Structural characterization of a poly(methacrylic acid)-poly(methyl methacrylate) copolymer by nuclear magnetic resonance and mass spectrometry

Mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been combined to achieve the complete microstructural characterization of a poly(methacrylic acid)-poly(methyl methacrylate) (PMAA-PMMA) copolymer synthesized by nitroxide-mediated polymerization. Various PMAA-PMMA species could be identified which mainly differ in terms of terminaisons. ¹H and ¹³C NMR experiments revealed the structure of the end-groups as well as the proportion of each co-monomer in the copolymers. These end-group masses were further confirmed from m/z values of doubly charged copolymer anions detected in the single stage mass spectrum. In contrast, copolymer composition derived from MS data was not consistent with NMR results, obviously due to strong mass bias well known to occur during electrospray ionization of these polymeric species. Tandem mass spectrometry could reveal the random nature of the copolymer based on typical dissociation reactions, i.e., water elimination occurred from any two contiguous MAA units while MAA-MMA pairs gave rise to the loss of a methanol molecule. Polymer backbone cleavages were also observed to occur and gave low abundance fragment ions which allowed the structure of the initiating end-group to be confirmed.     

Characterization of degradation products of poly[(3,3,3-trifluoropropyl)methylsiloxane] by nuclear magnetic resonance spectroscopy, mass spectrometry and gas chromatography

Poly[(3,3,3-trifluoropropyl)methylsiloxane] (PTFPMS) was treated with the solvents acetone, ethyl acetate and methanol and its degradation products were analyzed with multiple techniques. ¹H, ²⁹Si and ¹⁹F nuclear magnetic resonance (NMR) spectroscopy were useful for the characterization of the intact polymers and for the determination of cyclosiloxanes. Cyclosiloxanes with a ring size of up to 23 were quantified by gas chromatography. The only degradation products found were TFPmethyl-cyclosiloxanes. 1,3,5,7-Tetrakis(TFPmethyl)-cyclotetrasiloxane was predominant, and (TFPmethyl)cyclotri-, penta- and hexasiloxane could be detected at lower concentrations. The identity of cyclic degradation products with a ring size of up to 6 was unambiguously confirmed by direct infusion mass spectrometry. The TFPmethyl-cyclosiloxanes were successfully ionized by electrospray ionization in the negative mode. None of the techniques applied gave hints to other degradation products such as short linear oligomers. Almost complete degradation of PTFPMS occurred in acetone and methanol, while degradation is distinctly reduced in ethyl acetate.     

Selective separation and characterization of the stress degradation products of ondansetron hydrochloride by liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Ondansetron hydrochloride was subjected to forced degradation studies under various conditions of hydrolysis (acidic, basic, and neutral), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A simple, selective, precise, and accurate high‐performance liquid chromatography method was developed on a Waters Xterra C₁₈ (150 × 4.6 mm id, 3.5 μm) column using 10 mM ammonium formate (pH 3.0)/methanol as a mobile phase in gradient elution mode at a flow rate of 0.6 mL/min. The method was extended to liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry for identification and structural characterization of stress degradation products of ondansetron. The drug showed significant degradation in base hydrolytic and photolytic stress conditions in the liquid state, while it was found to be stable in neutral, acidic, thermal, and oxidative stress conditions. A total of five degradation products were characterized and most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H]⁺ ions of the drug and its degradation products. Finally, the developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonisation guideline Q2 (R1).     

Structural characterization of chelator-terminated dendrimers and their synthetic intermediates by mass spectrometry

Herein, we report the structural analysis of a novel family of iron-chelating dendrimers and their synthetic intermediates utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization ion trap (ESI IT) MS. These dendrimers share a benzene tricarbonyl core moiety attached to three tripodal branching units, each linking to three terminal groups, ranging from carboxyl, catechol and 3-hydroxy-6-methyl-pyran-4-one moieties and their protected analogs. In order to monitor the progression of dendrimer synthesis, all intermediates and final products were mass analyzed by conventional MALDI-TOF MS with and without alkali metal spiking. For structural characterization, interpretable post-source decay (PSD) and electrospray ionization ion trap MS/MS spectra were obtained from proton, sodium and potassium adducts of the dendrimers. One major route of dendrimer fragmentation was at or adjacent to the amide bonds either of the terminal chelating groups or near the core moiety. Fragmentation in the latter case was primarily at the N-terminal side of the amide bond and was directed by the proximity of a tertiary carbon of the branching unit. Furthermore, it was found that terminal ester, ether and amide linkages to the protecting and chelating groups could be sequentially broken in a single MS/MS spectrum through multiple cleavages resulting in product ions of decreasing intensity. Moreover, such cleavages could also be induced in a stepwise manner in a multistage ion trap MS(n) experiment.     

