Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.     

Site-Selective Real-Time Observation of Bimolecular Electron Transfer in a Photocatalytic System Using L-Edge X-Ray Absorption Spectroscopy**

Time-resolved X-ray absorption spectroscopy has been utilized to monitor the bimolecular electron transfer in a photocatalytic water splitting system. This has been possible by uniting the local probe and element specific character of X-ray transitions with insights from high-level ab initio calculations. The specific target has been a heteroleptic [Ir^III(ppy)₂(bpy)]⁺ photosensitizer, in combination with triethylamine as a sacrificial reductant and as a water reduction catalyst. The relevant molecular transitions have been characterized via high-resolution Ir L-edge X-ray absorption spectroscopy on the picosecond time scale and restricted active space self-consistent field calculations. The presented methods and results will enhance our understanding of functionally relevant bimolecular electron transfer reactions and thus will pave the road to rational optimization of photocatalytic performance.     

Radical Arylation Reactions of 4, 6, 8-Trimethylazulene

The synthesis of 1- and 2-aryl-substituted (aryl = Ph, 4-NO₂? C₆H₄, and 4-MeO? C₆H₄) 4, 6, 8-trimethylazulenes ( 4 and 3 , respectively) in moderate yields by direct arylation of 4, 6, 8-trimethylazulene ( 8 ) with the corresponding arylhydrazines 13 in the presence of Cu^IIions in pyridine (see Scheme 4) as well as with 4-MeO? C₆H₄Pb(OAc)₃ ( 16 ) in CF₃COOH (see Scheme 5) is described. With 13 , also small amounts of 1, 2- and 1, 3-diarylated azulenes (see 14 and 15 , respectively, in Scheme 4) are formed. The 4-methoxyphenylation of 8 with 16 yielded also the 1, 1′-biazulene 17 in minor amounts (see Scheme 5). 4, 6, 8-Trimethyl-2-phenylazulene ( 3a ) was also obtained as the sole product in moderate yields by the reaction of sodium phenylclopentadienide ( 1a ) with 2, 4, 6-trimethylpyrylium tetrafluoroborate ( 2 ) in THF (Scheme 1). The attempted phenylation of 8 as well as of azulene ( 9 ) itself with N-nitroso-N-phenylacetamide ( 10 ) led only to the formation of the corresponding 1-(phenylazo)-substituted azulenes 12 and 11 , respectively (Scheme 3).     

Roles of surface Te, Nb, and Sb oxides in propane oxidation to acrylic acid over bulk orthorhombic Mo-V-O phase

The outermost surfaces and subsurface layers of the orthorhombic (M1) Mo-V-O catalysts promoted with Te, Nb, and Sb oxide species at submonolayer surface coverage were examined by low-energy ion scattering (LEIS). This study indicated that the Nb oxide species was preferentially located at the topmost surface, while the subsurface Te and Sb concentrations declined gradually into the bulk. Although the original Mo-V-O catalyst was essentially unselective in propane oxidation to acrylic acid, significant improvement in the selectivity to acrylic acid was observed when Te, Nb, and Sb oxides were present as the surface species at submonolayer coverage. These findings further suggested that the formation of the surface V-O-M bonds (M = Nb, Te, or Sb) was highly beneficial for both the activity and selectivity of the orthorhombic Mo-V-O catalysts in propane oxidation to acrylic acid. The highest selectivity was observed when both Nb and Te (or Sb) oxide species were present at the surface. The selectivity trends established for the surface-promoted Mo-V-O catalyst parallel those found previously for the corresponding bulk Mo-V-M-O catalysts. These results further indicated that the introduction of surface metal oxide species is a highly promising method to prepare well-defined model catalysts for studies of the structure-activity/selectivity relationships as well as optimize the catalytic performance of the bulk mixed Mo-V-M-O catalysts for selective (amm)oxidation of propane.     

A new class of (mu-eta2:eta2-disulfido)dicopper complexes: synthesis, characterization, and disulfido exchange

Rare examples of (mu-eta2:eta2-disulfido)dicopper complexes have been prepared from Cu(I) and Cu(II) complexes of beta-diketiminate and anilido-imine supporting ligands. A novel byproduct derived from sulfur functionalization of the methine position of a beta-diketiminate ligand was identified. DFT calculations on [(LCu)2X2] (L = beta-diketiminate, X = O or S) complexes rationalize the absence of a bis(mu-sulfido)dicopper isomer, [Cu2(mu-S)2](2+), in the synthetic reactions, yet predict that a [Cu2(mu-S)2](0) core is a stable product of 2-electron reduction of the [Cu2(mu-eta2:eta2-S2)](2+) unit. Exchange of the disulfido ligand was discovered upon reaction of a (mu-eta2:eta2-disulfido)dicopper complex with a Cu(I) reagent.     

Electrochemical approach to the dynamics of molecular recognition of redox enzyme sites by artificial cosubstrates in solution and in integrated systems

Application of antigen-antibody technology allows the attachment to an electrode surface of an enzyme monolayer structure to which both the enzyme and the mediator are bound. As illustrated with the example of glucose oxidase and a ferrocene mediator, the enzyme preserves its full activity in such structures, which may be easily reproduced. In spite of their fixation to the structure, the mobility of the ferrocene heads is sufficient to ensure that its transport to the enzyme prosthetic group is not rate determining. The reaction is rather controlled by the prior formation of a complex between the ferrocenium ion and the flavin required for electron transfer to occur. The efficiency of this step is affected by steric hindrance and the various observations made with free-moving and attached ferrocene-ended poly(ethylene glycol) chains may be rationalized by the interplay of factors controlling their distribution and shape. Analyzing the dynamics of this system, in comparison with previous systems, was thus an occasion to shed further light on the recognition phenomenon. The enzyme monolayer integrated system is a good starting point for the step-by-step construction of spatially ordered multilayered assemblies with strong catalytic efficiencies. Fast responding systems are expected both in terms of electron transport and electron transfer between the mediator and the enzyme. The spatial order resulting from the step-by-step construction should allow a much more precise analysis of electron transport and electron transfer than in conventional assemblies of redox centers. Mastering both the construction and the functioning of such systems should help the design of more complex systems, integrating additional functionalities electrically controlled by means of their electron transport/electron transfer connection to the electrode surface.     

