Pyrrolidine alkaloids and their glycosylated derivatives from the root bark of Dichrostachys cinerea (L) Wight & Arn. (Fabaceae)

Phytochemical investigation of the methanol extract of the root bark of Dichostachys cinerea (Fabaceae) led to the isolation and characterization of 3 new pyrrolidine-derived compounds with 12 carbon side chain and their glycosides. The structures of these compounds (1–3) were established using spectroscopic analytical methods comprising NMR experiments, ESI and HR-ESIMS mass spectrometry, and mass tandem spectrometry. The compounds are related to some polyhydroxypyrrolidine type-iminosugars (broussonetinine and broussonetine) found in the restrictive plant families Moraceae, Campanulaceae and Hyacinthaceae, but with, no hydroxyl group on the pyrrolidine ring. Pyrrolidines with long-side chains have been found in the plant family, Araceae (Arisarum vulgare). The presence of compounds 1–3 in D. cinerea expands the range of distribution of pyrrolidine and its derivatives in plants, and thus presenting a new addition to the molecular diversity of pyrrolidine which could explain some of pharmacological properties of sourced plants.     

The scent of the reticulated giraffe (Giraffa camelopardalis reticulata)

The giraffe (Giraffa camelopardalis) emits a scent that can be detected by humans over considerable distances. Dichloromethane extracts of hair samples from adult male and female reticulated giraffes (G. c. reticulata) were analysed by gas chromatography–mass spectrometry. Two highly odoriferous compounds, indole and 3-methylindole, identified in these extracts appear to be primarily responsible for the giraffe’s strong scent. Other major compounds identified were octane, benzaldehyde, heptanal, octanal, nonanal, p-cresol, tetradecanoic acid, hexadecanoic acid, and 3,5-androstadien-17-one; the last compound has not previously been identified from a natural source. These compounds may deter microorganisms or ectoparasitic arthropods. Most of these compounds are known to possess bacteriostatic or fungistatic properties against mammalian skin pathogens or other microorganisms. The levels of p-cresol in giraffe hair are sufficient to repel some ticks.     

Identifying Bioaccumulative Halogenated Organic Compounds Using a Nontargeted Analytical Approach: Seabirds as Sentinels

Persistent organic pollutants (POPs) are typically monitored via targeted mass spectrometry, which potentially identifies only a fraction of the contaminants actually present in environmental samples. With new anthropogenic compounds continuously introduced to the environment, novel and proactive approaches that provide a comprehensive alternative to targeted methods are needed in order to more completely characterize the diversity of known and unknown compounds likely to cause adverse effects. Nontargeted mass spectrometry attempts to extensively screen for compounds, providing a feasible approach for identifying contaminants that warrant future monitoring. We employed a nontargeted analytical method using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS) to characterize halogenated organic compounds (HOCs) in California Black skimmer (Rynchops niger) eggs. Our study identified 111 HOCs; 84 of these compounds were regularly detected via targeted approaches, while 27 were classified as typically unmonitored or unknown. Typically unmonitored compounds of note in bird eggs included tris(4-chlorophenyl)methane (TCPM), tris(4-chlorophenyl)methanol (TCPMOH), triclosan, permethrin, heptachloro-1''-methyl-1,2''-bipyrrole (MBP), as well as four halogenated unknown compounds that could not be identified through database searching or the literature. The presence of these compounds in Black skimmer eggs suggests they are persistent, bioaccumulative, potentially biomagnifying, and maternally transferring. Our results highlight the utility and importance of employing nontargeted analytical tools to assess true contaminant burdens in organisms, as well as to demonstrate the value in using environmental sentinels to proactively identify novel contaminants.     

Identification of multiple antimicrobial peptides from the skin of fine-spined frog, Hylarana spinulosa (Ranidae)

In this study, peptidomics and genomics analyses were used to study antimicrobial peptides from the skin of Hylarana spinulosa. Twenty-nine different antimicrobial peptide precursors were characterized from the skin of H. spinulosa, which produce 23 mature antimicrobial peptides belonging to 12 different families. To confirm the actual presence and characteristics of these antimicrobial peptides in the skin tissue extractions from H. spinulosa, we used two distinct methods, one was peptide purification method that combined gel filtration chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the other was peptidomics approach based on liquid chromatography in conjunction with tandem mass spectrometry (LC–MS/MS). In the peptidomics approach, incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase peptidome coverage and reproducibility of peptide ion selection. Multiple species of microorganisms were chosen to test and analyze the antimicrobial activities and spectrum of these antimicrobial peptides.     