Structural characterization of poly(amino)ester dendrimers and related impurities by electrospray tandem mass spectrometry

An acid‐terminated poly(amino)ester dendrimer was studied by electrospray ionization tandem mass spectrometry to establish its fragmentation pathways, with the aim of using them to investigate the structure of any defective molecules generated during the dendrimer synthesis. This poly(amino)ester dendrimer could be ionized in both polarities but the most structurally relevant dissociation pathways were found from the deprotonated molecule in negative ion mode. The dissociation pattern of this dendrimer is fully described and supported by accurate mass measurements. The main dissociation reactions of the negatively charged polyacidic dendrimer were shown to consist of (i) the release of carbon dioxide and ethene within a branch, which proceeds as many times as intact neutral branches are available; and (ii) the elimination of an entire dendrimer arm. Monitoring the occurrence of these reactions together with any deviation from these two main routes allowed six major dendritic impurities to be structurally characterized. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of mixtures of biocidal oligoguanidines by capillary electrophoresis and high-performance liquid chromatography coupled to mass spectrometry

The polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) leads to various types of oligomeric guanidines exhibiting a broad spectrum of biocidal activities. In the present work a reversed-phase high-performance liquid chromatographic method with a gradient consisting of aqueous 0.1% trifluoroacetic acid and acetonitrile as mobile phase has been developed to separate these oligoguanidines according to type and chain length. The combination with electrospray mass spectrometry allowed the identification of the various compounds. By this technique, some structures already suggested in the literature could be confirmed, and several additional oligoguanidines not yet reported could be identified. As a complementary technique, capillary zone electrophoresis was investigated. Best results were obtained with carrier electrolytes consisting of phosphoric acid in water/acetonitrile mixtures. Although the number of peaks that could be separated by the electrophoretic method was considerably lower than in case of the chromatographic method, capillary electrophoresis in combination with UV detection at 195 nm may still be a fast method suitable for quantitation of some of the major compounds and for monitoring the reaction rate during the polycondensation reaction.     

Structural elucidation of two novel degradants of lurasidone and their formation mechanisms under free radical-mediated oxidative and photolytic conditions via liquid chromatography-photodiode array/ultraviolet-tandem mass spectrometry and one-dimensional/two-dimensional nuclear magnetic resonance spectroscopy

Lurasidone is an antipsychotic drug clinically used for the treatment of schizophrenia and bipolar disorder. During a mechanism-based forced degradation study of lurasidone, two novel degradation products were observed under free radical-mediated oxidative (via AIBN) and solution photolytic conditions. The structures of the two novel degradants were identified through an approach combining HPLC, LC-MSⁿ (n = 1, 2), preparative HPLC purification and NMR spectroscopy. The degradant formed under the free radical-mediated condition is an oxidative degradant with half of the piperazine ring cleaved to form two formamides; a mechanism is proposed for the formation of the novel N,N′-diformyl degradant, which should be readily applicable to other drugs that contain a piperazine moiety that is widely present in drug molecules. The degradant observed under the solution photolytic condition is identified as the photo-induced isomer of lurasidone with the benzisothiazole ring altered into a benzothiazole ring.     

Structural characterization of alachlor complexes with transition metal ions by electrospray ionization tandem mass spectrometry

Electrospray ionization mass (ESI-MS) spectrometry was used to investigate the nature of metal complexes of alachlor and their dissociations on activation. Ions of the first row transition metal series were employed to react with alachlor and the products were subjected to collision-induced dissociation (CID) for further structural characterization. The formation of diverse complex ions including doubly charged metal/alachlor complexes; [3L + M]²⁺ and [4L + M]²⁺ (L: alachlor and M: transition metal ions) were observed depending on the experimental conditions including the tube lens offset voltage (TLOV) and relative concentrations of alachlor and transition metal ions. It is clear that complexation with transition metal ions alters the reactive site of alachlor, promoting the loss of chlorine over the loss of CH₃OH that is the major reaction pathway in uncomplexed system. Direct elimination of chlorine from alachlor molecule was confirmed by the use of MnBr₂ instead of MnCl₂. These evidences clearly illustrate the catalytic activities of the metal ions through insertion mechanism. The function of transition metal ions in complexation was emphasized comparing the fragmentation patterns with those of protonated molecule. A change in the oxidation state of copper from + 2 to + 1 during the dissociation of metal complex was observed in company with elimination of radicals which is specific for the copper complex ions.     

