Multiplatform Metabolomics to Understand the Imidacloprid-Induced Toxicity in Drosophila.

Neonicotinoids, the class of insecticides used for crop protection, are subjected to vigilance due to their pernicious impacts. Imidacloprid (IMD) is one of the most representative insecticides of the neonicotinoid family, which has shown unfriendly consequences for non-target species. Metabolomics, a multidisciplinary approach, is being used in toxicological research to understand the metabolic responses to toxicant exposure by utilizing modern analytical techniques. Yet, no solitary analytical technique can cover the broad metabolite spectrum, but a multi-technique metabolomics platform can aid in analyzing the majority of the metabolites. In the present study, an effort has been made to identify the differential metabolites in Drosophila after exposure to IMD at 2.5 and 25 ng/mL using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), gas chromatography-MS (GC-MS), and NMR-based untargeted metabolomics. Multivariate pattern recognition analysis helped in identifying/recognizing 19 (LC-HRMS), 7 (GC-MS), and 13 (NMR) differential metabolites mainly belonging to the category of amino acids, sugars, fatty acids, and organic acids. The pathway analysis of differential metabolites predominantly showed impact on aminoacyl-tRNA biosynthesis, amino acid metabolism, and glycerophospholipid metabolism. Among these, arginine and proline metabolism was observed to be the common metabolic pathway perturbed in Drosophila due to IMD exposure. The multiplatform metabolomics based on LC-HRMS, GC-MS, and NMR analysis with an advanced level of statistical analysis can provide insights into potential perturbations in the metabolome of IMD-exposed Drosophila.

Degradants of Tenofovir Disoproxil Fumarate Under Forced Yet Mild Thermal Stress: Isolation, Comprehensive Structural Elucidation, and Mechanism.

In addition to understanding the mechanism of action for a specific drug candidate, information regarding degradation pathways/products under various stress conditions is essential to know about their short- and long-term effects on health and environment. In line with that, tenofovir disoproxil fumarate (TDF, a co-crystal form of the prodrug tenofovir with fumaric acid), particularly used as an antiretroviral drug for treatment of HIV and hepatitis-B among others, is subjected to primarily thermal and other ICH-prescribed forced degradation conditions and their various degradation products are identified. Upon thermal degradation at 60°C for 8 h, five different degradants (namely DP-1 to DP-5) are isolated, and their structures are unambiguously confirmed using advanced analytical and spectroscopic techniques including ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), high-resolution mass spectrometry (HRMS), state-of-the-art 1- and 2-dimensional nuclear magnetic resonance (1D and 2D NMR), and Fourier-transform infrared spectroscopic (FT-IR) techniques. Among fully characterized five degradants, two new degradants (DP-2 and DP-4) are identified which can potentially impact the stability of TDF via different pathways. Plausible mechanisms leading to all five thermal degradation products are also proposed including the generation of carcinogenic formaldehyde for some cases. The present systematic structural study especially combining MS and advanced NMR investigations unequivocally confirms the structures of the degradants and opens opportunities for connecting the various degradation pathways especially for the TDF-related pharmaceutical candidates.

Isolation and structural characterization of two novel oxidative degradation products of losartan potassium active pharmaceutical ingredient using advanced analytical techniques.

RATIONALE: Losartan potassium (losartan) is the most frequently utilized antihypertensive medication in the world. However, partial oxidation of losartan produces toxic by-products that could be harmful to living organisms. Therefore, it is necessary to degrade the losartan and identify the potential toxic oxidative degradation products to minimize their formation during manufacturing, formulation, storage, and packing conditions. METHODS: Oxidative degradation experiments of losartan were performed according to ICH guidelines. The degradation products were detected using ultra-high-performance liquid chromatography-mass spectrometry analysis, isolated by using preparative HPLC, and identified by using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopic techniques. RESULTS: The degradation products (DP-1, DP-2, and DP-3) were identified as (((2'-(2H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)amino)-2-oxoethylpentanoate, 5-(4'-((2 butyl-4-chloro-5-(hydroxymethyl)-1H-imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-yl)-1H tetrazol-1-ol, and 5-(4'-((2-butyl-4-chloro-5-(hydroxymethyl)-1H-imidazol-1 yl)methyl)-[1,1'-biphenyl]-2-yl)-2H-tetrazol-2-ol, respectively. CONCLUSIONS: Forced degradation of losartan potassium API under oxidative condition indicates the formation of two major novel oxidative degradation products (DP-2 and DP-3) and one minor known degradation product (DP-1).Preparative HPLC used for the isolation of the resultant DPs and their structures were successfully established using UHPLC-MS, 1H NMR, 13C NMR, HSQC, HMBC, and HRMS spectroscopic techniques.

Identification, isolation, and structural characterization of novel forced degradation products of apixaban using advanced analytical techniques.

