Fast atom bombardment and collisional activation mass spectrometry of retinyl phosphate mannose synthesized by liver membranes

Fast atom bombardment (FAB) and collisional activation dissociation (CAD) mass-analysed ion kinetic energy (MIKE) spectra have confirmed the structures of retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and guanosine 5'-diphospho-D-mannose (GDP-Man). Ret-P-Man was made in vitro while Ret-P and GDP-Man were chemically synthesized. Positive ion FAB mass spectrometry of Ret-P showed an observable short-lived spectrum with a mass ion at m/z 367 [M + H]+, and a major fragment ion at m/z 269 [M + H - H3PO4]+. Negative ion FAB mass spectrometry of Ret-P showed a strong stable spectrum with a parent ion at m/z 365 [M - H]-, a glycerol (G) adduct ion at m/z 457 [M - H + G]- and a dimer ion at m/z 731 [2M - H]-. GDP-Man showed an intense spectrum with parent ion at m/z 604 [M - H]- and cationized species at m/z 626 [M + Na - 2H]- and 648 [M + 2Na - 3H]-. Negative ion FAB mass spectrometry of Ret-P-Man showed a parent ion at m/z 527 [M - H]- and a fragment ion at m/z 259 [C6H12PO9]-. The CAD-MIKE spectra showed structurally significant fragment ions at m/z 442 and 361 for the [M - H]- ion of GDP-Man, and at m/z 509, 406, 364 and 241 for the [M - H]- ion of Ret-P-Man. FAB and CAD-MIKE spectra have been applied successfully to confirm the structure of Ret-P-Man made in vitro from Ret-P and GDP-Man.     

Simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application in pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the positive/negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of asperosaponin VI and its active metabolite hederagenin in rat plasma. After addition of internal standards diazepam (for asperosaponin VI) and glycyrrhetic acid (for hederagenin), the plasma sample was deproteinized with acetonitrile, and separated on a reversed phase C18 column with a mobile phase of methanol (solvent A)-0.05% glacial acetic acid containing 10 mM ammonium acetate and 30 μM sodium acetate (solvent B) using gradient elution. The detection of target compounds was done in multiple reaction monitoring (MRM) mode using a tandem mass spectrometry equipped with positive/negative ion-switching ESI source. At the first segment, the MRM detection was operated in the positive ESI mode using the transitions of m/z 951.5 ([M+Na](+))→347.1 for asperosaponin VI and m/z 285.1 ([M+H](+))→193.1 for diazepam for 4 min, then switched to the negative ESI mode using the transitions of m/z 471.3 ([M-H](-))→471.3 for hederagenin and m/z 469.4 ([M-H](-))→425.4 for glycyrrhetic acid, respectively. The sodiated molecular ion [M+Na](+) at m/z 951.5 was selected as the precursor ion for asperosaponin VI, since it provided better sensitivity compared to the deprotonated and protonated molecular ions. Sodium acetate was added to the mobile phase to make sure that abundant amount of the sodiated molecular ion of asperosaponin VI could be produced, and more stable and intensive mass response of the product ion could be obtained. For the detection of hederagenin, since all of the mass responses of the fragment ions were very weak, the deprotonated molecular ion [M-H](-)m/z 471.3 was employed as both the precursor ion and the product ion. But the collision energy was still used for the MRM, in order to eliminate the influences induced by the interference substances from the rat plasma. The validated method was successfully applied to study the pharmacokinetics of asperosaponin VI and its active metabolite hederagenin in rat plasma after oral administration of asperosaponin VI at a dose of 90 mg/kg.     

Generation of hydroxyeicosatetraenoic acids by human inflammatory cells: analysis by thermospray liquid chromatography-mass spectrometry

Arachidonic acid was converted to a series of hydroxyeicosatetraenoic acids (HETEs) by mixed human inflammatory cells following stimulation with the calcium ionophore A23187. HETEs were purified by a simple one-step extraction procedure followed by HPLC. The HPLC was coupled to a Finnigan quadrupole mass spectrometer using the now commercially available thermospray liquid chromatography-mass spectrometry interface. The HPLC eluant was monitored 'on line' by the mass spectrometer. Soft ionisation occurs, generating intense molecular ion species in the negative ion mode (M - H-:m/z 319) for each of the isomeric HETEs. The (M + H+ - H2O) ion at m/z 303 is the major species in the positive ion spectra of HETEs. Mass spectra were obtained on-line post-HPLC for HETEs formed by the human cells, and the HPLC-MS profile compared with that obtained from standards; species corresponding to the 11-, 9- and 5-HETEs were observed.     

