FAB Tandem Mass Spectrometry Research Articles

Structural determination of sildenafil and its analogues in dietary supplements by fast‐atom bombardment collision‐induced dissociation tandem mass spectrometry

Sildenafil and its analogues, which are used as illegal additives in several dietary supplements, were isolated by liquid-liquid extraction and column chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Structures of sildenafil and its derivatives were elucidated by FAB-tandem mass spectrometry (MS/MS) with exact mass measurement in the positive-ion mode. To find structurally diagnostic ions for the sildenafil analogues, authentic sildenafil was preferentially analyzed by high-energy collision-induced dissociation (CID)-MS/MS. The CID-MS/MS spectra of [M+H](+) precursor ions resulted in the formation of numerous characteristic ions via a series of dissociative processes. The product ions formed by CID provided important information on the modification of the piperazine ring, the phenylsulfonyl group and the pyrazolopyrimidine moiety of sildenafil. By interpreting their MS/MS spectra, the chemical structures of sildenafil analogues isolated from dietary supplements could be elucidated and fragmentation patterns were proposed. To clearly identify the sidenafil derivatives in dietary supplements, some of the derivatives such as acetildenafil, homosildenafil and hydroxyhomosildenafil which are not commercially available were synthesized and compared with their MS/MS spectra. In addition, high-resolution mass measurements were conducted to obtain the elemental compositions of sildenafil and its analogues.

Two new monogalactosyl diacylglycerols from brown alga Sargassum thunbergii.

Two new monogalactosyl diacylglycerols (MGDGs) along with two known glycolipids were isolated from the moderate polar fraction of the methanolic extract of the brown alga Sargassum thunbergii by using reversed silica flash chromatography. Two new MGDGs were identified as (2S)-1-O-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-2-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-beta-D-galactopyranosyl-sn-glycerol (1) and (2S)-1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-(6Z,9Z,12Z,15Z-octadecatetraenoyl)-3-O-beta-D-galactopyranosyl-sn-glycerol (2) by FAB tandem mass spectrometry, NMR techniques, and specific enzyme-catalyzed hydrolysis of the sn-1 fatty acyl linkage. The regiochemical attachment of the acyl chains in the glycerol moiety was established by 2D NMR correlations and confirmed by enzymatic hydrolysis.

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.

Structural determination of cyclitol derivatives by fast‐atom bombardment tandem mass spectrometry

Three cyclitol derivatives were isolated from the marine sponge Sarcotragus sp. by reversed-phase high-performance liquid chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Their structural elucidation was carried out with FAB tandem mass spectrometry (FAB-MS/MS). FAB-MS spectra produced a significant abundance of the sodium adducts [M+Na]+ and [M+2Na-H]+ from a mixture of m-NBA and NaI. In addition, trifluoroacetylation of the cyclitol derivatives was used for confirmation of the presence of the cyclitol ring. High abundance [M-5H+5CF3CO+Na]+ ions were observed in the FAB-MS spectra of the trifluoroacetyl-cyclitol derivatives. Collision-induced dissociation (CID) of the [M+Na]+ ions produced diverse product ions via a series of dissociative processes. Charge-remote fragmentation (CRF) patterns of [M+Na]+ ions were very useful for the identification of product ions which are characteristic for the cyclitol ring and long hydrocarbon chains substituted at the glycerol backbone. Moreover, the CID-MS/MS spectra of the [M+Na]+ ions yielded characteristic product ions at m/z 53, 83, 113, 155 and 171 for the cyclitol moiety, and at m/z 213, 229 and 245 for the glycerol backbone attached to the cyclitol ring.

Synthesis of 1-lyso-2-palmitoyl- rac-glycero-3-phosphocholine and its regioisomers and structural elucidation by NMR spectroscopy and FAB tandem mass spectrometry

Three regioisomers of a naturally occurring lysophosphatidylcholine were chemically synthesized from glycerol. The phosphocholine moiety of the molecule was introduced by sequentially reacting with ethylene chlorophosphite, bromine, water, and trimethyl amine. Removal of a silyl protecting group of the hydroxyl group in the glycerol backbone was achieved without any accompanying acyl migration in the final stage of the synthesis by using NBS in a dimethyl sulfoxide–water cosolvent system. Structures of all regioisomers were compared by NMR spectroscopy and FAB tandem mass spectrometry.

