FAB, ESI and MALDI Mass Spectrometric methods in the study of metallo-drugs and their biomolecular interactions.

This review surveys the soft ionisation mass spectrometric methods that are most commonly used for the investigation of the mechanism of interaction between metallo-drugs and biomolecules. In the first part of the review, an overview of the applications of transition metal complexes in the therapy of various diseases (arthritis, cancer, diabetes) is given, whereas the second part focuses on the obtained results dealing with various aspects of the interaction between metal complexes and different types of biomolecules. Possibilities and limitations of each mass spectrometric method-namely, fast atom bombardment (FAB), electrospray ionisation (ESI) and matrix-assisted laser desorption and ionisation (MALDI)--are discussed in the third part of this review along with the examples of their application for the analysis of metal complexes, as well as of the products of their interaction with biomolecules.

Detection and accurate identification of new or emerging bacteria in cystic fibrosis patients.

Respiratory infections remain a major threat to cystic fibrosis (CF) patients. The detection and correct identification of the bacteria implicated in these infections is critical for the therapeutic management of patients. The traditional methods of culture and phenotypic identification of bacteria lack both sensitivity and specificity because many bacteria can be missed and/or misidentified. Molecular analyses have recently emerged as useful means to resolve these problems, including molecular methods for accurate identification or detection of bacteria and molecular methods for evaluation of microbial diversity. These recent molecular technologies have increased the list of new and/or emerging pathogens and epidemic strains associated with CF patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells has also emerged recently as a powerful and rapid method for the routine identification of bacteria in clinical microbiology laboratories and will certainly represent the method of choice also for the routine identification of bacteria in the context of CF. Finally, recent data derived from molecular culture-independent analyses indicate the presence of a previously underestimated, complex microbial community in sputa from CF patients. Interestingly, full genome sequencing of some bacteria frequently recovered from CF patients has highlighted the fact that the lungs of CF patients are hotspots for lateral gene transfer and the adaptation of these ecosystems to a specific chronic condition.

Clinical steroid mass spectrometry: a 45-year history culminating in HPLC-MS/MS becoming an essential tool for patient diagnosis.

Automated rapid HPLC tandem mass spectrometry has become the method of choice for clinical steroid analysis. It is replacing immunoassay techniques in most instances because it has high sensitivity, better reproducibility, greater specificity and can be used to analyze multiple steroids simultaneously. Modern multiplex instruments can analyze thousands of samples per month so even with high instrument costs the price of individual assays can be affordable. The mass spectrometry of steroids goes back decades; the first on-line chromatography/mass spectrometry methods for hormone analysis date to the 1960s. This paper reviews the evolution of mass spectrometric techniques applied to sterol and steroid measurement There have been three eras: (1) gas chromatography-mass spectrometry (GC/MS), (2) Fast Atom Bombardment (FAB) and (3) HPLC/MS. The first technique is only suitable for unconjugated steroids, the second for conjugated, and the third equally useful for free or conjugated. FAB transformed biological mass spectrometry in the 1980s but in the end was an interim technique; GC/MS retains unique qualities but is unsuited to commercial routine analysis, while LC-MS/MS is rightly stealing the show and has become the dominant method for steroid analysis in endocrinology.

First solid state alkaline-earth complexes of monensic acid A (MonH): crystal structure of [M(Mon)2(H (2)O)2] (M = Mg, Ca), spectral properties and cytotoxicity against aerobic Gram-positive bacteria.

Alkaline-earth metal complexes of the monoanionic form of the polyether ionophore monensin A were isolated for the first time in solid state and were structurally characterized using various spectroscopic methods (IR, NMR, FAB-MS). The stoichiometric reaction of monensic acid (MonH) with M(2+) (M = Mg, Ca) in the presence of an organic base leads to the formation of mononuclear complexes of composition [M(Mon)(2)(H(2)O)(2)]. The structures of magnesium (1) and calcium (2) monensin complexes in the solid state were established by single crystal X-ray crystallography. The complexes crystallize as [Mg(Mon)(2)(H(2)O)(2)]x5MeCN (1) and [Ca(Mon)(2)(H(2)O)(2)]xH(2)Ox5MeCN (2) in the monoclinic P21 space group. The alkaline-earth metal ion is placed in a distorted octahedral environment, defined by two monensin anions acting as bidentate ligands in the equatorial plane of the complex as well as by two water molecules occupying the axial positions of the inner coordination sphere. The bactericidal activity of 1 and 2 was evaluated against aerobic Gram-positive microorganisms applying the double layer agar hole diffusion method.

