Secondary metabolites of Seseli rigidum: Chemical composition plus antioxidant, antimicrobial and cholinesterase inhibition activity.

Extracts of different polarity obtained from various plant parts (root, leaf, flower and fruit) of Seseli rigidum were studied by different antioxidant assays: DPPH and ABTS radical scavenging activity, by total reducing power method as well as via total content of flavonoids and polyphenols. Essential oils of all plant parts showed weak antioxidant characteristics. The inhibitory concentration range of the tested extracts, against bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and fungi Candida albicans and Aspergillus niger was 0.01-1.50 mg/mL and of a microbicidal 0.02-3.00 mg/mL. In the interaction with cholinesterase, all essential oils proved effective as inhibitors. The highest percentage of inhibition versus human and horse cholinesterase was shown by root essential oil (38.20% and 48.30%, respectively) among oils, and root hexane extract (40.56% and 50.65% respectively). Essential oils and volatile components of all plant parts were identified by GC, GC-MS and headspace/GC-MS. Statistical analysis of the ensemble of results showed that the root essential oil composition differed significantly from essential oils of other parts of the plant. Taking into account all of the studied activities, the root hexane extract showed the best overall properties. By means of high performance liquid chromatography coupled to high resolution mass spectrometry, the 30 most abundant constituents were identified in extracts of different polarity. The presence of identified constituents was linked to observed specific biological activities, thus designating compounds potentially responsible for each exhibited activity.

Assessment of the molecular weight distribution of tannin fractions through MALDI-TOF MS analysis of protein-tannin complexes.

An innovative mass spectrometry method was developed for determining mass distributions of tannin fractions that cannot be approached through direct MALDI-TOF analysis. It was applied to three procyanidin fractions with average degrees of polymerizations = 3, 9, and 28, respectively, and one gallotannin fraction (Tara tannin). The proposed approach consists of MALDI-TOF analysis of the soluble complexes formed between these tannin fractions and bovine serum albumin (BSA). Complexes were detected as an unresolved "hump" following the BSA signal, and spectra were mathematically processed to determine the parameters relative to the protein-tannin complexes, which are the number-average molecular weight (Mn), the weight-average molecular weight (Mw), and the polydispersity index (PI) for each tannin fraction. Regarding condensed tannins, results are consistent with those of the standard method (thiolysis followed by HPLC separation) for all tested fractions. The method was successfully applied to a hydrolyzable tannin fraction but no standard method is available for comparison.

Ranking of gas-phase acidities and chloride affinities of monosaccharides and linkage specificity in collision-induced decompositions of negative ion electrospray-generated chloride adducts of oligosaccharides.

Negative ion electrospray-tandem mass spectrometry has been employed to study chloride adducts of saccharide molecules. Decompositions of [M + Cl]- obtained under identical low-energy collision conditions allow the approximate ranking of chloride affinities and gas-phase acidities of a series of isomeric monosaccharides. The ketohexoses are found to be more acidic than the aldohexoses. Chloride adduct decompositions are examined for a glucopyranosyl fructose and a glucopyranosyl glucose series. For each disaccharide series, the linkage position is shown to markedly influence the favored pathways of [M + Cl]- decompositions, initiated either by loss of neutral HCl to form [M - H]- and possibly leading to further (consecutive) decompositions, or by loss of M to form Cl-. Upon formation of [M - H]-, both cross-ring cleavages and glycosidic bond decompositions were observed in varying degrees for the two series of disaccharides. Remarkably, for three non-reducing polysaccharides that each contain a terminal sucrose group at the "downstream" end, chlorine-containing product ions arising from cleavage of the Glcalpha-2Fru linkage have been observed. Apart from Cl-, chlorine-containing product ions are not observed for any of the other disaccharides investigated, and they appear to be specifically diagnostic of a terminal Glcalpha-2Fru linkage. Their appearance is rationalized based upon a substantially reduced tendency for HCl loss from these non-reducing polysaccharides.

SFE-plus-C(18) lipid cleanup and selective extraction method for GC/MS quantitation of polycyclic aromatic hydrocarbons in smoked meat.