Identification of a probable new adrenergic agonist by nuclear magnetic resonance and mass spectrometry

In animal production, it is consolidated the synthesis and the illegal use of growth promoters of new generation, able to skip routine screening and confirmatory analysis. In this work it is reported the nuclear magnetic resonance (NMR) and the mass spectrometry identification of a probable new adrenergic drug found in a feed premix. The substance was selectively purified on alpha 1 acid glycoprotein affinity columns; then its structure was first achieved by recording the 13C NMR spectrum that gave the total number of carbons of the molecule, successively sorted by DEPT experiments into quaternary, CH, CH2, and CH3 groups. However, the complete assignments of all resonances were derived from the bi-dimensional analysis and the crucial indications from the 1H-13C reverse experiments. Further characterisation was performed by atmospheric pressure chemical ionisation both in positive and negative ion mode, matching the molecular ion and the fragmentation pattern with those of most recently described new adrenergic agonists. After the loss of a ter-butylic group, the structure shows an internal symmetry along with the presence of Chlorine clusters. The proposed formula of the compound, the 8,8'-diamino-9,9'-dichloro-1-terbutyl-1,1',4,4-tetrahydro-5H,5'H-2,2'-bi-1-benzazepine-5,5'-dione, partially resembles that of Zilpaterol for the presence of a heterocyclic ring; Further work is in progress to characterise the structure-activity relationship.     

Separation and characterization of superoxide dismutase 1 (SOD-1) from human erythrocytes by capillary electrophoresis time-of-flight mass spectrometry

Cu, Zn-superoxide dismutase (SOD-1) is a homodimeric metalloenzyme that has been related to ALS (amyotrophic lateral sclerosis). The majority of ALS cases are sporadic while approximately 10% are inherited (familial ALS, FALS). Mutations in the amino acid sequence of human SOD-1 cause only 25% of the FALS cases, while the explanation for the rest is not clear yet. In this way, several authors have suggested the importance of posttranslational modifications or dimer dissociation on formation of the characteristic fatal intraneuronal SOD-1 aggregates. In this paper, we used capillary electrophoresis-electrospray mass spectrometry with an accurate mass and high-resolution time-of-flight mass spectrometer (CE-TOF-MS) for separation and characterization of standard bovine SOD-1 and human SOD-1 purified from erythrocytes. Two background electrolytes (BGEs) were used for CE-TOF-MS experiments in positive ion mode. An acidic BGE allowed detection of apo-monomer SOD-1, because the metal ions were completely released during the electrophoretic separation. The better sensitivity at acidic pH was especially interesting to detect different isoforms of human SOD-1. In contrast, a neutral BGE provided enhanced conditions for detection of the fully metalated dimeric and monomeric enzyme, but selecting an appropriate fragmentor voltage value in the TOF analyzer was critical to obtain reliable quantitative information. Anyway, only the metalated forms involving the main isoform of human SOD-1 were detected due to the lower sensitivity. Hence, the combination of both methodologies resulted necessary to obtain detailed structural information from the enzyme.     

Ultratrace analysis and isotope ratio measurements of long-lived radioisotopes by resonance ionization mass spectrometry (RIMS)

Resonance Ionization Mass Spectrometry (RIMS) is a sensitive and selective method for ultratrace analysis of long-lived radioisotopes and isotope ratio measurements. It provides extremely high isobaric suppression and good overall efficiency. The experimental limits of detection are as low as 10⁶ atoms per sample and isotopic selectivities of 5×10¹² have been obtained. The widespread potential of RIMS, using different experimental arrangements, is demonstrated for the determination of the radiotoxic isotopes Pu-238 to Pu-244 and Sr-89/Sr-90 in various environmental samples as well as for Ca-41 in nuclear reactor components and biomedical samples.     