Development of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of flurogestone acetate in ovine plasma

Flurogestone (FGA) is a synthetic progesterone, with a progestational action higher than that of progesterone itself. It is intended for vaginal use in large animals to induce oestrus synchronization. A quantitative method for the analysis of flurogestone acetate (FGA) in ovine plasma by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed. After the incorporation of megestrol acetate (MGA) as internal standard (IS) and followed by a liquid-liquid extraction from plasma, FGA and MGA were chromatographed using a reverse-phase HPLC column and detected by tandem mass spectrometry with a TurboIonSpray source. Multiple reaction-monitoring (MRM) mode was used for the quantitative determination of FGA in ovine plasma. The precursor ions [M+H](+) at m/z 407.2 and 385.1 for FGA and MGA, respectively, produced product ions at m/z 267.1/285.1 for FGA and m/z 267.1/224.0 for MGA. The validated concentration range was 0.2-5.0 ng/ml based on 500 microl plasma aliquots. The lower limit of quantitation was 0.2 ng/ml. Fully validated selectivity, accuracy, precision and reproducibility criteria for routine use in pharmacokinetic studies were demonstrated.     

Synthesis and spectroscopic analysis of modified bile salts

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.     

Sensitive HPLC-APCI-MS method for the determination of cyclovirobuxine D in human plasma

A sensitive high performance liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry (HPLC-APCI-MS) assay for determination of cyclovirobuxine D (CVB-D) in human plasma using mirtazapine as internal standard (I.S.) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C(18) column with a mobile phase of 30 mM ammonium acetate buffer solution containing 1% formic acid-methanol (48:52, v/v). CVB-D was determined with atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS). HPLC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at [M+H](+)m/z 403.4 for CVB-D and [M+H](+)m/z 266.2 for I.S. Calibration curves were linear over the range 10.11-4044 pg/ml. The lower limit of quantification was 10.11 pg/ml. The intra- and inter-run variability values were less than 9.5 and 12.4%, respectively. The mean plasma extraction recovery of CVB-D was in the range of 85.3-92.8%. The method was successfully applied to determine the plasma concentrations of CVB-D in Chinese volunteers.     

Quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry

The method describes quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS/MS). Samples were screened by enzyme-linked immunosorbent assay (ELISA) and only those samples presumptively declared positive were subjected to quantification and confirmation for the presence of flunixin by this method. The method is also readily adaptable to instrumental screening for the analyte. Flunixin was recovered from plasma by liquid-liquid extraction (LLE). The sample was diluted with 2 ml saturated phosphate buffer (pH 3.10) prior to LLE. The dried extract was reconstituted in acetonitrile:water:formic acid (50:50:0.1, v/v/v) and subsequently analyzed on a Q-TOF tandem mass spectrometer (Micromass) operated under electrospray ionization positive ion mode. The concentration of flunixin was determined by the internal standard (IS) calibration method using the peak area ratio with clonixin as the IS. The limits of detection (LOD) and quantification (LOQ) for flunixin in equine plasma were 0.1 and 1 ng/ml, respectively, whereas the limit of confirmation (LOC) was 2.5 ng/ml. The qualifying ions for the identification of flunixin were m/z 297 [M+H](+), 279 (BP), 264, 259, 239 and those for clonixin (IS) were m/z 263 [M+H](+), 245 (BP) and 210. The measurement uncertainty about the result was 8.7%. The method is simple, sensitive, robust and reliably fast in the quantification and confirmation of flunixin in equine plasma. Application of this method will assist racing authorities in the enforcement of tolerance plasma concentration of flunixin in the racehorse on race day.     

Determination of 11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostan oic acid and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoi c acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry

11 alpha-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid (PGE-M) and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m/z 349 and m/z 356 (2H7 standard) (daughter ions of m/z 637 and m/z 644 (2H7 standard] were used. The results of the PGE-M assay were compared with those of an assay using the [2H3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m/z 484 and m/z 491 (2H7 standard) with the parent ions m/z 682 and m/z 689 (2H7 standard) were chosen.     