In biological matrixes lipid material often poses an interference problem for determinations of nonpolar compounds, e.g., polycyclic aromatic hydrocarbons (PAHs). A newly developed supercritical fluid extraction plus adsorbent method, "SFE-plus-C(18)", offers selective extraction of PAHs in lipid-rich biological matrixes without the need for supplementary cleanup. This method eliminates the use of large volumes of toxic solvent and lengthy lipid removal procedures. This study reports the first application of the SFE-plus-C(18) method to the analysis of a genuine food product, i.e., smoked meat (beef). The procedure employs the addition of C(18) adsorbent beads to the initial sample slurry of pureed smoked meat prior to supercritical CO(2) extraction and GC/MS quantitation. During SF extraction, indigenous lipids are preferentially retained on the beads, and PAHs are selectively extracted with supercritical CO(2). In a comparison of determinations of PAHs by SFE-plus-C(18) vs the conventional SFE method, only 11-17% of the indigenous lipids observed by the conventional SFE method were co-extracted using the SFE-plus-C(18) method. The PAHs in smoked meat could thus be determined efficiently in the presence of a reduced background of co-extracted lipids. Out of 10 targeted PAHs, seven were detected with a range of 10.0-26.0 ng/g in the smoked meat sample. The other three PAHs were not present above the detection limit of the instrument (2.5-4.1 pg). The recoveries of PAHs obtained using the conventional SFE method were 63-94% lower than those achieved by SFE-plus-C(18).

Anticancer properties for 4,4'-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007)/3,7-diaminophenothiazin-5-ium double salts.

4,4'-Dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007) formed stable double salts with phenothiazin-5-ium salts (2a-d), which have improved in vitro anticancer activities, as compared to A-007 alone. The stable salt between methylene blue (2a) and A-007 allowed the latter to diffuse into the dermis layers of skin. It is anticipated that these new salts will allow A-007 to penetrate into the deep lymphatic/vascular channels of the dermis, which contain metastatic cancer cells, and improve in vivo anticancer activities.

A novel endo-beta-galactosidase from Clostridium perfringens that liberates the disaccharide GlcNAcalpha 1-->Gal from glycans specifically expressed in the gastric gland mucous cell-type mucin.

We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAcalpha1-->4Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-alpha-Glc. By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcalpha1-->4Gal. The specificity of this enzyme as an endo-beta-galactosidase was established by analyzing the liberation of GlcNAcalpha1-->4Gal from GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAcalpha1--> 4Galbeta1-->3)GalNAc-ol by mass spectrometry. Because this novel endo-beta-galactosidase specifically releases the GlcNAcalpha1-->4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcalpha1-->4Gal-releasing endo-beta-galactosidase (Endo-beta-Gal(GnGa)). Endo-beta-Gal(GnGa) was found to remove the GlcNAcalpha1-->4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with alpha1,4-N-acetylglucosaminyltransferase cDNA. Endo-beta-Gal(GnGa) should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAcalpha1-->4Gal epitope.

Monitoring of immune response by blood serum profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the analysis of blood serum leads to the observation of a large variety of reproducible mass spectral peaks corresponding to blood components. In this study, the use of MALDI-TOFMS was developed as a tool for monitoring immune response to bacterial infection. Employing the MALDI-TOFMS approach, the levels of many components of blood were found to be immune response independent whereas others were found to correlate directly with the response of the immune system to two known types of bacteria (Staphylococcus aureus and Enterococcus faecalis). The methodologies reported here should be useful for the rapid monitoring of blood, especially that of the immune response mechanisms in various animal species.

Some tenets pertaining to electrospray ionization mass spectrometry.

The field of electrospray ionization mass spectrometry is reviewed with emphasis placed upon advances in the elucidation of fundamental mechanistic aspects of the ionization process that have been reported over the past 10 years. The analytical consequences of these findings are also examined. Eight central conclusions or 'tenets' are presented, as deduced from the body of work contained in 80 references.

Electrospray ionization tandem mass spectrometry for structural elucidation of protonated brevetoxins in red tide algae.

Brevetoxins, the toxic components of "red tide" algae, all share one of two robust polycyclic ether backbone structures, but they are distinguished by differing side-chain substituents. Electrospray ionization mass spectrometry analyses of brevetoxins have shown that the polyether structure invariably has a very high affinity for sodium cations that results in the production of abundant (M + Na)+ ions even when sodium cations are only present as impurities. Because the ionic charge tends to remain localized on the sodium atom and because at least two bonds must be broken in order to produce polycyclic backbone fragmentation, it is extremely difficult to obtain abundant product ions (other than Na+) from (M + Na)+ brevetoxin precursor ions in low-energy collision-induced dissociation (CID) MS/MS experiments. This report establishes that acid additives (oxalic acid, trifluoroacetic acid, and particularly hydrochloric acid) in aqueous methanol solutions can promote high yields of protonated brevetoxin molecules (MH+ ions) for Btx-1, -2, and -9 brevetoxins. Most importantly, unlike their (M + Na)+ counterparts, MH+ precursor ions offer readily detectable product ions in CID MS/MS experiments, even under low-energy collisions. This direct structural characterization approach has provided decomposition information from brevetoxins that was previously inaccessible, including the identification of diagnostic product ions for "type A" brevetoxins (m/z 611) and "type B" brevetoxins (m/z 779, 473, 179) and characteristic ions for Btx-1 (m/z 221, 139), Btx-2 (m/z 153), and Btx-9 (m/z 157, 85). Precursor ion scans and constant neutral loss scans are proposed to enable screening of individual type A or type B brevetoxins present in naturally occurring mixtures.