Application of MALDI and PSD to the structure determination of adducts between DNA bases and the carcinogen 7 H-dibenzo[ c, g]carbazole

Dibenzo[ c, g]carbazole is the first heterocyclic aromatic compound that is known to be activated by one-electron oxidation; the resulting radical cation intermediate reacts with DNA to form adducts. We synthesized reference DNA adducts and studied them by MALDI and PSD, as well as by four-sector tandem mass spectrometry. The information content of PSD product-ion spectra compares favorably with those obtained by FAB or ESI and tandem sector mass spectrometry. The small differences that do occur are attributable to energy differences between the methods of ionization. We also assessed the reproducibility of PSD spectra using a statistical test called the ‘similarity index’ and found them to be as reproducible as product-ion spectra obtained on a four-sector tandem mass spectrometer.

A rapid screening by liquid chromatography/mass spectrometry and fast-atom bombardment tandem mass spectrometry of phenolic constituents with radical scavenging activity, fromKrameria triandra roots

Liquid chromatography/mass spectrometry (particle beam interface, electron impact ionization), fast-atom bombardment mass spectrometry (FAB-MS) (negative-ion mode) and FAB-tandem mass spectrometry (MS/MS) techniques were applied for the rapid detection/characterization of low-to-intermediate molecular weight polyphenol constituents of Krameria triandra roots with radical scavenging activity, this determined in a well established membrane model of lipid peroxidation (phosphatidylcholine liposomes). LC/MS analysis of the more lipophilic fractions of the drug (antioxidant activity: IC50, the concentration which gives 50% inhibition, 5.8 μg/mL petrol extract; IC50 3.7 μg/mL chloroform extract) evidences at least 9 components belonging to the class of lignans; FAB-MS/MS analysis of the butylacetate extract (IC50 = 0.23μg/mL) caffeoyl esters, catechin-glucosides, dimeric procyanidines and their corresponding phenylpropanoyd derivatives. © 1997 John Wiley & Sons, Ltd.

A fast screening method for tricyclic antidepressants and their urinary metabolites by FAB‐tandem mass spectrometry

A screening method for tricyclic antidepressants (TCA) and their urinary metabolites by FAB tandem mass spectrometry (FAB MS/MS) is presented. The collision induced dissociation (CID) behavior of pure dibenzocycloheptadienes (amitriptyline 1, nortriptyline 2), dibenzoxepines (doxepine 3, dosulepine 4), dibenzazepines (imipramine 5, desipramine 6, trimipramine 7) and the iminostilbene derivative opipramol 8 is described and used for the specific determination of these tricyclics by MS/MS.For the screening procedure a methanolic extract of the urine sample is passed through a XAD‐2 column and the eluate is examined without further purification after evaporation of the solvent. Identification of the TCA is achieved by constant neutral loss (CNL) scans for 173 u, 195 u and 193 u and subsequent daughter ion spectra of the molecular ions [M+H]+. Glucuronide conjugate metabolites of the tricyclic antidepressants 1, 2, 3, 6, 8 could be identified by CNL‐scans (176 u) and the assignments were confirmed by daughter ion mass spectra. A urine sample of a fatal 1, 2, 8 overdose and samples of three patients under therapeutic dosage with 1, 3 or 6 are presented as examples for application.

Structural characterization and location of disulphide linkages of a potent vasodilatory peptide, recombinant maxadilan, by a multiple mass spectrometric approach.

A multiple mass spectrometric strategy using fast-atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) has been used to confirm the sequence and to locate the disulfide linkages of recombinant maxadilan (r-maxadilan) (average molecular mass 7422.5 Da), a potent vasodilatory peptide from Lutzomyia longipalpis. MALDI measurements of intact r-maxadilan, its reduced form and its pyridylethylated form (p-maxadilan) indicated the presence of four Cys residues without major post-translational modifications. FAB and FAB-tandem mass spectrometry measurements of chymotryptic digests of p-maxadilan were sufficient to map the primary structure of p-maxadilan, though the complementary use of MALDI was necessary for complete mapping using Asp-N digestion due to a strong suppression observed in FAB. Assignment of the Cys-5-Cys-9 linkage was achieved by comparison of FAB mass spectra before and after reduction of tryptic digests of r-maxadilan. Since the molecular weight of the peptide fragment containing the Cys-18-Cys 55 linkage is more than 4000, MALDI measurement was indispensable for assignment of this linkage. The results fully support the value of the multiple mass spectrometric strategy in the structural characterization of peptides and proteins.