Structural determination of glucosylceramides isolated from marine sponge by fast atom bombardment collision-induced dissociation linked scan at constant B/E.

Five glucosylceramides (GlcCers) were isolated by reversed phase high-performance liquid chromatography from the MeOH extracts of a marine sponge, Haliclona (Reniera) sp., collected from the coast of Ulleung Island, Korea, and analyzed by fast atom bombardment mass spectrometry (FAB-MS) in positive-ion mode. FAB-mass spectra of these compounds included protonated molecules [M + H](+) and abundant sodiated molecules [M + Na](+) from a mixture of m-NBA and NaI. The structures of these GlcCers, which were similar, were elucidated by FAB-linked scan at constant B/E. To find diagnostic ions for their characterization, the GlcCers were analyzed by collision-induced dissociation (CID) linked scan at constant B/E. The CID-linked scan at constant B/E of [M + H](+) and [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 provided important information for the characterization of the fatty N-acyl chain moiety and the sphingoid base, commonly referred to as the long-chain base. The product ions at m/z 203 and 502 were diagnostic for the presence of a sodiated sugar ring and beta-D-glucosylsphinganine, respectively. For further confirmation of the structure of the fatty N-acyl chain moiety in each GlcCer, fatty acid methyl esters were obtained from the five GlcCers by methanolysis and analyzed by FAB-MS in positive-ion mode. On the basis of these dissociation patterns, the structures of the five GlcCers from marine sponge were elucidated. In addition, the accurate mass measurement was performed to obtain the elemental composition of the GlcCers isolated from marine sponge.

Differentiation of the isomeric o-(m- and p-)nitro-(chloro- and bromo-)benzyl-2,4-(and 2,1-) disubstituted 2-thiocytosinium halides by electron impact ionisation and fast atom bombardment mass spectrometry.

Electron ionisation (EI) and fast atom bombardment (FAB) mass spectral fragmentations of nine 2,4-(and 2,1-) disubstituted o-(m- and p-)nitro-(chloro- and bromo-)-2-thiocytosinium halides are investigated. Fragmentation pathways, whose elucidation is assisted by accurate mass measurements and metastable transitions [EI-mass spectrometry (MS)], as well as FAB/collision-induced dissociation (CID) mass spectra measurements are discussed. The correlations between the abundances of the (C(11)H(10)N(4)SO(2))(+) 1-3; (C(11)H(10)N(3)SCl)(+) 4-6 and (C(11)H(10)N(3)SBr)(+) 7-9 ions and the selected fragment ions (EI- MS), as well as (C(18)H(16)N(5)SO(4))(+) 1-3; (C(18)H(16)N(3)SCl(2))(+) 4-6 and (C(18)H(16)N(3)SBr(2))(+) 7-9 ions and the selected ions (C(7)H(6)NO(2))(+) 1-3; (C(7)H(6)Cl)(+) 4-6; (C(7)H(6)Br)(+) 7-9 (FAB-MS) are discussed. The data obtained can be used for distinguishing isomers.

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.

Chemical constituents from the leaves of Machilus zuihoensis Hayata var. mushaensis (Lu) Y.C. Liu.

One new lignan, machilolin-A (1), was isolated from the leaves of Machilus zuihoensis Hayata var. mushaensis (Lu) Y. C. Liu (Lauraceae), together with three known compounds, vanillin (2), beta-sitosterol (3) and stigmasterol (4). The structure of 1 was elucidated based on chemical analysis and spectral methods (IR, 1D and 2D NMR, HR-FAB-MS, EI-MS).