A new tripodal poly-imine indole-containing ligand: Synthesis, complexation, spectroscopic and theoretical studies

A novel flexible tripodal ligand derived from 3-methylindole, (“InTREN” L), and its mononuclear Zn(II), Cu(II), Ni(II), Hg(II) and Pd(II) complexes are described. All compounds gave analytically pure solid samples. Characterisation of the compounds was accomplished by ¹H NMR, IR and absorption spectroscopies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and elemental analysis and their geometry optimized using density functional theory (DFT).Time-dependent-density functional theory (TD-DFT) calculations have been used to assign the lowest energy absorption bands of the free ligand and the Zn(II) complex. The system is a very good candidate for in situ recognition/coordination effects by MALDI-TOF-MS spectrometry and absorption spectroscopy. The presence of three indole groups in InTREN opens up the possibility to synthesize new three-dimensional self-assembly supramolecular structures.     

Occurrence of cholesterol 7 alpha- and 7 beta-hydroperoxides in rat skin as aging markers

Evidence for presence of cholesterol 7 alpha- and 7 beta-hydroperoxides in rat skin was presented for the first time. The 7-hydroperoxides in rat skin were reduced with sodium borohydride and trimethylsilylated for identification with the authentic compounds by gas chromatography/mass spectrometry. A content of cholesterol 7-hydroperoxides in rat skin, determined by high performance liquid chromatography with a chemiluminescence detector, highly correlated with the age of rats (r = 0.874; between 1 and 45 weeks old), indicating that cholesterol 7 alpha- and 7 beta-hydroperoxides were good markers for aging.     

Low-molecular-weight compounds with anticoagulant activity from the scorpion <Emphasis Type="Italic">Heterometrus laoticus</Emphasis> venom

Low-molecular-weight compounds with anticoagulant activity were isolated from the scorpion Heterometrus laoticus venom. The determination of the structure of the isolated compounds by nuclear magnetic resonance and mass spectrometry showed that one of the isolated compounds is adenosine, and the other two are dipeptides leucyl-tryptophan and isoleucyl-tryptophan. The anticoagulant properties of adenosine, which is an inhibitor of platelet aggregation, is well known, but its presence in scorpion venom is shown for the first time. The ability of leucyl-tryptophan and isoleucyl-tryptophan to slow down blood clotting and their presence in scorpion venom are also established for the first time.     

MS-based metabolite profiling reveals time-dependent skin biomarkers in UVB-irradiated mice

We observed clinical and histological changes, including increased transepidermal water loss, epidermal thickness, and inflammatory cells, and changes in collagen fibers in the skin of mice chronically exposed to ultraviolet (UV) B radiation for 12 weeks. By using ultra-performance liquid chromatography-quadrupole time-of-flight (TOF) mass spectrometry (MS), gas chromatography (TOF–MS), and NanoMate tandem-MS-based metabolite profiling, we identified amino acids, organic compounds, fatty acids, lipids, nucleosides, carbohydrates, lysophosphatidylcholines, lysophosphatidylethanolamines, urocanic acids, and ceramides (CERs) as candidate biomarkers of the histological changes in mouse skin following UVB irradiation for 6 and 12 weeks. cis-Urocanic acid and cholesterol showed the most dramatic increase and decrease at 6 and 12 weeks, respectively. In addition, the changes in skin primary metabolites and lysophospholipids induced by UVB exposure were generally greater at 12 weeks than at 6 weeks. The results from primary metabolite, lysophospholipid, and CER profiles suggest that prolonged chronic exposure to UVB light has a great effect on skin by altering numerous metabolites. A comprehensive MS-based metabolomic approach for determining regulatory metabolites in UV-induced skin will lead to a better understanding of the relationship between skin and UV.     

Chemical constituents of flowers from Geoffroea spinosa Jacq. (Leguminosae), a plant species visited by bees

Phytochemical investigation of flowers from Geoffroea spinosa via ultra-performance liquid chromatography coupled with diode array and quadrupole time-of-flight mass spectrometry (UPLC-DAD-TOF-MS/MS) resulted in the characterization of fourteen compounds: six flavonoids, three hydroxycinnamic acid derivatives, and five hydroxycinnamic acid amide derivatives. This is the first report on the identification of hydroxycinnamic acid amide derivatives from the genus Geoffroea. The chemotaxonomic significance of the identified compounds is discussed.     

Mass-spectrometric evidence for quinonoid-lysine coupling products in cigar protein

On subjecting hydrolysates of hydrogenated cigar protein to derivatization and preparative GLC, two fractions were obtained which had volatilities and mass spectra consistent with an origin from oxidative-condensation products of chlorogenic acid with lysine residues. The mass-spectral evidence was chiefly from high-resolution mass spectrometry of the heaviest fragment ions; there was also high-resolution comparison of the lighter fragment ions with those from relevant model compounds. New compounds prepared were the hydantoin from 6-(1-trans-octahydroquinol-2-onyl)-DL-norleucine and the heptafluorobutyrylated n-propyl esters of four other less-common amino acids. Details of the mass spectra are provided in a Supplementary Publication 4 or have been sent to the Mass Spectrometry Data Centre.     

