Performance Characteristics of Mass Spectrometry-Based Analytical Procedures for Quantitation of Nitrosamines in Pharmaceuticals: Insights from an Inter-laboratory Study.

With the discovery of carcinogenic nitrosamine impurities in pharmaceuticals in 2018 and subsequent regulatory requirements for risk assessment for nitrosamine formation during pharmaceutical manufacturing processes, storage or from contaminated supply chains, effective testing of nitrosamines has become essential to ensure the quality of drug substances and products. Mass spectrometry has been widely applied to detect and quantify trace amounts of nitrosamines in pharmaceuticals. As part of an effort by regulatory authorities to assess the measurement variation in the determination of nitrosamines, an inter-laboratory study was performed by the laboratories from six regulatory agencies with each of the participants using their own analytical procedures to determine the amounts of nitrosamines in a set of identical samples. The results demonstrated that accurate and precise quantitation of trace level nitrosamines can be achieved across multiple analytical procedures and provided insight into the performance characteristics of mass spectrometry-based analytical procedures in terms of accuracy, repeatability and reproducibility.

Furan-oxidation-triggered inducible DNA cross-linking: acyclic versus cyclic furan-containing building blocks--on the benefit of restoring the cyclic sugar backbone.

Oligodeoxynucleotides incorporating a reactive functionality can cause irreversible cross-linking to the target sequence and have been widely studied for their potential in inhibition of gene expression or development of diagnostic probes for gene analysis. Reactive oligonucleotides further show potential in a supramolecular context for the construction of nanometer-sized DNA-based objects. Inspired by the cytochrome P450 catalyzed transformation of furan into a reactive enal species, we recently introduced a furan-oxidation-based methodology for cross-linking of nucleic acids. Previous experiments using a simple acyclic building block equipped with a furan moiety for incorporation into oligodeoxynucleotides have shown that cross-linking occurs in a very fast and efficient way and that substantial amounts of stable, site-selectively cross-linked species can be isolated. Given the destabilization of duplexes observed upon introduction of the initially designed furan-modified building block into DNA duplexes, we explore here the potential benefits of two new building blocks featuring an extended aromatic system and a restored cyclic backbone. Thorough experimental analysis of cross-linking reactions in a series of contexts, combined with theoretical calculations, permit structural characterization of the formed species and allow assessment of the origin of the enhanced cross-link selectivity. Our experiments clearly show that the modular nature of the furan-modified building blocks used in the current cross-linking strategy allow for fine tuning of both yield and selectivity of the interstrand cross-linking reaction.

Increased prevalence of cardiovascular and autoimmune diseases in periodontitis patients: a cross-sectional study.

BACKGROUND: Associations between periodontitis and cardiovascular and autoimmune diseases are most often assessed in patients with a particular cardiovascular or autoimmune disease. To prevent selection bias, this study assesses the existence of associations between periodontitis and cardiovascular and autoimmune diseases in patients attending a dental or periodontal clinic. METHODS: Data were collected from 1,276 randomly selected dental records from patients attending a dental (n = 588) or periodontal (n = 688) clinic. Data on the prevalence of cardiovascular and autoimmune diseases were obtained from a validated health questionnaire. Data on the presence of periodontitis were taken from patients' dental records. RESULTS: In uncontrolled analyses, the prevalence of hypertension, diabetes mellitus (DM), and rheumatoid arthritis (RA) is significantly increased in patients with periodontitis. Controlled for confounding, periodontitis was associated with DM, with an odds ratio of 4 (1.03 to 15.3), in the dental clinic. DM was not associated with periodontitis in periodontal clinics. Hypertension does not seem to be associated with periodontitis when controlling for confounders. Periodontitis may be associated with RA in both clinic types. CONCLUSIONS: The increased prevalence of cardiovascular and autoimmune diseases among patients with periodontitis attending dental or periodontal clinics may, at least in part, be influenced by confounding. However, the increased prevalence of DM and RA in patients with periodontitis could not be explained by confounding.

The recolonization hypothesis in a full-mouth or multiple-session treatment protocol: a blinded, randomized clinical trial.

AIM: To test recolonization of periodontal lesions after full-mouth scaling and root planing (FM-SRP) or multiple session-SRP (MS-SRP) in a randomized clinical trial and whether FM-SRP and MS-SRP result in different clinical outcomes. MATERIALS AND METHODS: Thirty-nine subjects were randomly assigned to FM-SRP or MS-SRP groups. At baseline and after 3 months, probing pocket depth (PPD), plaque index (PlI) and bleeding on probing (BoP) were recorded. At baseline, immediately after treatment, after 1, 2, 7, 14 and 90 days, paper point samples from a single site from the maxillary right quadrant were collected for microbiological analysis of five putative pathogens by polymerase chain reaction. RESULTS: FM-SRP and MS-SRP resulted in significant reductions in PPD, BoP and PlI and the overall detection frequencies of the five species after 3 months without significant differences between treatments. Compared with MS-SRP, FM-SRP resulted in less recolonization of the five species, significantly for Treponema denticola, in the tested sites. CONCLUSION: FM-SRP and MS-SRP result in overall clinically and microbiologically comparable outcomes where recolonization of periodontal lesions may be better prevented by FM-SRP.

