydroxyacetophenone with dimethyldipalmitylammonium bromide was determined to be the system of choice. Under optimum conditions all acidic carbohydrates gave molecular ions in the form of [M(Q(n))-Q]-, where M(Q(n)) is the molecular mass of a molecule containing n molecules of quaternary ions as salt.     

Fatty acid and sterol specificity in the biosynthesis of steryl esters by enzyme preparations from spinach leaves

Acetone powders prepared from the 20,000g participate fraction of spinach (Spinacia oleracea L.) leaves catalyzed the formation of steryl esters from free sterol and 1,2-diacylglycerol as the acyl donor. There was no sterol specificity when cholesterol, sitosterol, and campesterol were compared. When rates of sterol ester biosynthesis were compared using different 1,2-diacylglycerols it was found that the shorter chain fatty acids and the more unsaturated fatty acids were preferred. When the substrate concentration of diacylglycerol was varied, the maximal velocities obtained with the different substrates were dipalmitoleoyl- >dilinolenoyl- >dioleoyl- >dilinoleoyl-glycerol. It was demonstrated by silver nitrate thin-layer chromatography that the fatty acids of the supplied diacylglycerols were transferred to the sterol. When diacylglycerol mixtures were supplied, it was found that unsaturated diacylglycerols greatly stimulated conversion of saturated diacylglycerols to saturated steryl esters. For an equimolar mixture of dipalmitoyl-, dioleoyl-, dilinoleoyl-, and dilinolenoyl-glycerol, about equal amounts of the four steryl ester species were synthesized.     

Characterization of acyl-phosphatidylinositol from the opportunistic pathogen Corynebacterium amycolatum

The aim of the present study was to characterize a new lipid detected in the opportunistic pathogen Corynebacterium amycolatum. It was identified as acyl-phosphatidylinositol (acyl-PI), and revealed as a mixture of homologues compounds by electrospray ionization mass spectrometry, with pseudomolecular ions, (M-H)-, observed at 1099 (the major one) 1113, and 1127. Acyl-PI exclusively contained octadecenoyl on the inositol moiety (as 3-O-acyl), an unsaturated fatty acyl (mostly octadecenoyl) at sn-1 position of the glycerol and a saturated fatty acyl (mainly hexadecanoyl) at the sn-2 position. Acyl-PI constitutes a new natural substance and seems to be unique among the phospholipids of C. amycolatum. Other more complex molecules, previously undetected, and assigned in this work to several acyl forms of phosphatidylinositol trimannosides, lacked octadecenoyl in their polar heads. The present study reveals the existence of acyl-PI in C. amycolatum as rather unexpected finding and, additionally, gives evidence for the ability of this species to synthesize a great variety of inositol-containing phospholipids.     

Hydroquinone diglycoside acyl esters from the stems of Glycosmis pentaphylla

Four hydroquinone diglycoside acyl esters, glypentosides A-C (1-3) and seguinoside F (4), were isolated from the stems of Glycosmis pentaphylla. Glypentosides A-B (1-2) were identified as compounds and designated as methoxyquinol 4-O-[(5-O-trans-p-coumaroyl)-beta-d-apiofuranosyl-(1-->2)-beta-d-glucopyranoside] (1) and 4-demethylantiarol 4-O-[(3-methoxy-4-hydroxy-benzoyl)-beta-d-apiofuranosyl-(1-->2)-beta-d-glucopyranoside] (2). Glypentoside C (3) is a hydroquinone diglycoside acyl ester with a neolignan moiety in the acyl unit. Their structures were elucidated by the combination of one- and two-dimensional NMR analysis, mass spectrometry and chemical evidences.     

Rapid characterization and identification of steroidal alkaloids in Sarcococca coriacea using liquid chromatography coupled with electrospray ionization quadropole time-of-flight mass spectrometry

Rapid characterization of 23 pregnane-type steroidal alkaloids was studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M+H](+) which through low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis showed the characteristic loss of dimethylamine moiety [M+H-45](+) followed by the sequential lossess of attached substituents. Steroidal alkaloids having tigloyl or senecioyl group at C-3 produced diagnostic fragment ions at m/z 100 and 83. Our study also demonstrates the influence of unsaturation, and number and nature of substitutents on product ion abundance and fragment ions. Moreover, the generalization of the fragmentation pattern was linked with the structural features in steroidal skeleton. This strategy was successfully applied in LC-ESI-QqTOF-MS/MS analysis of Sarcococca coriacea extract to investigate and characterize pregnane-type steroidal alkaloids in complex mixture.