Reactive desorption electrospray ionization mass spectrometry (DESI-MS) of natural products of a marine alga

Presented here is the optimization and development of a desorption electrospray ionization mass spectrometry (DESI-MS) method for detecting natural products on tissue surfaces. Bromophycolides are algal diterpene-benzoate macrolide natural products that have been shown to inhibit growth of the marine fungal pathogen Lindra thalassiae. As such, they have been implicated in antimicrobial chemical defense. However, the defense mechanisms are not yet completely understood. Precise detection of these compounds on algal tissue surfaces under ambient conditions without any disruptive sample processing could shed more light onto the processes involved in chemical defense of marine organisms. Conventional DESI-MS directly on algal tissue showed relatively low sensitivity for bromophycolide detection. Sensitivity was greatly improved by the addition of various anions including Cl⁻, Br⁻, and CF₃COO⁻ into the DESI spray solvent. Chloride adduction gave the highest sensitivity for all assayed anions. Density functional optimization of the bromophycolide anionic complexes produced during DESI supported this observation by showing that the chloride complex has the most favorable binding energy. Optimized DESI protocols allowed the direct and unambiguous detection of bromophycolides, including A, B, and E, from the surface of untreated algal tissue. Figure Desorption Electrospray Ionization, a novel technique for mass spectrometric analysis under open air conditions reveals the presence of naturally-occurring antibiotics on the surface of marine algae. Ab-initio calculations and experimental results indicate that sensitiviity could be greatly enhanced by means of dynamic complexation of these antibiotics with various small anions during the dynamic desorption process. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Determination of fission products in nuclear samples by capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS)

Determination of fission products and of their isotopic composition is of high importance for the characterisation and complete inventory of nuclear fuels. Radiometric and mass spectrometric methods, which are currently used, enable only an incomplete determination of the fission products. Radiometric methods cannot be applied to all fission products and mass spectrometric methods are hindered by the existence of isobaric interferences, therefore a previous chemical separation is required before mass spectrometric analysis. Capillary electrophoresis (CE) has been coupled with inductively coupled plasma mass spectrometry (both ICP-QMS and ICP-SFMS). Typical detection limits of 6 ng/mL and 4 pg/mL for caesium as well as 8 ng/mL and 7 pg/mL for lanthanides have been obtained by CE-ICP-QMS and CE-ICP-SFMS, respectively. In addition to these very low detection limits, the procedures present the advantages to be fast (6 min for caesium and 13 min for lanthanides, respectively), to require a low microliter range sample volume and a nanoliter range injection volume. The radiation dose for the personnel as well as the volume of nuclear liquid wastes generated during the measurements are consequently reduced.The procedures have been applied to nuclear samples from PUREX process and leachates from MOX fuels.     

Structural deviations in poly(amidoamine) dendrimers: a MALDI-TOF MS analysis

A step-by-step synthesis/purification (CC, HILIC, HPLC) of poly(amidoamine) PAMAM dendrimers was performed. MALDI-TOF MS in the linear and reflectron mode was used to analyze the purified samples and byproduct samples of G0-G5 generations of the dendrimers up to the mass of 35 000 Da. DHB/fucose was found to give the best resolution, causing the least fragmentation of the samples. The precise mass number for the ideally branched dendrimers and their “structural errors” was obtained. The profile of the structural errors was established.     

Identification of 19 phthalic acid esters in dairy products by gas chromatography with mass spectrometry

A detection method for 19 kinds of phthalic acid ester compounds analyzed by n‐hexane/ether/acetonitrile 1:7:8 v/v/v mixed solvent extraction, quick, easy, cheap, effective, rugged, and safe purification and internal standard method of quantitative gas chromatography with mass spectrometry was established. This method can effectively remove interfering materials, such as lipids, fatty acids, and pigments, from dairy products. The 19 kinds of phthalic acid ester compounds were within a 0.025–0.2 mg/kg range, the recovery rate was 65.2–125.7%, relative standard deviation was 7.9–15.4% (n = 6), and the limit of detection was 0.005–0.02 mg/kg. Concentrations of the 19 kinds of phthalic acid ester compounds ranged between 0.01 and 0.12 mg/kg in ten dairy materials and 20 dairy products. The established method is simple, rapid, accurate, and highly sensitive.     