Mass spectrometry of oligonucleotides.

Expanding research in the field of modified oligonucleotides demands suitable analytical tools for size and purity verification of known compounds and accurate structure elucidation of unknowns. There is a need for characterization of the types and sites of modifications in oligonucleotides and to identify and sequence selected candidates originating from synthesis. The potential of electrospray tandem mass spectrometry (ESI-MS/MS) for structural characterization and sequencing of oligonucleotides is demonstrated. The fundamental behavior of DNA, RNA, and selected modified oligonucleotides in gas-phase is shown. Since gas-phase dissociation does not demand specific structural prerequisites, the method bears a great potential for rapid and most accurate characterization of modified oligonucleotides, e.g. from combinatorial libraries.

Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides.

Antisense oligonucleotides and aptamers are important candidates for future therapeutic applications. Different structural modifications are introduced into oligonucleotides to obtain high affinity and binding specificity. Sequence elucidation of oligonucleotides incorporating a wide variety of modifications presents an analytical challenge, as the standard protocols cannot be applied. Mass spectrometry has the potential to solve complex structural problems. However, a better understanding of the fundamental aspects of gas-phase dissociation of modified DNA and RNA is needed. In this work the influence of specific chemical modifications on backbone dissociation is pointed out. Biphenyl-modified oligo(deoxy)ribonucleotides, which incorporate C-glycosidic bound abasic nucleobase substitutes, were subjected to collision-induced dissociation in an electrospray tandem mass spectrometer, with the goal to investigate the role of nucleobase loss on backbone dissociation. DNA bearing biphenyl nucleobase substitutes show abundant [a-B]- and w-ions generated by cleavage of the 3'-C-O bonds, except for the phosphodiester groups adjacent to the biphenyl modifications. At these positions no dissociation was observed, demonstrating the dependence of DNA backbone dissociation on nucleobase loss. Also, no evidence for a base loss independent mechanism responsible for formation of w-ions was found. RNA incorporating biphenyl nucleobase substitutes fragment into c- and y-ions resulting from cleavage of the 5'-P-O bond. Adjacent to the biphenyl modifications no altered dissociation behavior was found. This leads to the conclusion that dissociation of RNA is independent of the 1'-modification and, therefore, independent of nucleobase loss.

Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry.

Oligoribonucleotides (RNA) and modified oligonucleotides were subjected to low-energy collision-induced dissociation in a hybrid quadrupole time-of-flight mass spectrometer to investigate their fragmentation pathways. Only very restricted data are available on gas-phase dissociation of oligoribonucleotides and their analogs and the fundamental mechanistic aspects still need to be defined to develop mass spectrometry-based protocols for sequence identification. Such methods are needed, because chemically modified oligonucleotides can not be submitted to standard sequencing protocols. In contrast to the dissociation of DNA, dissociation of RNA was found to be independent of nucleobase loss and it is characterized by cleavage of the 5'-P-O bond, resulting in the formation of c- and their complementary y-type ions. To evaluate the influence of different 2'-substituents, several modified tetraribonucleotides were analyzed. Oligoribonucleotides incorporating a 2'-methoxy-ribose or a 2'-fluoro-ribose show fragmentation that does not exhibit any preferred dissociation pathway because all different types of fragment ions are generated with comparable abundance. To analyze the role of the nucleobases in the fragmentation of the phosphodiester backbone, an oligonucleotide lacking the nucleobase at one position has been studied. Experiments indicated that the dissociation mechanism of RNA is not influenced by the nucleobase, thus, supporting a mechanism where dissociation is initiated by formation of an intramolecular cyclic transition state with the 2'-hydroxyl proton bridged to the 5'-phosphate oxygen.

CSTX-13, a highly synergistically acting two-chain neurotoxic enhancer in the venom of the spider Cupiennius salei (Ctenidae).

The survival of the spider Cupiennius salei depends on its hunting success, which largely relies on its immediately paralyzing multicomponent venom. Here, we report on the isolation and characterization of CSTX-13, a neurotoxic enhancer in the spider venom. De novo elucidation of the disulfide bridge pattern of CSTX-13 and the neurotoxin CSTX-1 by tandem MS revealed an identical arrangement. However, in contrast to CSTX-1, CSTX-13 is a two-chain peptide with two interchain and two intrachain disulfide bridges. Furthermore, the insecticidal activity of CSTX-13 is synergistically increased in the presence of K+ ions as well as of the cytolytic peptide cupiennin 1a. We demonstrated that the weakly neurotoxic CSTX-13 enhances the paralytic activity of the neurotoxin CSTX-1 by 65% when it is administered with the latter at its entirely nontoxic physiological concentration, which is 440 times below its LD50 concentration.