Solution reactivity of brevetoxins as monitored by electrospray ionization mass spectrometry and implications for detoxification.

The reactivities of brevetoxin compounds in acid and base and under oxidizing conditions were studied using electrospray ionization mass spectrometry (ESI-MS) to monitor reaction products. Brevetoxins are determined to be unstable in acidic and basic solutions. Under acidic conditions, brevetoxins containing an aldehyde functional group in the terminal "tail" side chain are easily converted to acetal structures, while "head" side lactone ring opening proceeds more slowly. Measurement of reaction rate constants indicates the following order of reactivity under acidic conditions: Btx-1 > Btx-2 > Btx-9. Under basic conditions, hydroxide ion attack at the head portion leads to lactone ring opening. Base hydrolysis (0.01 N NaOH in 50:50 methanol/water) goes to completion in 120 min for Btx-2 and Btx-9, but Btx-1 did not react to completion. Both acid and base hydrolyses can lead to reversible lactone ring opening, but base hydrolysis proceeds faster than acid hydrolysis under comparable conditions. Acid treatment is not an effective method for detoxifying brevetoxins. Base treatment can open the lactone ring (type B brevetoxins proceed faster than type A brevetoxins), leading to a product that is reportedly nontoxic, but the reaction is reversible. Brevetoxins are shown to be readily oxidized by permanganate in an irreversible and relatively fast reaction, likely through addition to double bonds followed by bond cleavage, suggesting that it is a viable method for detoxification.

Structural characterization of gangliosides isolated from mullet milt using electrospray ionization-tandem mass spectrometry.

Electrospray ionization (ESI) coupled with tandem mass spectrometry has been used in conjunction with microwave-mediated saponification, periodate oxidation, and clostridial sialidase hydrolysis to enable detailed structural characterization of gangliosides and their derivatives present in mullet milt. The gangliosides extracted from mullet milt were determined to be GM3, GM3 lactone, GM3 methyl ester, and 9-O-acetyl GM3. For the major ganglioside GM3 and all GM3 derivatives, the ceramide composition was revealed to be C18:1/C16:0. GM3 with a C18:0/C16:0 ceramide was also found as a minor ganglioside. Both the ganglioside intramolecular ester and the ganglioside methyl ester (lacking carboxylic acid groups) showed dominant chloride attachment peaks (M + Cl)- in negative ion ESI-MS in addition to low intensity peaks corresponding to (M-H)-. GM3 and O-acetyl GM3 bearing carboxylic acid functions showed only (M-H)-. In positive ion ESI, GM3 and O-acetyl GM3 revealed (M + 2Na-H)+ peaks in addition to (M + Na)+, indicating free exchange of the carboxylic acid proton with a sodium cation, while the ganglioside intramolecular ester and ganglioside methyl ester with no acidic protons yielded only (M + Na)+. The strategy of employing ESI-MS to detect products of established wet chemical reactions represents a general approach for elucidation of ganglioside structural details.

Rapid identification and speciation of Haemophilus bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Several species of the genus Haemophilus are well known etiological agents of pneumonia, meningitis, conjunctivitis, epiglottitis and chancroid. However, identification and speciation of Haemophilus is both time consuming and labor intensive. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) has been used by several investigators to profile proteins from intact and disrupted bacteria; consequently, MALDI/TOF-MS has emerged as a powerful tool in diagnostic bacteriology. This paper reports the use of MALDI/TOF-MS as a technique for the rapid identification and speciation of Haemophilus. This technique was used to not only identify the pathogen, H. ducreyi, but also to determine strain differences from different isolates. Mass spectral 'fingerprints' were obtained which permitted the rapid speciation of not only pathogenic forms of Haemophilus, but also those bacteria which are normally regarded as non-pathogenic and members of the normal flora. MALDI/TOF mass spectra can be acquired in 10 min, allowing the identification of Haemophilus spp. within 24 h rather than the 48 h or more needed for traditional bacteriological methods. In addition, these are the first mass spectral fingerprints available in the literature for many of these organisms.

Determination of chiral pharmaceutical compounds, terbutaline, ketamine and propranolol, by on-line capillary electrophoresis-electrospray ionization mass spectrometry.

