The characterisation of coumarins from selected structural classes by electrospray ionisation quadrupole time-of-flight tandem mass spectrometry.

Electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) has been used for characterisation of a selection of naturally occurring and synthetic coumarins from different structural classes. The product ions, suggested in earlier studies by electrospray ionisation ion trap mass spectrometry (ESI-MS(n)), are unequivocally established for the representative coumarins by virtue of accurate mass measurement. Synthetic coumarins that are unsubstituted in the heterocyclic ring give rise to a major product ion by loss of CO(2), whereas those substituted in the heterocyclic ring generally undergo alternative fragmentation releasing neutral species such as ketene or methyl ketene. Naturally occurring coumarins, unsubstituted in the heterocyclic ring and substituted in the benzene ring with chains or rings of hydrocarbons and oxygen, principally fragment at the side chain releasing unsaturated hydrocarbons. The ESI-QTOF-MS/MS behaviour of some naturally occurring and synthetic quinolines which are structurally similar or fragment similarly are included where appropriate.

Mass spectrometric characterisation and quantitation of selected low molecular mass compounds from the venom of Haplopelma lividum (Theraphosidae).

Arachnid venoms present a diverse and complex matrix for investigation, with their latent potential for innovative drug and pesticide design largely unrealised. The characterisation and quantification of selected low molecular mass compounds isolated from the crude venom of the Cobalt blue tarantula (Haplopelma lividum) were the objectives of this study. Fractionation of the crude venom was performed using reversed-phase high-performance liquid chromatography, with compound identification using both electrospray ionisation ion trap mass spectrometry and quadrupole time-of-flight mass spectrometry. Four compounds were identified, and quantification on a percentage dry weight basis was achieved by liquid chromatography/electrospray ionisation tandem mass spectrometry based on the formation of their corresponding product ions. Of these the most abundant component was glutamic acid, present at a level of 0.97%. Histamine and adenosine were detected at 0.14% and 0.10% dry weight, respectively, with the polyamine spermine noted in trace amounts at 0.002%. The limits of detection and quantification were established for each of the identified components. The fragmentation profile for histamine has also been proposed.

A study of the analytical behaviour of selected psycho-active drugs using liquid chromatography, ion trap mass spectrometry, gas chromatography and polarography and the construction of an appropriate database for drug characterisation.

This paper provides analytical chemical information on a range of psycho-active drugs. This analytical chemical information on liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS), ion trap mass spectrometry (ESI-MS(n)), gas chromatography-flame ionisation detection (GLC-FID) and polarographic behaviour is then incorporated into a database which is of use in drug characterisation. Application is found in the determination of selected drug compounds in hair samples.

The characterisation of selected antidepressant drugs using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their determination by high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry.

The electrospray ionisation ion trap tandem mass spectrometry (ESI-MS(n)) of selected antidepressant drugs, i.e., citalopram, fluoxetine, mirtazapine, paroxetine, sertraline, and venlafaxine, has been investigated. Sequential product ion fragmentation experiments (MS(n)) have been performed in order to elucidate the degradation pathways for the [M+H](+) ions and their predominant product ions. These MS(n) experiments show certain characteristic fragmentations in that functional groups are generally cleaved from the ring systems as molecules such as H(2)O, amines and phenols. When an aromatic entity is present in a drug molecule together with a nitrogen-containing saturated ring structure as with mirtazapine, fragmentation initially occurs at the latter ring with the former being predictably resistant to fragmentation. Also, when an amine-containing drug molecule such as fluoxetine also contains a functional group, which liberates a phenol with a significantly lower DeltaH(f) (0) value than that of the corresponding amine, the phenol is preferentially liberated. The structures of product ions proposed for ESI-MS(n) can be supported by electrospray ionisation quadrupole-time-of-flight tandem mass spectrometry (ESI-QToF-MS/MS). These molecules can be identified and determined in mixtures at low ng/mL concentrations by the application of high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS(2)), which can also be used for their analysis in hair samples.

Histamine-releasing and antimicrobial peptides from the skin secretions of the dusky gopher frog, Rana sevosa.

Seven novel peptides were isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa, on the basis of antimicrobial activity and histamine release from rat peritoneal mast cells. The peptides were purified to homogeneity using HPLC and characterized by electrospray ion-trap mass spectrometry, MALDI-TOF mass spectrometry and Edman sequencing. Bioinformatic analysis of primary structures revealed that the novel peptides could be assigned to four established families of ranid frog antimicrobial peptides, namely esculentin-1, esculentin-2, brevinin-1 and ranatuerin-2. The peptides were named in accordance with accepted terminology as ranatuerin 2SEa, etc., reflecting the peptide family name, the species of origin (SE for sevosa) and the isotype (a). Of major interest was the fact that brevinin 1SE displayed significant structural similarity to ponericin W5, an antibacterial venom peptide from the ant, Pachyconyla goeldii. This is a further example of amphibian skin defensive peptides showing striking structural similarities to peptides from insects. These data may shed some light on the functional biological relevance of defensive peptides that possess both antimicrobial and histamine-releasing activities.