Hydroxysteroid dehydrogenase transformations of 5β-scymnol and identification of oxoscymnol transformation products by liquid chromatography-tandem mass spectroscopy

A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS(2)) detection was developed to identify for the first time the oxidation products of 5β-scymnol [(24R)-(+)-5β-cholestan-3α,7α,12α,24,26,27-hexol] catalysed by bacterial hydroxysteroid dehydrogenase (HSD) reactions in vitro. The authentic scymnol (MW 468) standard yielded a protonated molecular ion [M+H](+) at m/z 469 Da, and higher mass adduct ions attributed to [M+NH(4)](+) (m/z 486), [M+H+CH(3)OH](+) (m/z 501) and [M+H+CH(3)COOH](+) (m/z 530). (24R)-(+)-5β-Cholestan-3-one-7α,12α,24,26,27-pentol (3-oxoscymnol, m/z 467 Da, relative retention time (RRT)=0.89) was identified as the principle molecular species of scymnol in the reaction with 3α-HSD pure enzyme. [S](0.5) for the reaction of 3α-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5β-cholestan-7-one-3α,12α,24,26,27-pentol (7-oxoscymnol, m/z 467 Da, RRT=0.79) and (24R)-(+)-5β-cholestan-12-one-3α,7α,24,26,27-pentol (12-oxoscymnol, m/z 467 Da, RRT=0.81) were similarly identified as principle molecular species in the respective 7α-HSD and 12α-HSD reactions. Polarity of the oxoscymnol species was established as 7-oxoscymnol>12-oxoscymnol>3-oxoscymnol>scymnol (in order from most polar to least polar). Confirmation that 5β-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated liquid chromatography-mass spectrometry (LC-MS) techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound.     

The combination effects of phenolic compounds and fluconazole on the formation of ergosterol in Candida albicans determined by high-performance liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC/MS) with atmospheric pressure chemical ionization (APCI) for the quantification of ergosterol, lanosterol, and squalene was developed to evaluate the combination effects of phenolic compounds with fluconazole on ergosterol biosynthesis in Candida albicans. The three analytes were separated by a column of C18 and were quantified without interference with each other using positive mode tandem mass spectrometry (MS/MS). Molecular ions of ergosterol and lanosterol were detected as the [M+H-H2O]+ ion species at m/z 380 and 410, whereas squalene appeared as the [M+H]+ ion species at m/z 412. On fragmentation of ergosterol, lanosterol, and squalene, the product ions at m/z 69, 149, and 109, respectively, were present as major fragments. These product ions were used for the quantification of them in multiple reaction monitoring acquisition mode. The relationship between signal intensity and the analytes' concentration was linear over the concentration range of 0.05-10 microg/ml. Following the treatment of C. albicans with fluconazole in combination with albicanyl caffeate, resveratrol, and 3,4'-difluorostilbene, respectively, the content of ergosterol in both the sensitive and resistant C. albicans showed depletion, whereas the squalene showed accumulation especially in the sensitive isolates determined with the method developed.     

Fast atom bombardment and tandem mass spectrometry for structure determination of steroid and flavonoid glycosides

The combination of fast atom bombardment (FAB) and tandem mass spectrometry (MS-MS) was tested for its applicability to generate useful structural information for steroid and flavonoid glycosides. The following compounds were investigated: quercetin, myricitrin, apigetrin, fraxin, rutin, neohesperidin, hesperidin, naringin, apiin, cymarin, digoxin, digitoxin, xanthorhamnin, and frangulin. Upon FAB, the sample molecules are desorbed as (M + H)+, (M - H)-, or as (M + Na)+ or (M + K)+. Collisional activation of (M + H)+ or (M - H)- ions in the MS-MS experiment leads to sequential losses of glycoside moieties in a manner which permits the sequence of glycosides to be established. Some glycosides occur as mixtures of homologs. Proper interpretation of the MS-MS or collisional activation decomposition spectra often allows the homology to be located. In addition to the simple and highly selective fragmentations observed in this combined experiment, FAB and MS-MS also remove interference caused by the ubiquitous matrix ions which are desorbed by FAB.     