On-line capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) has been employed for the determination of racemic mixtures of the chiral drugs, terbutaline, ketamine, and propranolol. Separation of the different chiral forms has been achieved by introducing cyclodextrins (CDs), which act as chiral selectors, into the CE operating electrolytes. Cyclodextrins function as chiral selectors in CE because of their ability to form host-guest complexes (inclusion complexes) of varying stability with an array of chiral drugs and other compounds. Derivatized forms of beta-CD (i.e., dimethyl-beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin) were used in this study due to their higher solubilities in the aqueous methanolic operating electrolyte than native beta-CD. Addition of minor quantities of methanol to the aqueous-based CE operating electrolytes improved the stability of electrospray ionization conditions and further enhanced CE resolution of the enantiomeric pairs relative to purely aqueous systems. Introduction of the CDs into the CE operating electrolytes caused suppression of analyte signals in ESI-MS, and the dependence of analyte signal intensities on the solution concentrations of the derivatized beta-CDs was examined. Under optimized conditions, the different enantiomeric forms of the compounds under investigation were successfully separated and detected by CE-ESI-MS.

Solvation energy and gas-phase stability influences on alkali metal cluster ion formation in electrospray ionization mass spectrometry.

The ability to observe abundant gas-phase metal cluster ions in electrospray ionization mass spectrometry (ESI-MS) is highly dependent on experimental conditions. Alkali halides (MX) and other alkali metal salts were used to investigate the formation of cluster ions in ESI-MS. All compounds were found to give cluster ions of the form (M(n)(+1)X(n))(+) and (M(n)X(n+1))(-), with only two alkali salts yielding doubly charged cluster ions. In homologous alkali halide series, the relative abundances of cluster ions increased with increasing size of either the cation (positive ion mode) or the anion (negative ion mode). Calculations using an electrostatic model show that the gas-phase stability of cluster ions is greater for smaller cations or anions when a fixed counterion is employed. This stability calculation goes in a direction just opposite to the trend in cluster ion abundances observed in ESI-MS. Studies of equimolar mixtures consisting of two alkali halides reveal two distinct trends. When the equimolar mixture was composed of differing ions that participate in the droplet charge excess with the same counterion, the less solvated ions were found to form more abundant cluster ions. When the ions participating in the charge excess were fixed, the preferred counterion in observed clusters was the one that is more solvated in solution and forms more stable clusters in the gas phase. These observations can be rationalized by an extended form of the charged residue model where the weakly solvated ions that are part of the charge excess are preferentially enriched in offspring droplets during uneven fission. By contrast, transfer of a particular counterion located in the bulk of the droplets to the offspring droplets is not disfavored when this counterion is strongly solvated.

SFE plus C18 lipid cleanup method for selective extraction and GC/MS quantitation of polycyclic aromatic hydrocarbons in biological tissues.

Lipid material represents a potential interference for determination of nonpolar compounds (e.g., polycyclic aromatic hydrocarbons) in biological tissue samples. This study reports the development of a selective extraction method using supercritical CO2 that allows the GC/MS quantitation of PAHs in the presence of a substantial lipid background. Selective extraction of PAHs relies upon addition of C18 adsorbent beads to the initial sample slurry. The dried mixture, including C18 adsorbent, is placed in the supercritical fluid extraction (SFE) chamber. During the SFE process, lipids are preferentially retained on the C18 beads. This "SFE plus C18" procedure was developed by first optimizing SFE conditions (100 degrees C, 350 bar) for recovery of PAH standards. PAHs containing added model lipid compounds (stearic acid and cholesterol) were then subjected to SFE plus C18 treatment followed by GC/MS analysis. Using this approach, a recovery of 94-100% of PAHs was obtained while only 9-17% of the lipid material present was coextracted from the same test sample. The developed method is demonstrated to permit efficient recovery and detection of PAHs spiked into crab tissue, a matrix with a high lipid content.

On-line linear sweep voltammetry-electrospray mass spectrometry.

