Development of an HPLC-UV Method to Assay Empagliflozin Tablets and Identification of the Major Photoproduct by Quadrupole Time-of-Flight Mass Spectrometry.

Diabetes is a set of metabolic disorders that affect >400 million individuals worldwide. Empagliflozin belongs to the gliflozin class and is used orally to treat type 2 diabetes. In this study, a simple stability-indicating HPLC-UV method was developed to assay empagliflozin tablets and its main photoproduct was identified by high-resolution mass spectrometry. The mobile phase, which was optimized by Central Composite Design, was composed of methanol, acetonitrile and purified water (60:5:35 v/v), at a flow rate of 1 mL min-1. The calibration curve was linear in the range of 5-150 µg mL-1. All the validation parameters were met and the method was specific, even in the presence of degradation products. In the forced degradation study, empagliflozin standard and empagliflozin tablets were submitted to several conditions (acidic, alkaline, neutral and oxidant media, thermal, photolytic and humidity), and empagliflozin showed instability under all these conditions. A degradation product generated after drug exposure to ultraviolet C radiation was isolated and analyzed by quadrupole time-of-flight mass spectrometry, and the results suggested that empagliflozin undergoes decomposition by a dechlorination pathway. In silico toxicity was predicted for the degradation product, which showed a high risk of genotoxicity and hepatotoxicity.

An in situ pre-concentration method for fluorine determination based on successive digestions by microwave-induced combustion.

A new strategy based on successive digestions by microwave-induced combustion (MIC) in the same reaction vessel with a single absorbing solution was proposed. As a proof of concept, F was determined by ion-selective electrode (ISE) in seafood digests. Samples were pressed as pellets (up to 0.7 g) and combusted in closed quartz vessels pressurized with oxygen. Sequential digestions were each performed (up to 4 combustion cycles) in the same vessel and using the same absorbing solution. In each cycle, a new filter paper, igniter and sample pellet (0.7 g of sample) were used. Ammonium hydroxide solutions (10-100 mmol L-1) were evaluated for F absorption. Accuracy of the proposed method was evaluated using certified reference material of oyster tissue (NIST 1566a) and also by comparison of results after pyrohydrolysis method. Up to 3 digestion cycles (total mass of 2.1 g) could be used with 50 mmol L-1 NH4OH as absorbing solution. Results were in agreement with those obtained using pyrohydrolysis and also with certified reference value; the coefficient of variation after 3 cycles was below 5%, which was considered as suitable for F determination even at low concentration. The residual carbon in digests was lower than 25 mg L-1, allowing F determination by ISE virtually free of interferences due to dissolved organic matter. The limit of quantification (LOQ) for F was 1.3 µg g-1 (using 2.1 g of seafood), which is almost 4 times lower than the LOQ obtained using the reference method (pyrohydrolysis). Contrary to the reference method, this relatively low LOQ allowed the determination of F in all the seafood samples analysed. Taking into account that only 6 mL of diluted NH4OH solution (50 mmol L-1) were used and the suitable LOQ, the proposed sequential digestion MIC method can be recommended for further F determination in trace levels in seafood, even using a low-cost technique such as ISE, instead of other, more powerful techniques, such as ion chromatography.

Mancozeb exposure results in manganese accumulation and Nrf2-related antioxidant responses in the brain of common carp Cyprinus carpio.

Manganese (Mn)-containing dithiocarbamates such as Mancozeb (MZ) have been shown to induce oxidative stress-related toxicity in rodents and humans. However, little is known about the neurotoxic effects induced by MZ in fish. In this study, carp (Cyprinus carpio) were exposed to non-lethal waterborne concentrations of MZ, and oxidative stress parameters as well as metal accumulation in fish brains were evaluated. The experimental groups were as follows: control, MZ 5 mg/L, and MZ 10 mg/L. Fish were exposed for 7 days, and then brain was removed and prepared for subsequent analysis of antioxidant enzymes, reactive oxygen species (ROS), and expression of Nrf2 and phosphoNrf2. In parallel, manganese (Mn) levels were evaluated in blood and brain tissues. Mn levels were significantly increased in blood and brain of MZ-exposed carps. In addition, a concentration-dependent increase (p < 0.05) in ROS levels was observed in parallel to increments (p < 0.05) in the activity of major antioxidant enzymes, such as GPx, GR, and GST. On the other hand, significant decreases (p < 0.05) in CAT and SOD activities were observed. The expression of total and phosphorylated forms of Nrf2 was significantly (p < 0.05) upregulated in the brain of carps exposed to Mz when compared to the control, indicating an activation of the Nrf2 antioxidant pathway. Our study showed for the first time the activation of the Nrf2/ARE pathway and bioaccumulation of Mn induced by MZ exposure in fish species, highlighting important mechanisms of action and its toxicological impacts to aquatic organisms.