Flunitrazepam and its metabolites exposure disturb the zebrafish gut-liver axis: Combined microbiome and metabolomic analysis.

Due to clinical treatment and illegal use, psychoactive substances have been widely detected in the aquatic environment. In this study, we investigated the effects of the benzodiazepine drug flunitrazepam (FLZ) and its metabolite 7-aminoflunitrazepam (7-FLZ) on the gut-liver axis of zebrafish. Zebrafish were exposed to two concentrations of FLZ and 7-FLZ (0.05 and 1 µg/L) for 30 days. Results showed that both FLZ and 7-FLZ exposure altered the relative abundance of Proteobacteria at the phylum level, with significant differences observed at the genus level for pathogenic bacteria such as Paracoccus, Shewanella, and Aeromonas. Metabolomics results showed both exposures significantly interfered with nucleotide and amino acid metabolism. The imbalance of gut microbiota and metabolic disorder increased the level of malondialdehyde, which in turn heightened the permeability of the gut mucosal barrier. FLZ and 7-FLZ induced oxidative stress in the liver via the gut-liver axis, leading to decreased levels of glucose, total cholesterol, and triglyceride, as well as the down-regulation of glycolipid metabolism-related genes (PPARα, PPARγ, FABP2, Fabp11, PFKFB3, and LDHA). Metabolomics results revealed that FLZ and 7-FLZ significantly affected the biosynthesis of amino acids and arginine, and other metabolic pathways such as nucleotide, nicotinate and nicotinamide, and purine in the liver. Our results unveiled the mechanisms behind the toxicological effects of psychoactive substances on the gut-liver axis, providing valuable data for ecological and environmental risk assessments.

UPLC-MS/MS Method for Detection of Etomidate and Its Metabolite Etomidate Acid Quantity in Blood.

OBJECTIVES: To establish a method for the simultaneous quantitative analysis of etomidate and its metabolite etomidate acid in blood, and to discuss its application value in actual cases. METHODS: Acetonitrile precipitate protein method was used, and C18 column was selected. Gradient elution was performed with acetonitrile and 5 mmol/L ammonium acetate within 6 min. Electrospray ionization source in positive ion mode was used. The internal standard etomidate acid-d5 was obtained by etomidate-d5 alkaline hydrolysis reaction. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for quantitative analysis. The methodological verification was conducted. RESULTS: Etomidate and etomidate acid in blood showed good linear relationship in the quantitative linear range (r>0.999), with the lower limit of quantification was 2.5 ng/mL and 7.5 ng/mL, respectively. The accuracy, precision, recovery rate, and matrix effect of the method met the professional verification standards. The practical application results showed that etomidate and etomidate acid could be detected in the blood of the abusers, and their mass concentrations ranged from 17.24 to 379.93 ng/mL. CONCLUSIONS: The method established in this study can simultaneously quantify etomidate and etomidate acid in blood, which is simple and convenient to operate with accuracy. It can meet the detection needs of actual cases and provide technical support for law enforcement to crack down on etomidate abuse.

A validated stability-indicating ultra performance liquid chromatography method for the determination of potential process-related impurities in eplerenone.

A simple, sensitive, and accurate stability-indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity-spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo-diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02-3.14 µg/mL), recovery (96.1-103.9%), limit of detection (0.01-0.02 µg/mL), limit of quantitation (0.03-0.05 µg/mL), and robustness. The correction factors of the process-related substances were calculated.