New polyacetylene and other compounds from Bupleurum chinense and their chemotaxonomic significance

Here, we isolated a new polyacetylene, (2Z,8E)-heptadecadiene-10-oxo-4,6-diyn-1-ol (1), an oxylipin (2), eight phenolic compounds (3–10), and 15 saponins (11–25) from the roots of Bupleurum chinense. We elucidated their structures by analysis of 1D and 2D-NMR spectroscopic data and mass spectrometry, and by comparison with those of related metabolites. This is the first report of the presence of compounds 2 and 6 in the Bupleurum spp.. Chemotaxonomic significance of these compounds is described herein.     

Structural and functional characteristics of various forms of red pigment of yeast Saccharomyces cerevisiae and its synthetic analog

Structural and functional characteristics of the yeast red pigment (product of polymerization of N¹-(β-D-ribofuranosyl)-5-aminoimidazole), isolated from ade1 mutant cells of Saccharomyces cerevisiae and its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N¹-methyl-5-aminoimidazole in vitro) were obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. All the studied compounds are able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in the presence of these compounds—they are merged into conglomerates more stable and resistant to the effects of ultrasound than are insulin aggregates grown without pigments. We suggest that all these compounds can cause coalescence of fibrils partially blocking the loose ends and, thereby, inhibit attachment of monomers and formation of new fibrils.     

Identification of maloyl glucans from Euphorbia tirucalli by ESI-(−)-FT-ICR MS analyses

Euphorbia tirucalli aerial parts are popularly used in Brazil for cancer treatment. The elution of the aqueous extract of the plant on silica gel C-18 cartridge furnished a water-soluble fraction, which was analyzed directly into the electrospray ionization (ESI) source combined with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and characterized as a mixture of malic acid glycosides 1–5. The compounds were detected in their deprotonated form [M−H]⁻, where their exact mass (mass error lower than 1 ppm), molecular formula (C_nH_hO_o), double bond equivalent (DBE) and connectivity were determined from ESI-(−)-MS and ESI-(−)-MS/MS experiments. The presence of malic acid and glucose, as part of the structures, could originate from crassulacean acid metabolism (CAM) of the plant.     

A comprehensive metabolite profiling of Isatis tinctoria leaf extracts

A broad-based characterisation of a pharmacologically active dichloromethane extract from Isatis tinctoria leaves was carried out. For a comprehensive picture we also included the polar constituents of I. tinctoria (MeOH extract) and for comparative purposes, the taxonomically closely related plant I. indigotica. Diode array detector, evaporative light scattering detector, atmospheric pressure chemical ionisation and electrospray ionisation mass spectrometry, and electrospray ionisation time-of-flight mass spectrometry detectors were used in parallel to ensure a wide coverage of secondary metabolites with highly diverging analytical properties. Off-line microprobe nuclear magnetic resonance spectroscopy after peak purification by semi-preparative high-pressure liquid chromatography served for structure elucidation of some minor constituents.More than 65 compounds belonging to various structural classes such as alkaloids, flavonoids, fatty acids, porphyrins, lignans, carotenoids, glucosinolates and cyclohexenones were unambiguously identified, and tentative structures were proposed for additional compounds. Numerous compounds were identified for the first time in the genus Isatis, and an indolic alkaloid was discovered.     

Combined gas chromatography-chemical ionization mass spectrometry of sphingolipids. I. Glucosyl sphingosine,ceramides and cerebrosides of the spleen in Gaucher's disease

Trimethylsilylated glucosyl sphingosine, ceramides and glucocerebrosides were analysed by combined gas chromatography (GC)-chemical ionization (CI) mass spectrometry. Isobutane, methane and ammonia were used as reactant gases for chemical ionization. Essentially the same fragment ions were detected in the spectra with the different reactant gases.Purified glucocerebrosides isolated from the spleen of a patient with Gaucher's disease were clearly separated into their 8 molecular species by gas chromatography. Three other minor components were detected by spectrometry, and these 11 molecular species of glucocerebrosides from the spleen of the patient with Gaucher's disease have been analysed.The ceramides obtained by periodate oxidation and then alkaline reduction of the glucocerebrosides were also separated into 11 molecular species by GC-CI mass spectrometry.Molecular weight could be determined using the major fragment ion of m/e (M⁺?90) in the spectra of ceramides and cerebrosides. The chemical ionization method is useful for structural analyses and determination of the molecular species of sphingoglycolipids.     

