Several lignin model compounds were examined to test whether gas-phase ion-molecule reactions of trimethylborate (TMB) in a FTICR can be used to differentiate the ortho-, meta-, and para-isomers of protonated aromatic compounds, such as those formed during degradation of lignin. All three regioisomers could be differentiated for methoxyphenols and hydroxyphenols. However, only the differentiation of the ortho-isomer from the meta- and para-isomers was possible for hydroxyacetophenones and hydroxybenzoic acids. Consideration of the previously reported proton affinities at all basic sites in the isomeric hydroxyphenols, and the calculated proton affinities at all basic sites in the three methoxyphenol isomers, revealed that the proton affinities of the analytes relative to that of TMB play an important role in determining whether and how they react with TMB. The loss of two methanol molecules (instead of one) from the adducts formed with TMB either during ion-molecule reactions, or during sustained-off resonance irradiated collision-activated dissociation of the ion-molecule reaction products, revealed the presence of two functionalities in almost all the isomers. This finding supports earlier results suggesting that TMB can be used to count the functionalities in unknown oxygen-containing analytes.

A mass spectrometric method has been developed for the identification of carbonyl and hydroxyl functional groups, as well as for counting the functional groups, in previously unknown protonated bifunctional oxygen-containing analytes. This method utilizes solution reduction before mass spectrometric analysis to convert the carbonyl groups to hydroxyl groups. Gas-phase ion-molecule reactions of the protonated reduced analytes with neutral trimethylborate (TMB) in a FT-ICR mass spectrometer give diagnostic product ions. The reaction sequence likely involves three consecutive steps, proton abstraction from the protonated analyte by TMB, addition of the neutral analyte to the boron reagent, and elimination of a neutral methanol molecule. The number of methanol molecules eliminated upon reactions with TMB reveals the number of hydroxyl groups in the analyte. Comparison of the reactions of the original and reduced analytes reveals the presence and number of carbonyl and hydroxyl groups in the analyte.

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.

Developing a pharmaceutical product has become increasingly difficult and expensive. With an emphasis on developing project knowledge at an earlier stage in development, the use of information-rich technologies (particularly MS) has continued to expand throughout product development. Continued improvements in LC/MS technology have widened the scope of utilizing MS methods for performing both qualitative and quantitative applications within product development. This review describes a multi-tiered MS strategy designed to enhance and accelerate the identification and profiling of both process- and degradation-related impurities in either the active pharmaceutical ingredient (API) or formulated product. Such impurities can be formed either during chemical synthesis, formulation, or during storage. This review provides an overview of a variety of orthogonal-mass spectrometric methodologies, namely GC/MS, LC/MS, and ICP-MS, in support of product development. This review is not meant to be all inclusive; however, it has been written to highlight the increasing use of hyphenated MS techniques within the pharmaceutical development area.

A mass spectrometric method is presented for the rapid identification of compounds that contain the aromatic N-oxide functional group. This method utilizes a gas-phase ion/molecule reaction with 2-methoxypropene that yields a stable adduct for protonated aromatic tertiary N-oxides (and with one protonated nitrone) in different mass spectrometers. A variety of protonated analytes with O- or N-containing functional groups were examined to probe the selectivity of the reaction. Besides protonated aromatic tertiary N-oxides and one nitrone, only three protonated amines were found to form a stable adduct but very slowly. All the other protonated analytes, including aliphatic tertiary N-oxides, primary N-oxides, and secondary N-oxides, are unreactive toward or react predominantly by proton transfer with 2-methoxypropene.

A mass spectrometric method has been developed for the identification of functional groups in unknown bifunctional oxygen-containing compounds and for the identification and counting of the hydroxyl groups in polyols. This method utilizes gas-phase ion–molecule reactions of protonated analytes with neutral trimethylborate (TMB) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The diagnostic reaction sequence involves proton abstraction from the protonated analyte by TMB, followed by the addition of the analyte to TMB and elimination of methanol. The functional groups in bifunctional oxygen-containing compounds are identified based on the total number of TMB molecules that have added to the protonated analyte and/or the number of methanol molecules lost, or by sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) of the reaction products. The number of hydroxyl groups in polyols is revealed by the number of methanol molecules lost during their reactions with TMB. Reactions of protonated carboxylic acids with TMB also lead to elimination of a methanol molecule. However, carboxylic acids can be differentiated from the isomeric hydroxyketones based on the loss of HO-B(OCH3)2 upon SORI-CAD of the reaction product. Reactions of protonated amides with TMB also lead to elimination of a methanol molecule. However, amides can be differentiated from bifunctional oxygen-containing compounds based on the loss of OBR from the reaction product upon SORI-CAD. Protonated amines do not react with TMB.

A mass spectrometry method is presented for the identification of compounds that contain the primary N-oxide functional group. This method utilizes a gas-phase ion-molecule reaction with dimethyl disulfide that rapidly and selectively derivatizes the protonated primary N-oxide functional group in a mass spectrometer to yield an ionic reaction product (with 31 Da higher mass than that of the protonated molecule) that is diagnostic for the presence of a primary N-oxide functionality. A variety of protonated analytes containing different functional groups were tested in Fourier transform ion-cyclotron resonance and triple quadrupole mass spectrometers to probe the selectivity of the reaction. Only molecules containing the protonated primary N-oxide functional group yielded the diagnostic reaction product; all other protonated molecules gave protonated dimethyl disulfide or no reaction products. The feasibility of this method for compound screening was tested by examining six analytes with the same molecular formula but different atom connectivity. The one analyte that contained the primary N-oxide functional group was readily differentiated from the other analytes.