Liquid chromatography–fast atom bombardment mass spectrometry for detection and determination of pentazocine in human tissues

A reliable and sensitive method was developed for the detection and determination of pentazocine in human solid tissues using liquid chromatography–dynamic fast atom bombardment (FAB) mass spectrometry, combined with a three-step liquid–liquid extraction procedure. Levallorphan tartrate served as an internal standard. The extract was evaporated to dryness and dissolved in the mobile phase, acetonitrile–10 mM ammonium acetate solution (20:80, pH 4.0) containing 0.5% glycerol as FAB matrix. The eluent was pumped at a flow rate of 25 μl/min and split before introduction to FAB mass spectrometer. Quantitative analysis was carried out by means of monitoring quasi-molecular ions with m/z 286 for pentazocine and m/z 284 for levallorphan. The lower limit of detection of pentazocine in each tissue tested was 1 ng/g with scan mode and 0.1 ng/g with SIM mode. Using this method, the concentrations of pentazocine were determined in the tissues of an autopsied individual to perform toxicological evaluation.     

Isolation and structural elucidation of 4-(beta-D-glucopyranosyldisulfanyl)butyl glucosinolate from leaves of rocket salad (Eruca sativa L.) and its antioxidative activity

A structurally unique glucosinolate (GSL) was identified to be 4-(beta-D-glucopyranosyldisulfanyl)butyl GSL in rocket leaves. The positive-ion electrospray ionization mass spectrometry (ESI-MS) data indicated that the new GSL had a molecular weight of 521 (m/z 522, [M+H](+), as desulfo-GSL). The molecular formula of the substance was determined to be C(17)H(32)O(11)NS(3) (m/z 522.1143, [M+H](+)) based on its positive-ion high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) data. For the further confirmation, desulfated GSL of 4-(beta-D-glucopyranosyldisulfanyl)butyl GSL was prepared by commercial 1-thio-beta-D-glucose and dimeric 4-mercaptobutyl desulfo-GSL, which was also isolated from rocket leaves, and its chemical structure was then confirmed by MS data and nuclear magnetic resonance (NMR) spectroscopy. In addition, the antioxidative activity of 4-(beta-D-glucopyranosyldisulfanyl)butyl desulfo-GSL was measured by means of chemiluminescence (CL) for evaluating the functional properties. The antioxidative activity (2.089 unit/g) was relatively higher than that of dimeric 4-mercaptobutyl desulfo-GSL (1.227).     

Fast atom bombardment mass spectrometry and tandem mass spectrometry in antibiotics: identification of nucleoside antitumor antibiotic toyocamycin in fermentation broth

The presence of the nucleoside antitumor antibiotic toyocamycin in the fermentation broth was determined by a combination of negative and positive ion fast atom bombardment (FAB) mass spectrometry, high resolution FAB mass spectrometry and mass-analysed ion kinetic energy spectrometry (MIKES). A reasonable limit of detection for toyocamycin in the whole broth was obtained by combining the specificity of mass spectrometry/mass spectrometry (also called tandem mass spectrometry) to FAB. The role played by the fermentation matrix upon the production and the observation of characteristic ions by FAB using xenon atoms was examined. High-performance liquid chromatography (HPLC) and FAB mass spectrometry were used to monitor toyocamycin at all stages of strain development, fermentation and recovery.     

Determination of the platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) in human urine by liquid chromatography-tandem mass spectrometry

A validated stable isotope dilution liquid chromatography-tandem mass spectrometry assay for the novel platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) (ZD0473) in human urine has been developed. This method uses selected reaction monitoring on the transition of m/z 393 [M+NH(4)](+) to m/z 304 [M+NH(4)-NH(3)-2 x H(35)Cl](+) for ZD0473, and m/z 400 [M+NH(4)](+) to m/z 310 [M+NH(4)-NH(3)-H(35)Cl-(2)H(35)Cl](+) for the internal standard [(2)H(7)]ZD0473. Standard curves were prepared over the range from 0.15 to 50 microg/ml. The lower limit of quantitation was 0.2 microg/ml for 100 microl of urine. This simple, rapid, reliable, and sensitive method of quantitation displayed acceptable accuracy and precision over the 3 days of assay validation. A novel platinum adduct was formed during the storage of ZD0473 in human urine. The adduct did not correspond to any of the typical sulfhydryl adducts that have been identified previously for platinum drugs. Formation of the adduct was prevented by the addition of 50% (w/v) sodium chloride to the urine. The assay can be used to quantify intact ZD0473 in the urine of subjects dosed with this new platinum drug.     

