Simultaneous selected reaction monitoring, MS/MS and MS3 quantitation for the analysis of pharmaceutical compounds in human plasma using chip-based infusion

An assay method with mass spectrometric detection was developed for the quantitative analysis of a pharmaceutical compound and its major metabolite in human plasma using chip-based infusion. Liquid-liquid extraction sample preparation was found to be essential to minimize matrix suppression and to achieve a limit of quantitation (LOQ) of 2.5 ng/mL using a 100 microL plasma aliquot. The potential for simultaneous quantitation in selected reaction monitoring (SRM), tandem mass spectrometry (MS/MS) (enhanced product ion), and MS(3) was investigated and found to be very beneficial in improving assay selectivity. A novel concept for monitoring quantitative assay performance using a SRM/MS(3) ratio is proposed.     

Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics

Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a ‘grind-and-find’ study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research.

Antimicrobial resistance is a major public concern and novel antibiotics are largely based on natural products. We summarize recent analytical and genome based technological developments that gain increasing importance in the natural products field.     

Liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry of the Alternaria mycotoxins alternariol and alternariol monomethyl ether in fruit juices and beverages

Alternariol (AOH) and alternariol monomethyl ether (AME) are among the main mycotoxins formed in apples and other fruits infected by Alternaria alternata. For determination of AOH and AME by LC, apple juice and other fruit beverages were cleaned up on C18 and aminopropyl solid-phase extraction columns. Positive and negative ion mass spectra of AOH and AME under electrospray (ESI) and atmospheric pressure chemical ionization (APCI) conditions were obtained. Collision-induced dissociation of the [M+H]+ and [M-H]- ions for both compounds were also studied. The phenolic anions of both compounds are more stable with less fragmentation. In quantitative analysis, negative ion detection also offers lower background and better sensitivity. Sensitive LC-MS and LC-MS-MS confirmatory procedures based on APCI with negative ion detection were applied to confirm the natural occurrence of AOH in nine samples of apple juice and in single samples of some other clear fruit beverages--grape juice, cranberry nectar, raspberry juice, red wine, and prune nectar (which also contained 1.4 ng AME/ml)--at levels of up to 6 ng AOH/ml. Electrospray LC-MS-MS with negative ion detection and in multiple reaction monitoring mode offers higher sensitivity and specificity. Absolute detection was better than 4 pg per injection for both compounds.     

Reactivity of the Coordinated Hydroperoxo Ligand

The reactivity of the hydroperoxo complex [Co(CN)(5)OOH](3)(-) has been studied in aqueous solution. The complex undergoes acid-catalyzed aquation (k = 1.89(5) x 10(-)(2) s(-)(1), pK(a) = 5.21(4), T = 20 degrees C, I = 0.1 M). Assuming an I(d) mechanism, this allows the relative affinity for Co(III) to be deduced as H(2)O(2) < H(2)O < HO(2)(-) and implies H(2)O(2) to be a very weak ligand. At neutral pH the hydroperoxo complex effects efficient oxygen atom transfer to L-methionine to give an intermediate identified as [Co(CN)(5)(L-methionine S-oxide)](2)(-), which then dissociates to [Co(CN)(5)OH(2)](2)(-) and L-methionine S-oxide. The reaction is acid catalyzed and is proposed to take place via nucleophilic attack of sulfur on the proximal oxygen of the hydroperoxo ligand with concerted loss of water. The significance of these results for the interaction of hydrogen peroxide with labile metal ions is discussed.     

Photodissociation and electronic spectroscopy of [Re(H)(CO)(3)(H-dab)] (H-dab=1,4-diaza-1,3-butadiene): quantum wavepacket dynamics based on ab initio potentials

The photodissociation dynamics of [Re(H)(CO)(3)(H-dab)] (H-dab=1,4-diaza-1,3-butadiene) were studied by means of wavepacket propagations on CASSCF/MR-CCI potentials calculated for the electronic ground state and low-lying excited states as a function of two coordinates, q(a) and q(b), that correspond to the Re-H bond homolysis and to the axial CO loss, respectively. The theoretical absorption spectrum is characterized by two bands, one intense peak centered at lambda=500 nm (21,000 cm(-1)) and one broad band centered at 310 nm (32,500 cm(-1)). The visible band was assigned to the low-lying metal-to-ligand charge-transfer (MLCT) states with a main contribution of the a(1)A'-->c(1)A' transition corresponding to the 3d(xz)-->pi*(dab) excitation. The second band calculated in the UV energy domain was assigned to the d(1)A' (sigma(Mn-H)-->pi*(dab)) state corresponding to a sigma-bond-to-ligand charge-transfer (SBLCT) state. The photodissociation dynamics of the low-lying (1)MLCT and (3)SBLCT states following irradiation in the visible energy domain was simulated by wavepacket propagation on the two-dimensional diabatic potentials V(q(a), q(b)) coupled by the spin-orbit. In contrast to what was found for the manganese analogue, the (1)MLCT state is nonreactive and a rather slow (beyond the ps time scale), nontotal and indirect homolysis of the Re-H bond occurs through (1)MLCT-->(3)SBLCT intersystem crossing.     

Synthesis and characterization of alkyl-, alkenyl-, acyl- and nitrogen-substituted derivatives of the potent phototoxin α-terthiophene

Some alkyl-, alkenyl-, acyl- and nitrogen- derivatives of α-terthienyl 1 have been synthesized in an effort to expand the potential insecticidal activity profile of this compound. The introduction of substituents was carried out directly on the α-terthienyl molecule. Three compounds were isolated in the nitration reaction and the compounds have been identified as 5-nitro-2,2′:5′,2″-terthiophene 5 , 3′-nitro-2,2′:5′,2″-terthiophene 6 and 3-nitro-2,2′:5′,2″-terthiophene 7 respectively. Preliminary results of biological acitivity are reported.     

Photoelectrochemical oxidation of water at transparent ferric oxide film electrodes

The fabrication of thin-film Fe(2)O(3) photoanodes from the spray pyrolysis of Fe(III)-containing solutions is reported along with their structural characterization and application to the photoelectrolysis of water. These films combine good performance, measured in terms of photocurrent density, with excellent mechanical stability. A full investigation into the effects that modifications of the spray-pyrolysis method, such as the addition of dopants or structure-directing agents and changes in precursor species or carrier solvent, have on the performance of the photoanodes has been realized. The largest photocurrents were obtained from photoanodes prepared from ferric chloride precursor solutions, simultaneously doped with Ti(4+) (5%) and Al(3+) (1%). Doping with Zn(2+) also shows promise, cathodically shifting the onset potential by approximately 0.22 V.     

A new biotinylated tris bipyridinyl iron(II) complex as redox biotin-bridge for the construction of supramolecular biosensing architectures

The bioaffine immobilization of several avidin layers on an electrode modified by a biotinylated polymer was accomplished by the first biotinylated redox bridge consisted of a tris(bipyridyl)iron(II) complex bearing six pre-oriented biotin groups.