A mass spectrometric method is described for the identification and counting of hydroxyl groups in an analyte. Analytes introduced into a FT-ICR mass spectrometer and ionized by positive mode ESI were allowed to react with the neutral reagent diethylmethoxyborane. This results in derivatization of the hydroxyl groups of the analytes by replacement of a proton with a diethylborenium ion. Protonated polyols react by consecutive derivatization reactions, wherein all, or nearly all, of the hydroxyls are derivatized. The polyol derivatization products are separated by 68 mass units in the mass spectrum. This 68 Da mass shift, along with 30 Da mass shifts arising from intramolecular derivatization of the primary derivatization products, makes it easy to count the number of functional groups present in the analyte. The utility of this method for the analysis of polyols as single-component solutions, as mixtures, or in HPLC effluent (LC-MS analysis) is demonstrated.

Protonated oxygen-containing monofunctional compounds react with selected methoxyborane reagents by proton transfer followed by nucleophilic substitution of methanol at the boron atom in a Fourier transform ion cyclotron resonance mass spectrometer. The derivatized oxygen functionality can be identified by H/D exchange, collision-activated dissociation, or both. This information on the identity of the functionalities in the analyte, in conjunction with molecular formula information obtained from exact mass measurements on either the protonated or derivatized analyte, facilitates structure elucidation of unknown organic compounds in a mass spectrometer.

Bacterial peptidoglycan is composed of a network of beta-[1,4]-linked glyan strands that are cross-linked through pendant peptide chains. The final product, the murein sacculus, is a single, covalently closed macromolecule that precisely defines the size and shape of the bacterial cell. The recent increase in bacterial resistance to cell wall active agents has led to a resurgence of activity directed toward improving our understanding of the resistance mechanisms at the molecular level. The biosynthetic enzymes and their natural substrates can be invaluable tools in this endeavor. While modern experimental techniques have led to isolation and purification of the biosynthetic enzymes utilized in peptidoglycan biosynthesis, securing useful quantities of their requisite substrates from natural substrates has remained problematic. In an effort to address this issue, we report the first total synthesis of lipid II (4), the final monomeric intermediate utilized by Gram positive bacteria for peptidoglycan biosynthesis.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

[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.

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.

Commercial mass spectrometers have been available for more than 50 years. Mass spectrometry (MS) has been applied widely to organic analysis, and during the last 20 years applications for the characterization of complex biological macromolecules have steadily spiraled upwards. In sharp contrast, today only a handful of polymer laboratories rely on MS. Why? Here we show what modern MS can offer to polymer chemistry and engineering. This review describes MS results on polymers that have been obtained with newer ionization techniques — matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) and by using time-of-flight and Fourier transform mass spectrometers. Examples illustrating structure and molecular mass determination are presented.

High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (Mr 12,231) and porcine pepsin (Mr 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.

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 (> 105 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.

A hybrid mass spectrometer consisting of a magnetic sector, two electric sectors, and a quadrupole mass filter (BEEQ) has been built for the study of polyatomic ion/surface collision phenomena over the energy range of a few electron volts to several keV. Primary ions are generated by electron ionization or by chemical ionization, and the first two sectors are used to deliver a monoenergetic beam of ions, of a selected mass-to-charge m/z ratio, to a decelerator which sets the desired collision energy. The target, which can be introduced into the system without breaking vacuum, is mounted on a goniometer and situated in an electrically shielded region in the center of a large scattering chamber which contains an electric sector and a quadrupole mass analyzer used for kinetic energy and mass measurements on the ejected ions. These analyzers rotate around the scattering center to allow selection of the scattering angle of ions leaving the surface. Ultimate pressures attainable in the main scattering chamber are below 10−9 Torr allowing molecular targets, such as self-assembled monolayers of alkyl thiols on gold, to be examined without surface contamination. Low-energy (20–100 eV) collisions of polyatomic ions are reported, and examples are given of the effects of collision energy and scattering angle on surface induced dissociation mass spectra. The kinetic energy of the inelastically scattered ions is also measured, and in some cases, the internal energy can be estimated, the two measurements together providing information on energy partitioning associated with surface collisions. For example, it is shown that n-butylbenzene molecular ions of 25 eV colliding with ferrocenyl-terminated self-assembled monolayer surfaces, rebound with 10 eV of recoil energy and 3 eV of internal energy. The remainder of the energy goes into the surface. The capability of the BEEQ instrument to provide data on ion/surface reactive collisions is also illustrated with reactions such as alkyl group transfer at self-assembled monolayer surfaces. In addition, data are given showing the ability of the system to provide information on the kinetic energy distributions of ions generated in the course of high-energy collisions at the surface. Mass analysis of the sputtered products provides the instrument with secondary-ion mass spectrometry capabilities.