A three-electrode electrochemical cell coupled to an electrospray (ES) mass spectrometer has been developed and employed for on-line linear sweep voltammetry-ES mass spectrometry and electrochemical reaction studies. Generated in this type of electrochemistry/mass spectrometry (EC/MS) experiment are ion abundance vs potential profiles that provide molecular weight and structural information pertaining to intermediates and products of anodic oxidation of diphenyl sulfide, anodic pyridation of 9,10-diphenylanthracene, and reduction of nitrobenzene. Anodic oxidation of diphenyl sulfide in methylene chloride media at the platinum electrode yielded diphenyl sulfoxide and pseudodimer sulfonium ion along with products of further oxidation. The obtained ion abundance vs potential profiles offer additional evidence to corroborate previously proposed reaction mechanisms for pseudodimer sulfonium ion generation from diphenyl sulfide, as well as pyridine addition to DPA radical cation also in methylene chloride at the platinum electrode. From the latter experiment, a newly found reaction product, [DPA(py) - H](+), is reported. Despite the inherent tendency of ES devices to induce oxidation processes in the positive ion mode, intermediates and products of nitrobenzene reduction in nonaqueous and organic/water media were detected in both negative and positive ES modes.

Silicosis presenting as bilateral hilar lymphadenopathy.

Classical radiographic features of patients presenting with silicosis are diffuse interstitial shadowing with subsequent enlargement of hilar nodes, sometimes with "eggshell" calcification. Five case histories are described of workers who were exposed to silica and presented initially with bilateral hilar lymphadenopathy without radiographic evidence of interstitial lung disease. One case progressed to show features of silicosis.

Acid and base hydrolysis of lipid A from Enterobacter agglomerans as monitored by electrospray ionization mass spectrometry: pertinence to detoxification mechanisms.

Lipopolysaccharides (LPS), which are endotoxins found in the cell wall of Gram-negative bacteria, are common components of organic dusts that cause or contribute to symptoms associated with organic dust diseases. The lipid A subgroup within LPS is believed to be responsible for the toxicity. Acid and base treatments, which can be effective detoxification methods, were performed on lipid A from Enterobacter agglomerans (EA), a bacterium commonly found in field cotton. Negative-ion electrospray ionization mass spectrometry was employed to characterize the post-treatment structural changes to lipid A. Acid treatment (1% acetic acid, 100 degrees C) hydrolyzed the ester side-chains of lipid A. It was found that the ester-linked palmitoyl group was the most labile to acid hydrolysis. Hydrolysis of the palmitoyl moiety conformed to pseudo-first-order chemical reaction kinetics with a rate constant for decomposition of heptacyl-lipid A from Enterobacter agglomerans of approximately 3.3 x 10(-3) min-1. An order of lability of lipid A acyl side-chains to acid hydrolysis was also deduced: R4' (palmitoyl) > R1' (myristoyl or hydroxymyristoyl) > R3 (hydroxymyristoyl at position 3) > R1 (oxymyristoyl group at position 3') > R2' (lauroyl). Base treatment (0.05 M NaOH in 95% EtOH, 65 degrees C) was shown to be more effective at cleaving ester-linked side-chains. In addition, mass spectral evidence suggests that opening of the pyranose rings of the disaccharide backbone of lipid A and/or removal of the phosphoryl groups may be occurring during base treatment. This study sheds light on mechanistic aspects of treatment procedures leading to the detoxification of endotoxins.

Analysis of tamoxifen and its metabolites by on-line capillary electrophoresis-electrospray ionization mass spectrometry employing nonaqueous media containing surfactants.

On-line capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESMS) has been employed for the analysis of metabolites of the anticancer drug tamoxifen. Nonaqueous (methanol) CE electrolyte provided better resolution and detection sensitivity compared to aqueous systems or highly aqueous water-methanol electrolyte mixtures. Nonaqueous methanol also permitted the use of lower ES voltages presumably owing to its lower surface tension, which facilitated droplet breakup. This decreased the tendency to produce electric discharges, thus improving the stability of electrospray conditions. The relative ease of methanol solvent evaporation may contribute to an improved yield of protonated analytes as compared to highly aqueous solutions. Enhanced CE resolution can be at least partially attributed to the improved solubility of analytes in methanol relative to water. Higher solubility implies less aggregation of hydrophobic analytes, thus improving homogeneity in solution. Moreover, electroosmotic flow toward the detector decreased in methanol relative to water. The reduction of this force pushing all analytes through the capillary, but not aiding in separation, implies that other factors such as slight differences in electrophoretic mobilities are more apt to lead to successful separations. Surfactants were employed as nonaqueous CE-ESMS buffer additives. An SDS concentration of 7 mM lowered the ESMS signal response for N-desmethyltamoxifen by a factor of approximately 3. However, separation of tamoxifen metabolites using 7 mM SDS was augmented relative to the unadulterated methanol electrolyte. This enabled the separation of alpha-hydroxytamoxifen and 4-hydroxytamoxifen, which were not resolvable in methanol electrolyte devoid of SDS. The methanol-surfactant electrolyte system has been successfully used to determine metabolites formed after incubation of tamoxifen with mouse hepatocytes.