Identification of a New Sulfonic Acid Metabolite of Metolachlor in Soil

An ethanesulfonic acid metabolite of metolachlor (metolachlor ESA) was identified in soil-sample extracts by negative-ion, fast-atom bombardment mass spectrometry (FAB-MS) and FAB tandem mass spectrometry (FAB-MS/MS). Product-ion fragments from MS/MS analysis of the deprotonated molecular ion of metolachlor ESA in the soil extract can be reconciled with the structure of the synthesized standard. The elemental compositions of the (M − H)- ions of the metolachlor ESA standard and the soil-sample extracts were confirmed by high-resolution mass spectrometry. A dissipation study revealed that metolachlor ESA is formed in soil under field conditions corresponding to a decrease in the concentration of the parent herbicide, metolachlor. The identification of the sulfonated metabolite of metolachlor suggests that the glutathione conjugation pathway is a common detoxification pathway shared by chloroacetanilide herbicides.

Identification of an N-acetylated microsomal glutathione S-transferase by mass spectrometry.

Microsomal glutathione S-transferase (mGST) was purified to homogeneity from male Sprague-Dawley rat liver, as determined by SDS-PAGE. Removal of Triton X-100 and further separation by reversed phase HPLC revealed two proteins, mGST 1 and mGST 2, in a 1:3 ratio. Analysis of mGST 1 and mGST 2 by electrospray ionization mass spectrometry determined their molecular weights to be 17,354.2 +/- 6.6 and 17,397.9 +/- 6.6, respectively. mGST 1 was in close agreement with the calculated molecular weight of 17,348, as predicted by the previously reported cDNA sequence. Cyanogen bromide digestion and peptide mapping by fast atom bombardment mass spectrometry (FAB-MS) localized the mass increase to the N-terminal peptide, 1-7. FAB-tandem mass spectrometry of this peptide in conjunction with Edman reactions on the intact protein demonstrated the N-terminal alanine to be acetylated.

Improving the Sensitivity of FAB Tandem Mass Spectrometry for PAH-DNA Adducts

Abstract The mass spectrometric detection limits for guanine, adenine, and an adeninddimethylbenthracene adduct were greatly improved by converting the materials to pentafluorobenzyl derivatives, as well as by doing coaxial continuous flow-FAB (coaxial cf-Fast Atom Bombardment). The lowest detection limit (approximately 10 pg) was achieved when the derivatized sample was introduced by coaxial cf-FAB and the determination was by fill-scan mass spectrometry. The lowest detection limit achieved by tandem mass spectrometry was 50 pg obtained after derivatization, coaxial cf-FAB introduction and detection with an extended mass range array.

α,N-Acetyl β-Endorphin [1-8] Is the Terminal Product of Processing of Endorphins in the Melanotrope Cells of Xenopus laevis, as Demonstrated by FAB Tandem Mass Spectrometry

The major N-terminal acetylated endorphin of the pars intermedia of Xenopus laevis was purified and submitted to fast-atom bombardment tandem mass spectroscopy. The collisionally induced dissociation MS/MS spectrum of the [M+H]+ ion revealed sufficient fragment ions to determine unambiguously the identity of the peptide as α,N-acetyl β-endorphin [1-8], the sequence of which was predicted on the basis of the nucleotide sequence of Xenopus POMC cDNA. The determination was confirmed by showing that the synthetic peptide of this structure had identical FAB tandem mass spectrometric characteristics as the endogenous endorphin. We conclude that α,N-acetyl β-endorphin [1-8] is the terminal product of processing of endorphins in the melanotrope cell of Xenopus laevis.