Adenosine in the venoms from viperinae snakes of the genus Bitis: identification and quantitation using LC/MS and CE/MS.

Snake venoms are rich sources of toxic proteins and small molecules. This study was directed at molecules of molecular mass below 1 kDa. Thirty different venoms, of either neurotoxic or haemorrhagic type, were fractionated using size-exclusion chromatography. Only venoms of the Puff adder (Bitis arietans), Gaboon viper (Bitis gabonica), and Rhinoceros viper (Bitis nasicornis) exhibited large absorbance peaks at lambda(280 nm) in the total volume range of the chromatographic column indicating the presence of abundant low molecular mass material. Analysis of fractions containing this material using both HPLC and capillary electrophoresis interfaced with electrospray ion-trap mass spectrometry unequivocally established that the bioactive nucleoside, adenosine, was the major component. The concentrations of adenosine found (Puff adder--97.7 x 10(-6) mol L(-1); Gaboon viper--28.0 x 10(-6) mol L(-1); and Rhinoceros viper-56.8 x 10(-6) mol L(-1)) were above those required to activate all known sub-types of adenosine receptors. Adenosine may thus act at the site of envenomation causing local vasodilatation and may play a role in the subsequent systemic hypotension observed.

The characterisation of selected drugs with amine-containing side chains using electrospray ionisation and ion trap mass spectrometry and their determination by HPLC-ESI-MS.

The electrospray ionisation-ion-trap mass spectrometry (ESI-MS(n)) of selected drug compounds with amine-containing side chains has been investigated. Certain characteristic in-source fragmentations have been observed for these molecules. Sequential product ion fragmentation experiments (MS(n)) have been performed in order to elucidate the degradation pathways for the [M + H](+) ions and their predominant fragment ions. These MS(n) experiments also show certain characteristic fragmentations with respect to the amine-containing side chains. QTOF-MS/MS has been used to support the identity of the proposed fragments. The data presented in this paper therefore provides useful information on the structure of these compounds with amine-containing side chains and can be used in the characterisation of such drugs, their structurally related metabolites and unknown molecules of pharmaceutical significance extracted from animal and plant sources, for example. Amphetamine, clenbuterol, flurazepam and methadone can be identified and determined in mixtures at low ng/ml concentrations by the application of HPLC-ESI-MS which can also be used for their analysis in saliva samples.

Characterisation of selected drugs with nitrogen-containing saturated ring structures by use of electrospray ionisation with ion-trap mass spectrometry.

The electrospray ionisation-ion-trap mass spectrometry (ESI-MS(n)) of selected drugs with nitrogen-containing saturated ring structures has been investigated. Sequential product-ion fragmentation experiments (MS(n)) have been performed to elucidate degradation pathways for the [M+H](+) ions and their predominant fragment ions. These MS(n) experiments result in characteristic fragmentations in which functional groups are generally cleaved from the ring systems as neutral molecules such as H(2)O, amines, alkenes, esters, carboxylic acids, etc. When such a nitrogen-containing drug molecule also contains a functional group, such as an ester, that on liberation as a neutral molecule has a significantly lower -Delta H(f) degrees value than that of the corresponding amine then the former is preferentially liberated. Furthermore, when an aromatic entity is present in these drug molecules together with the nitrogen-containing saturated ring structure fragmentation of the latter ring occurs with the former, predictably, being resistant to fragmentation. The structures of fragment ions proposed for ESI-MS(n) can be supported by electrospray ionisation-quadrupole time-of-flight mass spectrometry (ESI-QTOFMS). The data presented in this paper therefore provide useful information on the structure of these heterocyclic compounds which could be used to characterise unknown drug compounds isolated from natural sources, for example.

Cloning of maximakinin precursor cDNAs from Chinese toad, Bombina maxima, venom.

Using a novel technique that we have developed for cloning of amphibian skin secretion peptide cDNAs from lyophilized samples, we report here that maximakinin (DLPKINRKGP-bradykinin) is encoded by two different cDNAs, named BMK-1 and BMK-2, containing either four tandem repeat sequences or a single copy. The open reading frames of both precursor cDNAs were found to be 152 and 116 amino acid residues, respectively. These data provide evidence that the structural diversity of peptides in amphibian skin secretions arising from molecular evolutionary events, can be mediated by parallel diversity in encoding mRNAs that in itself may reflect serial gene duplications.

Isolation of scorpion (Androctonus amoreuxi) putative alpha neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom.

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60-70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.