A nonanuclear copper(II) polyoxometalate assembled around a mu-1,1,1,3,3,3-azido ligand and its parent tetranuclear complex

Reaction of Cu(II), [gamma-SiW10O36]8-, and N3- affords three azido polyoxotungstate complexes. Two of them have been characterized by single-crystal X-ray diffraction. Complex KNaCs10[{gamma-SiW10O36Cu2(H2O)(N3)2}2].26H2O (1) is obtained as crystals in few hours after addition of CsCl. This linear tetranuclear Cu(II) complex consists in two [gamma-SiW10O36Cu2(H2O)(N3)2]6- units connected through two W=O bridges. When the filtrate is left to stand for one night, a new complex is obtained. From both elemental analysis and IR spectroscopy, it has been postulated that this compound could be formulated K(1.5)Cs(5.5)[SiW10O37Cu2(H2O)2(N3)].14 H2O (1 a), showing the loss of one azido ligand per polyoxometalate unit. Finally, when no cesium salt is added to the reaction medium, the nonanuclear complex K12Na7[{SiW8O31Cu3(OH)(H2O)2(N3)}3(N3)].24 H2O (2) is obtained after three days. Compound 2 crystallizes in the R3c space group and consists in three {Cu3} units related by a C3 axis passing through the exceptional mu-1,1,1,3,3,3-azido bridging ligand. Each trinuclear Cu(II) unit is embedded in the [gamma-SiW8O31]10- ligand, an unprecedented tetravacant polyoxometalate, showing that partial decomposition of the [gamma-SiW10O36]8- precursor occurs with time in such experimental conditions. Magnetically, complex 1 behaves as two isolated {Cu2(mu(1,1)-N3)2} pairs in which the metal centers are strongly ferromagnetically coupled (J = +224 cm(-1), g = 2.20), the coupling through the W=O bridges being negligible. The magnetic behavior of complex 2 has also been studied. Relatively weak ferromagnetic couplings (J1 = +1.0 cm(-1), J2 = +20.0 cm(-1), g=2.17) have been found inside the {Cu3} units, while the intertrimeric magnetic interactions occurring through the hexadentate azido ligand have been found to be antiferromagnetic (J3 = -5.4 cm(-1)) and ferromagnetic (J4 = +1.3 cm(-1)) with respect to the end-to-end and end-on azido-bridged Cu(II) pairs, respectively.     

Hydrogen bonding: Part 19. IR and NMR study of the lower hydrates of choline fluoride and acetylcholine chloride

Acetylcholine chloride, like choline chloride, forms a liquid salt dihydrate, and a crystalline monohydrate that only exists at reduced pressure; at atmospheric pressure the monohydrate disproportionates into liquid dihydrate and anhydrous acetylcholine chloride. Both choline and acetylcholine chlorides give endothermic dissolution in water. In contrast, choline fluoride gives exothermic dissclution in water, and forms an extra-ordinarily stable monohydrate in which choline cation hydroxyls form strong hydrogen bonds to an H₄O₂F^2?₂ cluster anion. Since the hydration behavior of choline fluoride is like that of unsubstituted tetraalkylammonium fluorides, the unusual hydration behavior of choline and acetyline chlorides results from the presence of chloride ion, and is not an intrinsic property of cholinergic cations.     

Magnetic field induced dipolar couplings in the pretransitional region of a nematic liquid crystal

13C and 1H NMR spectroscopy have been used to study the orientational order which develops when a nematogenic compound, 4-pentyl-4'-cyanobiphenyl (5CB), approaches the transition from the isotropic to the nematic phase at T(NI). The experiments yield values of field induced dipolar couplings, (1), between all of the directly bonded carbon and hydrogen nuclei in the molecule, and 2D(HH)B, the geminal dipolar coupling between protons in the first methylene group in the alkyl chain. The temperature dependence of these couplings shows that in each case they follow a divergence behavior governed by (T - T*)(-1),where T* is a temperature determined from the experimental data and which is close to but less than T(NI). Experiments performed at spectrometer field strengths of 11.75 and 18.79 T confirm the prediction that the induced couplings should depend on the square of the applied field strength. It was found that, within experimental error, T* is the same for each field-induced coupling, and that T(NI) - T* is the same at 11.75 and 18.79 T. It is shown that the set of field-induced couplings 1D(CHi)B obtained at a temperature close to T(NI) can be used to derive a conformer distribution for 5CB in the isotropic phase.     

Nitropyridyl isocyanates

We hereby report the first preparation of 3‐nitro‐4‐pyridyl isocyanate 9 and 5‐nitro‐2‐pyridyl isocyanate 18 . They were formed by Curtius rearrangement of the corresponding acyl azides 8 and 17 , prepared from methyl 3‐nitro‐4‐pyridinecarboxylate 6 via the hydrazide 7 and 5‐nitro‐picolinic acid 16 , respectively. The substrates 6 and 16 were generated by nitration of methyl 4‐pyridinecarboxylate 5 and nitration and oxidation of 2‐picoline 14 . 3‐Nitro‐4‐pyridyl isocyanate 9 can be stored in dry solution and is stable at room temperature for several weeks while 5‐nitro‐2‐pyridyl isocyanate 18 was less stable and should be used for synthetic purposes immediately.