Sensitive quantification of diphemanil methyl sulphate in human plasma by liquid chromatography-tandem mass spectrometry

A simple detection system with a high-performance liquid chromatography (HPLC) with positive ionisation-tandem mass spectrometry (ESI-MS/MS) for determining diphemanil methylsulphate (DMS) levels in human plasma using 4-diphemanylmethylene,1-methylpiperidine as an internal standard (I.S.), is proposed. The acquisition was performed with the multiple reactional monitoring (MRM) mode, by monitoring the transitions: m/z 278>262 for DMS and m/z 263>247 for the I.S. The method involved a simple single-step deproteinisation with acetonitrile. The analyte was chromatographed on a Zorbax C18 reversed-phase chromatographic column by isocratic elution with 10(-3)M ammonium acetate and 10(-3)M hexafluorobutyric acid, adjusted to pH 7.0 with ammoniac/acetonitrile (40/60, v/v). The results were linear over the studied range (0.5-50.0 ng mL(-1)) and the total analysis time for each run was 10 min. The mean extraction apparent recoveries expressed at the 95% intervals of confidence were 94-104% for DMS and 92-106% for the I.S. The intra- and inter-assay precisions were 4.6-8.4% and 2.9-10.6%, respectively. The limit of quantification was 0.15 ng mL(-1). The devised assay was successfully applied to the residual concentrations monitoring in infant.     

Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry

The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.     

The determination of huperzine A in European Lycopodiaceae species by HPLC-UV-MS

A rapid qualitative HPLC-UV-MS method was developed for the detection of huperzine A in Lycopodiaceae species. HPLC coupled with mass spectrometry experiments allowed the identification of the alkaloid and enabled targeted analysis of complex matrices such as herbal extracts. Huperzine A was detected by single ion monitoring of the pseudomolecular ion [M + H]+ at m/z 243.2. The alkaloid was also detected on TLC in a bioautographic enzyme assay with acetylcholinesterase to show the activity of the compound. Four Lycopodiaceae species collected in Switzerland were tested by these methods and Huperzia selago (L.) Bernh. ex Schrank et Martius was the only species found to contain huperzine A.     

New approach for characterization of lysosulfatide by TLC, fast atom bombardment mass spectrometry and NMR spectroscopy

Thin layer chromatography of lysosulfatide showed anomalous Rf-values in contrast with such lysosphingolipids as glucopsychosine and galactopsychosine with neutral, acidic, and alkaline developing solvents. This was thought to be due to the presence of oppositely charged sulfate and amino groups in the lysosulfatide. In the negative mode of fast atom bombardment mass spectrometry, the lysosulfatide showed the pseudo molecular ion (M-H)- peak at m/z 540 and sulfate ion peak at m/z 97, whereas in the positive mode, it showed not only the pseudo molecular ion (M+H)+ peak at m/z 542, but also the major peaks of protonated psychosine at m/z 462 and fragment ions of dehydrated sphingosine at m/z 282 and 264, 13C-NMR signals of all carbons of lysosulfatide were determined by using distortionless enhancement by polarization transfer. The difference in chemical shifts of ring carbons of galactose residue between lysosulfatide and galactopsychosine was largest at C-3 (downfield shift), thereby indicating the location of the sulfate group to be at C-3 of galactose. This conclusion is supported by the 1H-NMR spectra of the lysosulfatide and galactopsychosine. Thus, the chemical structure of lysosulfatide was confirmed by fast atom bombardment mass spectrometry and 13C- and 1H-NMR spectroscopy. Furthermore, 13C-NMR signals of C-1 to C-5 of the sphingosine moiety showed significantly different chemical shifts between the lysosulfatide and galactopsychosine. These differences suggested that C-1 to C-5 of sphingosine might be influenced by intramolecular or intermolecular interaction between the sulfate group of the galactose residue and the amino group of sphingosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.