Ixazomib is the only orally active proteasome inhibitor used in clinical practice as an anticancer drug. The novel, rapid UHPLC-UV assay for ixazomib was developed and applied to the forced degradation study followed by HRMS identification of the main degradation products. Oxidative deboronation and hydrolysis of the amid bond were found to be the principal degradation pathways. The chemical standards of the main degradation products were prepared. The method was validated for the simultaneous assay of ixazomib and its degradation products within the concentration ranges of 2.50-100.00 μg/mL (ixazomib); 0.75-60.00 μg/mL (Impurity A and B) and 1.25-60.00 μg/mL (Impurity C). The stability study revealed that ixazomib in solution is: 1) relatively stable in neutral and acidic environments, 2) its decomposition is accelerated at higher pH, 3) it is sensitive to the effects of oxidants and light, and 4) the degradation of ixazomib follows the first-order kinetics under neutral, acidic, alkaline, and UV stress. Contrary, the solid substance of ixazomib citrate was relatively resistant to heat (70 °C), heat/humidity (70 °C/75 % RH), and UV irradiation for 24 h. This study presents the first MS-compatible UHPLC method for the quantification of ixazomib and its degradation products. Furthermore, it provides data about the inherent stability and kinetics of degradation of ixazomib in a solution that may be useful in further investigation of this drug, or the development of novel proteasome inhibitors based on the ixazomib structure.
In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and β2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits. The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20-40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied. Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method.
The inhibition and eradication of oral biofilms is increasingly focused on the use of plant extracts as mouthwashes and toothpastes adjuvants. Here, we report on the chemical composition and the antibiofilm activity of 15 methanolic extracts of Iris species against both mono-(Pseudomonas aeruginosa, Staphylococcus aureus) and multi-species oral biofilms (Streptococcus gordonii, Veillonella parvula, Fusobacterium nucleatum subsp. nucleatum, and Actinomyces naeslundii). The phytochemical profiles of Iris pallida s.l., Iris versicolor L., Iris lactea Pall., Iris carthaliniae Fomin, and Iris germanica were determined by ultra-high performance liquid chromatography-high-resolution tandem mass spectroscopy (UHPLC-HRMS/MS) analysis, and a total of 180 compounds were identified among Iris species with (iso)flavonoid dominancy. I. pallida, I. versicolor, and I. germanica inhibited both the quorum sensing and adhesion during biofilm formation in a concentration-dependent manner. However, the extracts were less active against maturated biofilms. Of the five tested species, Iris pallida s.l. was the most effective at both inhibiting biofilm formation and disrupting existing biofilms, and the leaf extract exhibited the strongest inhibitory effect compared to the root and rhizome extracts. The cytotoxicity of the extracts was excluded in human fibroblasts. The inhibition of bacterial adhesion significantly correlated with myristic acid content, and quorum sensing inhibition correlated with the 7-β-hydroxystigmast-4-en-3-one content. These findings could be useful for establishing an effective tool for the control of oral biofilms and thus dental diseases.
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Currently, the interest in microalgae as a source of biologically active components exploitable as supplementary ingredients to food/feed or in cosmetics continues to increase. Existing research mainly aims to focus on revealing and recovering the rare, cost competitive components of the algae metabolom. Because these components could be of very different physicochemical character, a universal approach for their isolation and characterization should be developed. This study demonstrates the systematic development of the extraction strategy that represents one of the key challenges in effective algae bioprospecting, which predefines their further industrial application. By using of Trachydiscus minutus as a model microalgae biomass, following procedures were tested and critically evaluated in order to develop the generic procedure for microalgae bioprospecting: (i) various ways of mechanical disintegration of algae cells enabling maximum extraction efficiency, (ii) the use of a wide range of extraction solvents/solvent mixtures suitable for optimal extraction yields of polar, medium-polar, and non-polar compounds, (iii) the use of consecutive extractions as a fractionation approach. Within the study, targeted screening of selected compounds representing broad range of polarities was realized by ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometric detection (UHPLC-HRMS/MS), to assess the effectiveness of undertaken isolation steps. As a result, simple and high-throughput extraction-fractionation strategy based on consecutive extraction with water-aqueous methanol-hexane/isopropanol was developed. Moreover, to demonstrate the potential of the UHPLC-HRMS/MS for the retrospective non-target screening and compounds identification, the collected mass spectra have been evaluated to characterize the pattern of extracted metabolites. Attention was focused on medium-/non-polar extracts and characterization of lipid species present in the T. minutus algae. Such detailed information on the composition of native (non-hydrolyzed) lipids of this microalga has not been published yet.