The current research explains the stress degradation behavior of Apixaban, which is an anticoagulant or blood thinner. The degradation was conducted using hydrolytic, oxidative, thermal, and photolytic conditions. Apixaban is relatively stable in oxidative, thermal, and photolytic conditions; however, considerable degradation was observed in acid and base hydrolysis. Degradation products were identified using ultra-high performance liquid chromatography-mass spectrometry, isolated using semi-preparative high-performance liquid chromatography, and structural characterization by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. A total of five degradation products were identified and isolated in acid and base degradation. Degradation products 1, 2, and 3 were observed in acid conditions, whereas in base conditions, along with those three, two more degradation products 4 and 5 were identified. The representative thing was that among the five degradation products, two sets of positional isomers 1, 4, and 2, 5 were observed; out of which 2 and 5 are novel. The remaining degradation products 1, 3, and 4 are already reported tentatively using a single analytical technique of mass analysis without any evidence from nuclear magnetic resonance spectroscopy. Hence, the present study focused on using high-resolution mass, and nuclear magnetic resonance spectroscopy data for concrete confirmation of structures for degradation products.

Oxidative degradation profile studies of tavaborole by a validated stability indicating RP-UPLC method: Isolation and characterization of novel degradant using 2D-NMR and HRMS.

The current research work reports a study on the degradation profile of tavaborole, which is an oxaborole antifungal drug used to treat infections in the toenails. This work also reports the chemical stability of tavaborole in different stress conditions along with the isolation and characterization of degradation products by high-resolution mass spectrometry and two-dimensional nuclear magnetic resonance techniques. A sensitive and reproducible stability-indicating ultra-performance liquid chromatography method was developed and validated for quantification of tavaborole bulk drug in the presence of degradation products. Significant degradation was observed during oxidative stress conditions using H2 O2 . It was observed that the drug was highly unstable under oxidation stress conditions and thus degradation profiles with various oxidizing reagents were studied. One unknown impurity (DP-1) was formed during peroxide degradation, which was isolated by reverse-phase preparative chromatography. The structure of this degradant was characterized by high-resolution mass spectrometry and multidimensional nuclear magnetic resonance techniques. The structure of this novel impurity DP-1 was identified as [4-fluoro-2-(hydroxymethyl)phenol], which was not reported as a degradant in the literature. An Acquity BEH C18 , 100 × 2.1 mm, 1.7 µm column was used to achieve the desired separation within a shorter runtime of 4.0 min. The method was validated for specificity, precision, linearity and accuracy over the concentration range of 5.0-400 µg ml-1 (r2 -0.9999) and limit of quantitation 5.0 µg ml-1 . This method is compatible with LCMS analysis which enables to identify the unknown impurities formed in the process.

Degradation studies of Ibrutinib under stress conditions: Characterisation and structural elucidation of novel degradants.

The aim of the research work is to study the degradation behaviour of Ibrutinib (IBN) which is performed under different stress conditions according to International Conference on Harmonization guidelines (ICH). The study included monitoring degradation of the Ibrutinib drug under acidic, base, oxidation, thermal and photolytic conditions followed by isolation and characterisation of degradation products (DP) by Liquid Chromatography Mass Spectrometry (LCMS), High resolution Mass Spectrometry (HR-MS/MS) and Nuclear Magnetic Resonance (NMR) studies. The IBN drug is stable under oxidation, thermal and photolytic conditions. The degradation of drug is observed under acidic and basic conditions. Two novel degradation products are formed which are not reported in the literature. The LCMS method has been developed for chromatographic separation of drug and its degradation products which were attained on C18 BEH UPLC column (50 mm X 2.1 mm, 1.7 µm). The combination of 0.05% Acetonitrile in water and 0.05% Formic acid in water are used as a mobile phase. The flow rate is 0.6 ml/min and UV wavelength monitored at 215 nm. Acetonitrile and water are used as diluents.

The Disappearing Director: The Case of Directed N-Arylation via a Removable Hydroxyl Group.

A facile and broadly applicable method for the regiospecific N-arylation of benzotriazoles is reported. Copper-mediated reactions of diverse 1-hydroxy-1H-benzotriazoles with aryl boronic acids lead to 1-aryl-1H-benzotriazole 3-oxides. A N1-OH → N3 prototropy in the 1-hydroxy-1H-benzotriazoles is plausibly the underlying basis, where the tautomer is captured by the boronic acid, leading to C-N (not C-O) bond formation. Because the N-O bond in amine N-oxides and 1-hydroxy-1H-benzotriazoles can be easily reduced by diboron reagents such as (pinB)2 and B2(OH)4, exposure of the 1-aryl-1H-benzotriazole 3-oxides to B2(OH)4 then leads to facile reduction of the N-O bond resulting in diverse, regiospecifically-arylated benzotriazoles. Thus, the N-hydroxyl group in 1-hydroxy-1H-benzotriazoles acts as a disposable arylation director.