Volatile antimicrobial compounds in the pelage of the Mexican free-tailed bat, Tadarida brasiliensis mexicana

Gas chromatography–mass spectrometry (GC–MS) showed that CH₂Cl₂ extracts of the pelage from the Mexican free-tailed bats, Tadarida brasiliensis mexicana, contained only seven volatile compounds. They include two hydrocarbons, 1-octene and octane; three aldehydes, heptanal, octanal and nonanal; a carboxylic acid, nonanoic acid; and urea. It is likely that nonanal, at an average concentration 0.62 μg/mg hair, and the two homologues, heptanal and octanal found at 0.059 and 0.066 μg/mg hair, respectively, function as antimicrobial agents against mammalian skin bacteria and fungi. These three aldehydes may also act as a chemical protection against bat ectoparasites.     

Diffusion-controlled reaction kinetics of the binding of carbon monoxide to the heme undecapeptide of cytochrome c (microperoxidase 11) in high viscosity solvents

Glycosphingolipids from human plasma with Le^a, Le^b, and H-type 1 (Le^dH) Lewis-blood-group activity have been analyzed after permethylation by electron impact mass spectrometry using an indirectly heated direct insertion probe. The spectra obtained are compared with that of permethylated neo-lactotetraosyl ceramide (Gl-3) from human plasma. The fragmentation patterns presented show clearly, that Le^a and H-type 1 glycosphingolipids are ceramide pentasaccharides while Le^b is a ceramide hexasaccharide. All Lewis-blood-group-active compounds investigated produced ions specific for type 1 carbohydrate chains. It is therefore concluded, that all compounds are derivatives of lacto-N-tetraose. The obtained spectra support the following sequences: Hexose-1→3-hexosamine[4←1-deoxyhexose]-hexose-hexose ceramide for the Le^a derivatives; deoxyhexose-hexose-1→3-hexosamine4←1-deoxyhexose]-hexose-hexose ceramide for the Le^b derivatives; and deoxyhexose-hexose-1→3-hexosamine-hexose-hexose ceramide for all H-type 1 (Le^dH) derivatives. In the case of the H-type 1 glycosphingolipids four subfractions were analyzed separately. While all four fractions contained the same carbohydrate sequence, significant differences were observed in the ceramide residues. Specific fragmentation patterns indicate the presence of sphingosine, icosasphingosine, and 4-hydroxysphinganine besides normal, unsaturated, and hydroxylated fatty acids in all Lewis-blood-group-active glycolipids.     

Determination of l-carnitine,acetyl-l-carnitine and propionyl-l-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene

A new sensitive high-performance liquid chromatographic procedure for the determination of l-carnitine (LC), acetyl-l-carnitine (ALC) and propionyl-l-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C₁₈). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%–95.4% for all 3 compounds. Good linearity (R≈0.99) was observed within the calibration ranges studied: 5–160 nmol/ml for LC, 1–32 nmol/ml for ALC and 0.25–8 nmol/ml for PLC. Precision was in the range 0.3–16.8% and accuracy was always lower than 10.6%.     

Recent advances in applications of liquid chromatography-tandem mass spectrometry to the analysis of reactive drug metabolites

Biotransformation of chemically stable compounds to reactive metabolites which can bind covalently to macromolecules, such as proteins and DNA, is considered as an undesirable feature of drug candidates. As part of an overall assessment of absorption, distribution, metabolism and excretion (ADME) properties, many pharmaceutical companies have put methods in place to screen drug candidates for their tendency to generate reactive metabolites and as well characterize the nature of the reactive metabolites through in vitro and in vivo studies. After identification of the problematic compounds, steps can be taken to minimize the potential of bioactivation through appropriate structural modifications. For these reasons, detection, structural characterization and quantification of reactive metabolites by mass spectrometry have become an important task in the drug discovery process. Triple quadrupole mass spectrometry is traditionally employed for the analysis of reactive metabolites. In the past 3 years, a number of new mass spectrometry methodologies have been developed to improve the sensitivity, selectivity and throughput of the analysis. This review focuses on the recent advances in the detection and characterization of reactive metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in drug discovery and development, especially through the use of linear ion trap (LTQ), hybrid triple quadrupole-linear ion trap (Q-trap) and the high resolution LTQ-Orbitrap instruments.     

Enhancement of protonated molecular ions and ammonium·molecular ion complexes in direct-liquid-introduction-mass spectrometry of carbohydrates

Addition of ammonium acetate to the mobile phase in direct-liquid-introduction mass spectrometry enhances the abundance of the protonated molecular ion or ammonium·molecular ion complex for compounds of biological interest. The efficacy of the method was investigated by comparing mass spectra obtained, with and without ammonium acetate, for a variety of underivatized, per-O-acetylated, and per-O-alkylated carbohydrates, and for several underivatized peptides. The mass spectra of the per-O-alkylated carbohydrates obtained by direct-liquid-introduction mass spectrometry with ammonium acetate were also compared to those obtained by thermospray mass spectrometry.