Artifactual formylation of the secondary amine of duloxetine hydrochloride by acetonitrile in the presence of titanium dioxide: implications for HPLC method development.

Duloxetine hydrochloride, a secondary amine containing pharmaceutical, currently marketed as Cymbalta, is shown to undergo N-formylation as an artifact of sample preparation prior to HPLC analysis for impurities. The reaction was discovered as a result of an investigation into variability in the levels of N-formyl duloxetine observed upon HPLC analysis. The reaction is catalyzed by sonication and/or light in the presence of titanium dioxide and is proposed to occur via a radical-initiated mechanism. The mechanism is supported by controlled sample preparation studies with deuterium-labeled acetonitrile and LC/MS studies that showed incorporation of one deuterium into N-formyl duloxetine. This discovery is broadly relevant because sonication is commonly used to aid dissolution of pharmaceuticals in acetonitrile for HPLC analysis, titanium dioxide is a commonly used excipient, the amount of light found in modern analytical laboratories is sufficient to cause the reaction to occur, and secondary amines are present in the structures of many pharmaceuticals. The artifactual reaction was effectively eliminated by changing the sample solvent to methanol and replacing sonication with shaking to aid sample dissolution.

High-nuclearity close-packed palladium-nickel carbonyl phosphine clusters: heteropalladium.

[Pd(16)Ni(4)(CO)(22)(PPh(3))(4)](2)(-) (1) and [Pd(33)Ni(9)(CO)(41)(PPh(3))(6)](4)(-) (2) were obtained as the two major products from the reduction of PdCl(2)(PPh(3))(2) with [Ni(6)(CO)(12)](2)(-). Their crystal structures as [PPh(4)](+) salts were unambiguously determined from CCD X-ray crystallographic analyses; the resulting stoichiometries were ascertained from elemental analyses. Infrared, multinuclear (1)H, (31)P[(1)H] NMR, UV-vis, CV, variable-temperature magnetic susceptibility, and ESI FT/ICR mass spectrometric measurements were performed. The Pd(16)Ni(4) core of 1 ideally conforms to a ccp nu(3) tetrahedron of pseudo-T(d)() (4 3m) symmetry. Its geometry normal to each tetrahedral Pd(7)Ni(3) face (i.e., along each of the four 3-fold axes) may be viewed as a four-layer stacking of 20 metal atoms in a ccp [a(Ni(1)) b(Pd(3)) c(Pd(6)) a(Pd(7)Ni(3))] sequence. A comparative analysis of the different ligand connectivities about the analogous metal-core geometries in 1 and the previously reported [Os(20)(CO)(40)](2)(-) has stereochemical implications pertaining to the different possible modes of carbon monoxide attachment to ccp metal(111) surfaces. The unique geometry of the Pd(33)Ni(9) core of 2, which has pseudo-D(3)(h)() (6 2m) symmetry, consists of five equilateral triangular layers that are stacked in a hcp [a(Pd(7)Ni(3)) b(Pd(6)) a(Pd(7)Ni(3)) b(Pd(6)) a(Pd(7)Ni(3))] sequence. Variable-temperature magnetic susceptibility measurements indicated both 1 and 2 to be diamagnetic over the entire temperature range from 5.0 to 300 K. Neutral Pd(12)(CO)(12)(PPh(3))(6) (3) and [Pd(29)(CO)(28)(PPh(3))(7)](2)(-) (4) as the [PPh(4)](+) salt were obtained as minor decomposition products from protonation reactions of 1 and 2, respectively, with acetic acid. Compound 3 of pseudo-D(3)(d)() (3 2/m) symmetry represents the second highly deformed hexacapped octahedral member of the previously established homopalladium family of clusters containing uncapped, monocapped, bicapped, and tetracapped Pd(6) octahedra. The unprecedented centered 28-atom polyhedron for the Pd(29) core of 4 of pseudo-C(3)(v)() (3m) symmetry may be described as a four-layer stacking of 29 metal atoms in a mixed hcp/ccp [a(Pd(1)) b(Pd(3)) a(Pd(10)) c(Pd(15))] sequence.

