Development and characterization of a small electromembrane extraction probe coupled with mass spectrometry for real-time and online monitoring of in vitro drug metabolism

A small and very simple electromembrane extraction probe (EME-probe) was developed and coupled directly to electrospray ionization mass spectrometry (ESI-MS), and this system was used to monitor in real time in vitro metabolism by rat liver microsomes of drug substances from a small reaction (incubation) chamber (37 °C). The drug-related substances were continuously extracted from the 1.0 mL metabolic reaction mixture and into the EME-probe by an electrical potential of 2.5 V. The extraction probe consisted of a 1-mm long and 350-μm ID thin supported liquid membrane (SLM) of 2-nitrophenyl octyl ether. The drugs and formed metabolites where extracted through the SLM and directly into a 3 μL min^?1 flow of 60 mM HCOOH inside the probe serving as the acceptor solution. The acceptor solution was directed into the ESI-MS-system, and the MS continuously monitored the drug-related substances extracted by the EME-probe. The extraction efficiency of the EME-probe was dependant on the applied electrical potential and the length of the SLM, and these parameters as well as the volume of the reaction chamber were set to the values mentioned above to avoid serious depletion from the reaction chamber (soft extraction). Soft extraction was mandatory in order not to affect the reaction kinetics by sample composition changes induced by the EME-probe. The EME-probe/MS-system was used to establish kinetic profiles for the in vitro metabolism of promethazine, amitriptyline and imipramine as model substances.     

Spectral properties and absolute rate constants for beta-scission of ring-substituted cumyloxyl radicals. A laser flash photolysis study

A laser flash photolysis study of the spectral properties and beta-scission reactions of a series of ring-substituted cumyloxyl radicals has been carried out. All cumyloxyl radicals display a broad absorption band in the visible region of the spectrum, which decays on the microsecond time scale, leading to a strong increase in absorption in the UV region of the spectrum, which is attributed to the corresponding acetophenone formed after beta-scission of the cumyloxyl radicals. The position of the visible absorption band is red-shifted by the presence of electron-donating ring substituents, while a blue-shift is observed in the presence of electron-withdrawing ring substituents, suggesting that + R ring substituents promote charge separation in the excited cumyloxyl radical through stabilization of the partial positive charge on the aromatic ring of an incipient radical zwitterion. Along this line, an excellent Hammett-type correlation between the experimentally measured energies at the visible absorption maxima of the cumyloxyl radicals and sigma(+) substituent constants is obtained. A red-shift is also observed on going from MeCN to MeCN/H(2)O for all cumyloxyl radicals, pointing toward a specific effect of water. The ring substitution does not influence to a significant extent the rate constants for beta-scission of the cumyloxyl radicals, which varies between 7.1 x 10(5) and 1.1 x 10(6) s(-1), a result that suggests that cumyloxyl radical beta-scission is not governed by the stability of the resulting acetophenone. Finally, k(beta) increases on going from MeCN to the more polar MeCN/H(2)O 1:1 for all cumyloxyl radicals, an observation that reflects the increased stabilization of the transition state for beta-scission through increased solvation of the incipient acetophenone product.     

Role of nearby charges on the electronic structure of π-conjugated molecules: symmetric versus asymmetric charge distributions in oligo(p-phenyleneethynylene)

Oligo(p-phenyleneethynylene)s (OPEs) are conjugated oligomers of great interest within materials science and molecular electronics on account of their highly applicable electronic and optical properties. Here we use gas-phase action spectroscopy to elucidate how the intrinsic electronic properties of these chromophores are affected by nearby charges. An OPE3 chromophore with two nearby ammonium groups was synthesized. This molecule and a related OPE3 with only one amine protonation site were transferred to the gas phase by electrospray ionization and subjected to action spectroscopy. Ions were bunched in a 14-pole ion trap, accelerated to 50-keV kinetic energies, mass-to-charge selected by a magnet, and photoexcited in a crossed-beam configuration. Fragment ions were finally mass-analyzed by an electrostatic analyzer. The setup enables photodissociation mass spectrometry and action spectroscopy on the microsecond time scale. The gas-phase absorption of the mono- and dication was measured and compared to that of neutral chromophores in solution. Similar absorption was found for neutral chromophores (in solution) and the dication (in gas phase or solution), whereas the monocation absorbs at lower energies in the gas phase. Simple electrostatic considerations lead to an energy difference like the one found from the experiment. The work presented here addresses how the electronic properties of a π-conjugated system are affected by nearby charges, a question of fundamental interest in, for example, molecular electronics.     