Phosphatidyl ethanolamine with increased polyunsaturated fatty acids in compensation for plasmalogen defect in the Zellweger syndrome brain.

To elucidate the neuropathological mechanism of Zellweger syndrome (ZS), we studied changes in the molecular species of glycerophospholipids in the cerebral tissue by thin-layer chromatography (TLC) and fast atom bombardment mass spectrometry (FABMS). First, we estimated the amount of plasmalogens by TLC. Plasmalogen-type phosphatidyl ethanolamine (PE) accounted for 30% of the total PE in the control brain, but was absent in the ZS brain. Plasmalogen-type phosphatidyl choline (PC) was undetectable in both control and ZS brains. Next, we analyzed plasmalogen-type PE by FABMS. Oleic (18:1), arachidonic (20:4) and docosapentanoic (22:5) acids were present in the control gray matter, but not in the ZS gray matter. In compensation for the defect of plasmalogen, the level of diacyl PE with polyunsaturated fatty acids, 20:4, 22:4, 22:5 and 22:6, was higher in the ZS brain than that in the control brain. These results indicate an alteration in the molecular species of PE, which may cause abnormal neural membrane fluidity and excessive vulnerability to oxygen stress.

[Progress in the determination of glucosinolates].

The glucosinolates are a large group of hydrophilic, sulphur-containing plant secondary metabolites, occurring mainly in cruciferous family, which have recently attracted intense research interest because of their cancer chemoprotective attributes. Developing the analytical methods is the premises for studying the property of glucosinolates, so that much effort has been devoted to developing methods for efficient isolation and identification of them. In this article, the recent progress on analyzing of glucosinolates was reviewed.

Charge-remote metastable ion decomposition of free fatty acids under FAB MS: evidence for biradical ion structures.

Classical charge-remote fragmentation (CRF) of a series of long-chain saturated and monounsaturated fatty acid anions, a well-known phenomenon under collisional activation conditions, is observed for the first time during fast atom bombardment of the analyte-matrix mixture without collisional activation. The process is efficient enough to allow collision-induced dissociation and metastable ion decomposition MS/MS spectra of any charge-remote [M-H2-(CH2)n]- fragments as well as spectra of neutral losses to be recorded. The results obtained are in contradiction to the generally accepted theory that CRF results exclusively in terminally unsaturated carboxylate anions. The new results indicate that a multistep radical mechanism is involved in CRF ion formation. The first step of the process appears to be accompanied by hydrogen elimination that occurs randomly throughout the molecule. The primary fragment radical ions formed can decompose further with the formation of the next generation of CRF ions.

Alterations in the molecular species of plasmalogen phospholipids and glycolipids due to peroxisomal dysfunction in Chinese hamster ovary-mutant Z65 cells by FABMS method.

Changes in the molecular species of lipids associated with Pex2 gene-mutation were investigated to elucidate the pathogeneses of peroxisome biogenesis disorders. Although no differences were observed in the concentrations of cholesterol and phosphatidyl choline between mutated Z65 and control CHO-K1 cells, the amounts of cholesterol esters and glycolipids in Z65 cells were twice those in CHO-K1 cells, but phosphatidyl ethanolamine (PE), particularly 1-O-octadec-1'-enyl-2-oleoyl PE, was absent in Z65 cells by FABMS. Enhanced synthesis of glycolipids in Z65 cells was associated with an abundance of lignoceric acid-containing ones, suggesting a role of glycolipids in the retention of longer saturated fatty acids.

Distinguishing between cis/trans isomers of monounsaturated fatty acids by FAB MS.

Fast-atom bombardment (FAB) mass spectrometry in the negative ion mode can be used to unambiguously distinguish between cis and trans isomers of monounsaturated fatty acids by the relative signal strengths of an intense pair of ion signals. Under normal FAB ionization/desorption conditions, the deprotonated molecules (i.e., [M - H]-) of six fatty acids underwent charge remote fragmentation. A characteristic fragmentation pattern of two intense peak clusters of peaks with three weak intervening clusters of peaks are used in each case to identify the position of the double bond. The possibility of resonance electron capture occurring during the FAB process is discussed.