Evaluation of false positive responses by mass spectrometry and ion mobility spectrometry for the detection of trace explosives in complex samples

Secondary electrospray ionization-ion mobility-time of flight mass spectrometry (SESI-IM-TOFMS) was used to evaluate common household products and food ingredients for any mass or mobility responses that produced false positives for explosives. These products contained ingredients which shared the same mass and mobility drift time ranges as the analyte ions for common explosives. The results of this study showed that the vast array of compounds in these products can cause either mass or mobility false positive responses. This work also found that two ingredients caused either enhanced or reduced ionization of the target analytes. Another result showed that an IMS can provide real-time separation of ion species that impede accurate mass identifications due to overlapping isotope peak patterns. The final result of this study showed that, when mass and mobility values were used to identify an ion, no false responses were found for the target explosives. The wider implication of these results is that the possibility exists for even greater occurrences of false responses from complex mixtures found in common products. Neither IMS nor MS alone can provide 100% assurance from false responses. IMS, due to its low cost, ease of operation, rugged reliability, high sensitivity and tunable selectivity, will remain the field method of choice for the near future but, when combined with MS, can also reduce the false positive rate for explosive analyses.     

Synthesis and studies of Rh(I) catalysts within and across poly(alkyl aryl ether) dendrimers

In order to study the efficiencies of catalytic moieties within and across dendrimer generations, partially and fully functionalized dendrimers were synthesized. Poly(alkyl aryl ether) dendrimers from zero to three generations, presenting 3 to 24 peripheral functionalities, were utilized to prepare as many as 12 catalysts. The dendrimer peripheries were partially and fully functionalized with triphenylphosphine in the first instance. A rhodium(I) metal complexation was performed subsequently to afford multivalent dendritic catalysts, both within and across generations. Upon synthesis, the dendritic catalysts were tested in the hydrogenation of styrene, in a substrate-to-catalyst ratio of 1:0.001. Turn-over-numbers were evaluated for each catalyst, from which significant increases in the catalytic activities were identified for multivalent catalysts than monovalent catalysts, both within and across generations.     

Study of the forced degradation behavior of prasugrel hydrochloride by liquid chromatography with mass spectrometry and liquid chromatography with NMR detection and prediction of the toxicity of the characterized degradation products

Prasugrel was subjected to forced degradation studies under conditions of hydrolysis (acid, base, and neutral), photolysis, oxidation, and thermal stress. The drug showed liability in hydrolytic as well as oxidative conditions, resulting in a total of four degradation products. In order to characterize the latter, initially mass fragmentation pathway of the drug was established with the help of mass spectrometry/time‐of‐flight, multiple stage mass spectrometry and hydrogen/deuterium exchange data. The degradation products were then separated on a C₁₈ column using a stability‐indicating volatile buffer method, which was later extended to liquid chromatography‐mass spectrometry studies. The latter highlighted that three degradation products had the same molecular mass, while one was different. To characterize all, their mass fragmentation pathways were established in the same manner as the drug. Subsequently, liquid chromatography‐nuclear magnetic resonance (NMR) spectroscopy data were collected. Proton and correlation liquid chromatography with NMR spectroscopy studies highlighted existence of diastereomeric behavior in one pair of degradation products. Lastly, toxicity prediction by computer‐assisted technology (TOPKAT) and deductive estimation of risk from existing knowledge (DEREK) software were employed to assess in silico toxicity of the characterized degradation products.     

Determination of pyrethroids and their degradation productds in indoor air and on surfaces by HRGC-ECD and HRGC-MS(NCI)

Air and surface samples taken following indoor applications of natural pyrethrins and pyrethroids have been analyzed by HRGC-ECD and HRGC-MS using negative chemical ionization (NCI). It has been found that pyrethroids persist on surfaces as long as sixty hours after application. Primary pyrethroid degradation products have been characterized by mass spectrometry; their identities and route of formation by ozonolysis have been verified by degradation studies and NMR.     

Synthesis and reactivity profiles of phosphinated poly(alkyl aryl ether) dendrimers

Poly(alkyl aryl ether) dendrimers were utilized to synthesize a series of new triphenylphosphine functionalized dendrimers. Zero, first, second and third generation dendrimers, carrying 3, 6, 12 and 24 triphenylphosphine units, were prepared and characterized. The new triphenylphosphine containing dendrimers were assessed for their reactivity profiles and in this instance, the dendrimers were used as reagents to mediate Mitsunobu etherification reaction between phenol and various primary, secondary and benzylic alcohols. In addition, dendritic poly-phenols were also tested in an O-benzylation reaction. A monomeric methoxy group attached triphenylphosphine acted as a control for comparison of reactivity profiles of dendrimers. It was observed that the etherification reaction was mediated efficiently by the dendritic reagent, and in addition, the dendritic phosphine oxide reagents could be recovered quantitatively by precipitation methods. The recovered dendritic phosphine oxides were reduced subsequently to the corresponding phosphines and used as reagents for the Mitsunobu reaction, repetitively.