Mass spectrometric fragmentation of cyclic peptides belonging to the polymyxin and colistin antibiotics studied by ion trap and quadrupole/orthogonal-acceleration time-of-flight technology

Electrospray ionization linked to quadrupole/orthogonal-acceleration time-of-flight (Q/oaTOF) and ion trap equipment was used to study the fragmentation behavior of the linear side-chain cyclized peptides of the polymyxin B and E antibiotics. This study exemplifies both the benefits and the drawbacks of mass spectrometric techniques for the determination of the sequence of such complex linear side-chain cyclized peptides. Q/oaTOF accurate mass measurements did not help sufficiently to assign the product ions observed in the product ion spectra. An ion trap mass spectrometer providing MS(n) capability was used to eliminate ambiguities encountered with a single MS/MS approach. The complex fragmentation behavior of these compounds of well-established structure is described which could be useful for structural characterization of unknown substances related to polymyxin B and E in the future.     

Application of liquid chromatography-ion trap mass spectrometry to the characterization of the 16-membered ring macrolide josamycin propionate

Coupled liquid chromatography and ion trap mass spectrometry (LC/MS) was used for the characterization of the semi-synthetic 16-membered ring macrolide josamycin propionate. On-line identification of impurities in this antibiotic complex was performed with an ion trap mass spectrometer without recourse to time-consuming isolation and purification procedures. Ion trap mass spectrometry is ideally suited to identification of impurities because it provides MSn capability, enabling multiple stages of mass spectrometry to obtain the maximum amount of structural information for a given molecule. The ion trap was used with an electrospray ionization source operated in the positive ion mode or with an atmospheric pressure chemical ionization source operated in the negative ion mode. The identity of the unknown compounds was deduced using the MS/MS and MSn collision-induced dissociation spectra of reference substances or structural analogs as interpretative templates, combined with knowledge about the nature of functional group fragmentation behavior. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing josamycin propionate. The knowledge of the fragmentation behavior is also of importance in further research on other 16-membered macrolides.     

Analytical use of immobilized glucose oxidase

With the aim of immobilizing glucose oxidase (GO) for routine determination of glucose, a covalent bond immobilization method on titanium (IV) chloride activated silica supports was used (1). Several parameters were studied in order to optimize the residual activity upon immobilization and during operation. The immobilized enzyme can be reutilized at 25°C for several h a day alternating with storage (4°C) for at least 3,300 h.     

Liquid chromatography-ion trap tandem mass spectrometry for the characterization of polypeptide antibiotics of the colistin series in commercial samples

A selective reversed-phase liquid chromatography-tandem mass spectrometry method is described for the characterization of related substances in the colistin complex. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ionization probe operated in the positive ion mode. The main advantage of this technique is the characterization of novel related substances without time-consuming isolation and purification procedures. Using this method seven new related substances were partially identified in colistin bulk sample and tablets. Four components were assigned as isomers of the main components of colistin.     

Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation

Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.     

1,4-Dihydropyridine/BF3OEt2 for the reduction of imines: Influences of the amount of added BF3OEt2 and the substitution at N-1 and C-4 of the dihydropyridine ring

We have evaluated four 1,4-dihydropyridines (DHPs 1a, 1b, 1c and 1d) as reducing agents, which presented free (hydrogenated) or phenyl-substituted N-1 and C-4 positions of the DHP ring. Reactions combining each of the DHP and different amounts of BF₃OEt₂ were evaluated for the reduction of imine 2a (N-benzylideneaniline). DHP simultaneously substituted at N-1 and C-4 (1a), and DHP substituted at C-4 (1b) gave lower yields for reduction of 2a in comparison with DHPs 1c and 1d (both unsubstituted at the C-4 position). By evaluating the amount of added BF₃OEt₂ to the reaction mixture, we have found that DHP 1c (substituted at N-1) provided its best yield for amine 3a (82%) when associated with stoichiometric amounts BF₃OEt₂, while DHP 1d (N-1- and C-4-unsubstituted derivative) was more effective (90% yield) with catalytic quantities of the Lewis acid. The reaction system using DHP 1c under stoichiometric BF₃OEt₂ could also be successfully applied with additional imine examples and under reductive amination conditions.     

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