Characterization of combinatorial peptide libraries by electrospray ionization Fourier transform mass spectrometry.

The advantages of Fourier transform mass spectrometry (FTMS) are precision high mass accuracy measurements and the capability of high resolution, multistage mass spectrometry together with a number of other advanced features. These powerful facilities can be used to rapidly screen complex mixtures without the necessity of chromatographic separations. The example shown here illustrates the use of the high resolving power and accurate mass capabilities of FTMS for the rapid, direct analysis of a complex mixture, which had been ionized by direct infusion electrospray ionization.

Proton transfer reaction studies of multiply charged proteins in a high mass-to-charge ratio quadrupole mass spectrometer.

Proton transfer reactions of multiply charged ions at high mass-to-charge ratios were explored with a low frequency quadrupole mass spectrometer. This instrument enabled a qualitative comparison of proton transfer reaction rates at low charge states for ions generated by electrospray ionization (ESI) from different solution conformations and for disulfide-linked versus disulfide-reduced protein ions. Proton transfer reactions that efficiently reduced the number of charges for ESI-generated ions to approximately the number of arginines in the polypeptide sequence were observed. No significant differences in gas-phase reaction rates were noted between different solution conformers. Differences in reaction rates between "native" and disulfide-reduced proteins were much smaller than those observed below m/z 2000 with lower proton affinity reagents or by using lower reagent concentrations. These smaller differences in reaction rates are thought to reflect the reduced electrostatic contributions from widely spaced charge sites and thus, the reduced sensitivi ty to an ion's three-dimensional structure or U compactness.

New developments in microscale separations and mass spectrometry for biomonitoring: capillary electrophoresis and electrospray ionization mass spectrometry.

In this article, we briefly highlight the use of capillary electrophoresis for sampling, manipulating, and separating extremely small sample sizes. The extraordinary sensitivity that can be obtained by combined capillary electrophoresis-mass spectrometry is then demonstrated using recent results. We briefly describe the ability to detect noncovalently associated complexes (e.g., double-stranded DNA) by electrospray ionization-mass spectrometry, and conclude with recent results that show the potential for using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry for characterization of biomolecules.

Observation and implications of high mass-to-charge ratio ions from electrospray ionization mass spectrometry.

High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (M r 12,231) and porcine pepsin (M r 34,584), by using a quadrupole mass spectrometer with an m/z 45,000 range. The ESI mass spectrum for cytochrome c in an aqueous solution gives a charge state distribution that ranges from 12 + to 2 +, with a broad, low-intensity peak in the mass-to-charge ratio region corresponding to the [M + H](+) ion. the negative ion ESI mass spectrum for pepsin in 1% acetic acid solution shows a charge state distribution ranging from 7- to 2-. To observe the [M - H](-) ion, harsher desolvation and interface conditions were required. Also observed was the abundant aggregation of the protens with average charge states substantially lower than observed for their monomeric counterparts. The negative ion ESI mass spectrum for cytochrome c in 1-100 mM NH4OAc solutions showed greater relative abundances for the higher mass-to-charge ratio ions than in acuidic solutions, with an [M - H](-) ion relative abundance approximately 50% that of the most abundant charge state peak. The observation that protein aggregates are formed with charge states comparable to monomeric species (at fower mass-to-charge ratios) suggests that the high mass-to-charge ratio monomers may be formed by the dissociation of aggregate species. The observation of low charge state and aggregate molecular ions concurrently with highly charged species may serve to support a variation of the charged residue model, originally described by Dole and co-workers (Dole, M., et al. J. Chem. Phys. 1968, 49, 2240; Mack, L. L., et al. J. Chem. Phys. 1970, 52, 4977) which involves the Coulombically driven formation of either very highly solvated molecular ions or lower ananometer-diameter droplets.

High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer.

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10(-9) torr. The increased pumping speed attainable with cryopumping (> 10(5) L/s) allowed brief pressure excursions to above 10(-4) torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10-25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (Mr 5734) and ubiquitin (Mr 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H3PO4 adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4(+) charge state (m/z 1434) of insulin.

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions.

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H](15+) ion being the most intense peak. With the addition of H2O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.