PRIMARY DNA DAMAGE, HPRT MUTATION AND CELL INACTIVATION PHOTOINDUCED WITH VARIOUS SENSITIZERS IN V79 CELLS

DNA strand breaks and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants were measured in parallel in photochemically treated (PCT) cells and compared at the same level of cell survival. Chinese hamster fibroblasts (V79 cells) were either incubated with the lipophilic dyes tetra(3—hydroxyphenyl)porphyrin (3THPP) and Photofrin II (PII), the anionic dye meso -tetra(4—sulfonatophenyl)porphine (TPPS₄) or the cationic dye meso -tetra( N -methyl-4-pyridyl)porphine ( p -TMPyPH₂ before light exposure. In the cells, the lipophilic dyes were localized in membranes, including the nuclear membrane, while the hydrophilic dyes were taken up primarily into spots in the cytoplasm. In addition, the hydrophilic TPPS₄ was distributed homogeneously throughout the whole cytoplasm and nucleoplasm. According to the HPRT mutation test, the mutagenicity of light doses survived by 10% of the cells was a factor of six higher in the presence of 3THPP than of PII, whereas for X-rays it was a factor of three higher than for PCT with 3THPP. Light exposure in the presence of the hydrophilic dyes TPPS₄ and p -TMPyPH₂ was not significantly mutagenic. There was no correlation between the induced rates of HPRT mutants and of DNA strand breaks. Thus, TPPS₄ was the most efficient sensitizer with regard to DNA strand breaks when compared at the same level of cell survival, followed by 3THPP, PII and p -TMPyPH₂. Hence, the rate of DNA strand breaks cannot be used to predict the mutagenicity of PCT.     

Absorption Spectra of 4‐Nitrophenolate Ions Measured in Vacuo and in Solution

From blue to red: While four π‐conjugated nitrophenolates absorb within a relatively narrow region in solution, they cover the entire visible spectrum when isolated in vacuo (see picture). The work combines gas‐ and solution‐phase spectroscopy and provides the first benchmark of theoretical excitation energies for nitrophenolates.

     

Chemical ionization of phenyl n-propyl ether and methyl substituted analogs: Propene loss initiated by competing proton transfer to the oxygen atom and the aromatic ring

The mechanism of propene loss from protonated phenyl n-propyl ether and a series of mono-, di-, and trimethylphenyl n-propyl ethers has been examined by chemical ionization (CI) mass spectrometry in combination with tandem mass spectrometry experiments. The role of initial proton transfer to the oxygen atom and the aromatic ring, respectively, has been probed with the use of deuterated CI reagents, D₂O, CD₃OD, and CD₃CN (given in order of increasing proton affinity), in combination with deuterium labeling of the β position of the n-propyl group or the phenyl ring. The metastable [M + D]⁺ ions of phenyl n-propyl ether—formed with D₂O as the CI reagent—eliminate C₃H₅D and C₃H₆ in a ratio of 10:90, which indicates that the added deuteron is incorporated to a minor extent in the expelled neutral species. In the experiments with CD₃OD as the CI reagent, the ratio between the losses of C₃H₅D and C₃H₆ from the metastable [M + D]⁺ ions of phenyl n-propyl ether is 18:82, whereas the ratio becomes 27:73 with CD₃CN as the reagent. A similar trend in the tendency to expel a propene molecule that contains the added deuteron is observed for the metastable [M + D]⁺ ions of phenyl n-propyl ether labeled at the β position of the alkyl group. Incorporation of a hydrogen atom that originates from the aromatic ring in the expelled propene molecule is of negligible importance as revealed by the minor loss of C₃H₅D from the metastable [M + H]⁺ ions of C₆D₅OCH₂CH₂CH₃ irrespective of whether H₂O, CH₃OH, or CH₃CN is the CI reagent. The combined results for the [M + D]⁺ ions of phenyl n-propyl ether and deuterium-labeled analogs are suggested to be in line with a model that assumes that propene loss occurs not only from species formed by deuteron transfer to the oxygen atom, but also from ions generated by deuteron transfer to the ring. This is substantiated by the results for the methyl-substituted ethers, which reveal that the position as well as the number of methyl groups bonded to the ring exert a marked effect on the relative importances of the losses of C₃H₅D and C₃H₆ from the metastable [M + D]⁺ ions of the unlabeled methyl-substituted species.