Determination of genotoxic phenylhydrazine agaritine in mushrooms using liquid chromatography-electrospray ionization tandem mass spectrometry.

A new method with good sensitivity and specificity for detecting and quantifying genotoxic hydrazines, agaritine and 4-(hydroxymethyl)phenylhydrazine (HMPH), was developed using liquid chromatography-electrospray tandem mass spectrometry (MS). Synthetic agaritine and HMPH were structurally assigned by 1H-, 13C- and two-dimensional nuclear magnetic resonance (NMR) analysis (HMBC and HMQC), high-resolution fast-atom bombardment (HR-FAB) MS and time of flight (TOF) MS. The polar molecule agaritine was separated on an ODS column using 0.01% AcOH-MeOH (99:1, v/v) as an eluent with a simple solid-phase extraction cleanup. There were no interference peaks for any of the mushrooms. Agaricus spp. contained 1247 and 2017 microg g-1 agaritine. Other species of mushroom had no agaritine. Recoveries of agaritine from spiked mushroom samples were 60.3-114%. Intra-day precision values were 5.5 and 4.2%, and the inter-day precision values were acceptable (15.0 and 23.0%), as agaritine is unstable. The limit of quantification was 0.003 microg g-1. Even a trace amount of agaritine in mushrooms can, therefore, be determined using this method. We also directly analysed HMPH, an active free hydrazine form of genotoxic agaritine, and obtained direct evidence of its absence from mushrooms. A precursor ion scan confirmed that agaritine derivatives, which could exert similar toxicity, were absent. The results indicate that this specific and sensitive analytical method for detecting and quantifying agaritine and its derivatives could help evaluate the risk of mushroom hydrazines to humans.

Three 1-thio-beta-D-glucopyranosides from the seeds of Afrostyrax lepidophyllus Mildbr.

Three new 1-thioglycosides namely methylthiomethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside A), methylsulfonylmethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside B) and methylsulfonylmethylthiomethyl 1-thio-beta-D-glucopyranoside (Afrostyraxthioside C) were isolated from the seeds of Afrostyrax lepidophyllus Mildbr. Their structures were mainly elucidated by using one- and two-dimensional NMR and mass spectroscopies and also by an efficient one-step synthesis. Moreover, Afrostyraxthiosides A, B and C constitute a new subclass of 1-thioglycosides isolated from natural sources.

Generation of a novel fluorescent product, monochlorofluorescein from dichlorofluorescin by photo-irradiationdagger.

Dichlorofluorescin (DCFH), a widely used fluorescent probe for reactive oxygen species (ROS) was decomposed completely and generated two distinct fluorescent products by photo-irradiation at 254 nm for 30 min. In the previous study, we had shown that one was dichlorofluorescein (DCF), a well known oxidized product of DCFH. In this study we investigated the other product and identified it as monochlorofluorescein (MCF) by 1H-NMR and fast atom bombardment/mass spectrum (FAB/MS) analyses. MCF was generated by photo-irradiation, but not by ROS. On the other hand, DCF was produced by both photo-irradiation and ROS. MCF showed similar fluorescent emission spectrum to DCF, however, its fluorescence intensity was more than that of DCF. The kinetic study suggested that MCF was not generated from DCF but from monochlorofluorescin, which might be generated from DCFH by photo-irradiation.

5-Hydroxy-seco-carotenoids from Pittosporum tobira.

Three 5-hydroxy-seco-carotenoids were isolated from seeds of Pittosporum tobira. These structures were determined to be (3S,3'S,5'?)-3,3'-di(tetradecanoyloxy)-5'-hydroxy-5,6,5',6'-diseco-beta,beta-carotene-5,6,6'-trione (1), (3S,5?,3'S,5'R,6'S,9'Z)-3-tetradecanoyloxy-5',6'-epoxy-5,3'-dihydroxy-5',6'-dihydro-5,6-seco-beta,beta-caroten-6-one (2), and (3S,5?,3'S,5'R,6'R)-3-tetradecanoyloxy-5,3',5'-trihydroxy-6',7'-didehydro-5',6'-dihydro-5,6-seco-beta,beta-caroten-6-one (3) based on analysis of UV-vis, IR, FAB MS, and NMR spectroscopic data.