Analysis of an Egyptian mummy resin by mass spectrometry

Fast atom bombardment combined with mass spectrometry (FAB/MS), high resolution FAB/MS, FAB tandem mass spectrometry (MS/MS), and gas chromatography/mass spectrometry (GC/MS) were used to determine the composition of the resinous material recovered from the wrapping of an Egyptian mummy believed to be from the Roman period (100–350 A.D.). FAB/MS and MS/MS studies identified several oxidation products of abietic acid as the principal resin components, indicating that one or more species of coniferous trees were used by the Egyptians as a source of the resin. GC/MS studies also identified several n-alkanes with carbon numbers from C 19 to C 33 in the sample. The relative amounts of these n-alkanes, along with characteristic trace metals, indicate that bitumen, an asphalt native to the region, was added to the resin. The presence of this ancient source of carbon in this sample explains the inconsistent data assigned to the mummy by carbon-14 analysis.

High and Low Energy FAB Tandem Mass Spectrometry of a Series of Anomeric and Regioisomeric 2′-Deoxyribofuranosyl Imidazole Nucleosides

Abstract Positive ion fast atom bombardment mass spectrometry has aided the structural characterisation of a novel series of anomeric and regioisomeric ethyl 5- and 4-amino-2′ -deoxy-D-erythro-pentofuranosylimidazole-4- and 5 carboxylate nucleosides. Daughter ion MS/MS analyses (high energy MIKE and low energy MS/MS CAD), of the precursor protonated molecular ions for this series of anomeric and regioisomeric nucleosides have provided distinct and reproducible fingerprint patterns which have permitted discrimination between the individual configurations of the various anomers and regioisomers.

Differentiation of Anomeric & Positional Isomers of Acyclic & Cyclic Nucleosides by FAB Tandem Mass Spectrometry & NMR

Abstract We have been interested in developing routine methods, using standard 1H and 13C NMR spectroscopy of assigning anomeric configuration for both acyclic and cyclic nucleosides of the types (1) & (2) respectively. In the latter class of compounds ve have also sought to determine the site of glycosidation (viz N1 or N3 with respect to the imidazole ring). We have extended this study using FAB tandem mass spectrometry1 to identify stereochemical differences.

Adipokinetic hormone controls lipid metabolism in adults and carbohydrate metabolism in larvae of Manduca sexta

The peptide hormone which controls activation of fat body glycogen phosphorylase in starving larvae of Manduca sexta was isolated from larval corpora cardiaca and sequenced by FAB tandem mass spectrometry. It was found to be identical with Manduca AKH. This, together with earlier observations, demonstrates that in M. sexta AKH controls glycogen phosphorylase activation in starving larvae while in adults it controls lipid mobilization during flight. Larval corpora cardiaca contain about 10 times less AKH than the corpoca cardiaca of adults. The corpora cardiaca of M. sexta appear to contain only one AKH.

Chemical synthesis and cytotoxic properties of N-alkylcarbamic acid thioesters, metabolites of hepatotoxic formamides

The S-linked cysteine and glutathione conjugates of N-methylformamide and N-ethylformamide, together with a series of methyl ester derivatives thereof, have been synthesized and characterized by 1H NMR, 13C NMR, FAB-MS, and FAB tandem mass spectrometry. In vitro cytotoxicity assays showed that all of the title conjugates were toxic to isolated mouse hepatocytes when incubated at concentrations in excess of 1 mM and that they served as potent growth inhibitors of murine TLX5 lymphoma cells when present at levels of 10-100 microM. Both of these effects were reversed by the addition to incubation media of glutathione (10 mM). The possibility is raised that N-alkylcarbamic acid thioester conjugates, which are formed during the metabolism of N-alkylformamides in mammalian systems, may act as important mediators of the antineoplastic and/or hepatotoxic activity of the parent formamides, possibly through their ability to liberate methyl isocyanate at cell membranes.

Differentiation of anomeric acyclic nucleosides by FAB tandem mass spectrometry

Differentiation of anomeric acyclic nucleosides by FAB tandem mass spectrometry

Rapid and efficient sequence determination of a 4 nmol sample of adipokinetic hormone I (AKH I) using FAB tandem mass spectrometry.

Fast atom bombardment mass spectrometery in conjunction with tandem mass spectrometry provided the unambiguous sequence determination of the adipokinetic hormone I. The amount of sample needed is of the order less than or equal to 4 nmol, and the complete analysis is performed in less than 4 h.