Pyrolysis mass spectrometry for distinguishing potential hoax materials from bioterror agents.

Pyrolysis mass spectrometry (PyMS) was investigated as a rapid tool to distinguish potential bioterror hoax materials from samples containing pathogenic bacteria. A pyrolysis time-of-flight (TOF) mass spectrometer equipped with an alternative ionization technique, metastable atom bombardment (MAB), was used to produce sample spectra. These spectra were analyzed by principal component and discriminant analysis for pattern recognition. Materials investigated were two strains of Vibrio parahaemolyticus, one of which produced the tdh toxin, two Salmonella enterica serotypes, a biological mosquito control product containing spores of Bacillus thuringiensis, and several white to off-white powders (which could be used as hoax materials), such as flour, corn starch, methyl cellulose, and xanthan gum. PyMS distinguished bacterial samples from hoax materials. Furthermore, pattern analysis differentiated Vibrios from Salmonellae, Salmonella enterica Anatum from S. enterica Heidelberg, and the two V. parahaemolyticus strains from each other. The B. thuringiensis mixture was distinguished from other bacteria and powders, suggesting that PyMS with pattern recognition may differentiate samples containing pathogens, including Bacillus spp., from nonbiological agents and that it can be a rapid method for detection of bacteria. MS data acquisition took only 7 min for each sample.

[Preparative separation of aloin diastereoisomers by high-speed countercurrent chromatography combined with silica gel column chromatography].

Aloin, naturally a mixture of two diastereoisomers, aloin A and aloin B, is the major anthraquinone in aloe, and now served as one of the important control constituents in most of the commercial aloe products. High-speed countercurrent chromatography (HSCCC) combined with silica gel column chromatography was developed for the preparative separation of the two individual aloins. Aloin A (98%) and aloin B (96%) were obtained. Fast atom bombardment mass spectrometry (FAB-MS), 1H nuclear magnetic resonance (1H NMR) and GOESY (gradient-enhanced nuclear Overhauser effect spectroscopy) were employed for the elucidation of their structure conformation. The developed method is of high preparative capacity and high efficiency in resolution.

Studies on high-energy collision-induced dissociation of endogenous cannabinoids: 2-arachidonoylglycerol and n-arachidonoylethanolamide in FAB-mass spectrometry.

Analysis of 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (anandamide) via alkali or alkaline earth metal-adduct high-energy collision-induced dissociation (CID) in fast-atom bombardment (FAB) ionization-mass spectrometry (MS) is described. The CID-MS/MS of the [2-AG+Li](+) or [2-AG+Na](+) ion undergoes charge-remote fragmentation (CRF), which is useful for the determination of the double-bond positions in the hydrocarbon chain, while the CID-MS/MS of the [2-AG-H+Cat](+) (Cat = Mg(2+), Ca(2+), Ba(2+)) ion provides an abundant fragment ion of the cationized arachidonic acid species, which is derived from cleaving the ester bond via a McLafferty-type rearrangement in addition to structurally informative CRF ions in small amounts. On the other hand, the CID-MS/MS spectra of anandamide cationized with both alkali metal (Li(+) or Na(+)) and alkaline earth metal (Mg(2+), Ca(2+), or Ba(2+)) show CRF patterns: the spectra obtained in lithium or sodium adduct are more clearly visible than those in magnesium, calcium, or barium adduct. The McLafferty rearrangement is not observed with metal-adduct anandamide. The characteristics in each mass spectrum are useful for the detection of these endogenous ligands. m-Nitrobenzyl alcohol (m-NBA) is the most suitable matrix. A lithium-adduct [2-AG+Li](+) or [anandamide+Li](+) ion is observed to be the most abundant in each mass spectrum, since the affinity of lithium for m-NBA is lower than that for other matrices examined.