[Determination of 12 prohibited veterinary drug residues in pig urine by ultra high performance liquid chromatography-tandem mass spectrometry].

A method was established for the simultaneous detection of 12 prohibited veterinary drugs, including ß2-receptor agonists, nitrofuran metabolites, nitroimidazoles, chlorpromazine, and chloramphenicol, in pig urine. The sample was pretreated by enzymolysis, acid hydrolysis/derivatization, and liquid-liquid extraction combined with solid-phase extraction. Detection was performed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Ammonium acetate solution (0.2 mol/L, 4.5 mL) and ß-glucuronidase/aryl sulfatase (40 µL) were added to the sample, which was subsequently enzymolized at 37 ℃ for 2 h. Then, 1.5 mL of 1.0 mol/L hydrochloric acid solution and 100 µL of 0.1 mol/L o-nitrobenzaldehyde solution were added to the sample. The mixture was incubated at 37 ℃ for 16 h, and the analytes were extracted with 8 mL of ethyl acetate by liquid-liquid extraction. The lower aqueous phase obtained after extraction was extracted and purified using a mixed cation-exchange solid-phase extraction column. The extracts were combined, the extraction solution was blow-dried with nitrogen, and the residue was redissolved for determination. The samples were analyzed under multiple-reaction monitoring mode with both positive and negative electrospray ionization, and quantified using an isotope internal standard method. The correlation coefficients (r) of the 12 compounds were >0.99. The limits of detection (LODs) and quantification (LOQs) of chloramphenicol were 0.05 and 0.1 µg/L, respectively, and the LODs and LOQs of the other compounds were 0.25 and 0.5 µg/L, respectively. The mean recoveries and RSDs at 1, 2, and 10 times the LOQ were 83.6%-115.3% and 2.20%-12.34%, respectively. The proposed method has the advantages of high sensitivity, good stability, and accurate quantification; thus, it is suitable for the simultaneous determination of the 12 prohibited veterinary drug residues in pig urine.

Cinobufagin inhibits tumor progression and reduces doxorubicin resistance by enhancing FOXO1-mediated transcription of FCGBP in osteosarcoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufagin (Huachansu), an aqueous extract from the dried skin of the toad Bufo bufo gargarizans Cantor (frog skin), is a biologically active ingredient of a traditional Chinese medicine cinobufacini that can treat multiple bone pathological conditions such as bone pain, bone tumors, and osteosarcoma. AIM OF THE STUDY: The study aimed to explore the roles and molecular mechanisms of cinobufagin underlying osteosarcoma development and doxorubicin (ADR) resistance. MATERIALS AND METHODS: Cell viability, migration, and invasion were examined by CCK-8, wound healing, and Transwell invasion assays, respectively. RNA sequencing analysis was performed in MNNG/HOS cells treated with or without cinobufagin. The relationships of cinobufagin, forkhead box O1 (FOXO1), and Fc fragment of IgG binding protein (FCGBP) were examined by luciferase reporter, immunofluorescence (IF), RT-qPCR, and chromatin immunoprecipitation (ChIP) assays together with weighted gene co-expression network analysis (WGCNA) analysis. Epithelial-mesenchymal transition (EMT) marker levels were examined through the Western blot assay. The function and molecular basis of cinobufagin in osteosarcoma were further investigated by mouse xenograft experiments. RESULTS: Cinobufagin reduced cell viability, weakened ADR resistance, and inhibited cell migration/invasion/EMT in osteosarcoma cells. Cinobufagin enhanced FOXO1-mediated transcription of downstream genes including FCGBP. FCGBP knockdown partly abrogated the effect of cinobufagin on osteosarcoma cell development. Cinobufagin inhibited the growth of mouse osteosarcoma xenografts in vivo. Cinobufagin reduced the expression of Ki-67 and MMP9 and facilitated caspase-3 expression in osteosarcoma xenografts. CONCLUSION: Cinobufagin suppressed tumor progression and reduced ADR resistance by potentiating FOXO1-mediated transcription of FCGBP in osteosarcoma.

A simplified sample pretreatment for the rapid determination of 22 ß-agonist residues in swine muscle and liver tissues by ultra-high-performance liquid chromatography tandem mass spectrometry.

A reliable ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous identification and quantification of 22 ß-agonist residues in swine muscle and liver tissues. A special focus was given to sample the pretreatment procedures and was crucial for an accurate and reliable analytical method. With regard to the high complexity of food matrices, the extraction solvent, time, and temperature, as well as clean-up cartridges, were optimized to improve the extraction efficiency and reduce matrix effects. The UHPLC-MS/MS method was further validated by determining the linearity (R2 ≥ 0.9900), sensitivity (limit of detection varying from 0.05 µg/kg to 0.8 µg/kg, limit of quantitation ranging from 0.2 µg/kg to 2.5 µg/kg), recovery (ranging from 76.4 to 111.7%) and precision (≤22.9%) in swine muscle and liver tissues. The UHPLC-MS/MS method was proven to be suitable for both screening and the confirmatory determination of 22 ß-agonist residues in swine muscle and liver tissues.

[Determination of fenbutatin oxide residue in orange products by gas chromatography].

An analytical method for the determination of fenbutatin oxide (FBT) residue in oranges by capillary gas chromatography-flame photometric detection (GC-FPD) was developed. The FBT was extracted with acetone-acetic acid (99:1, v/v) and hexane, filtered and evaporated by nitrogen evaporator in a water bath at 35 degrees C. The residue was dissolved in hexane. The FBT in the solvent was derivatized with ethyl magnesium bromide for 15 min, 1 mol/L hydrochloride was added, the supernatant was collected and the solvent was evaporated to get dry supernatants, then the supernatant was dissolved in hexane and cleaned up with a silica solid phase extraction column, eluted with 5 mL hexane-dichloromethane (4:1, v/v), determined by GC. The standard curve was linear in the range of 0.2-2.0 mg/L. The correlation coefficients (r) were more than 0. 999 5, the average recoveries were 79.6%-109.6% with the relative standard deviations (RSDs) of 3.60%-9.04% at the spiked levels of 0.1-0.4 mg/kg, and the detection limit of fenbutatin oxide was 0.1 mg/kg. This method is suitable for the analysis of fenbutatin oxide residue in orange products.

[Simultaneous determination of ten sulfonylurea herbicide residues in soybeans by high performance liquid chromatography].

A reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the simultaneous determination of ten sulfonylurea herbicide (oxasulfuron, thifensulfuron-methyl, metsulfuron-methyl, triasulfuron, chlorsulfuron, bensulfuron-methyl, prosulfuron, pyrazosulfuron-methyl, chlorimuron-ethyl and primisufuron-methyl) residues in soybeans. Sulfonylurea herbicides were extracted with acetonitrile, followed by hexane partitioning. After the extract was cleaned up with a Florisil column, sulfonylurea herbicides were analyzed by HPLC-photo diode array detector (DAD) and quantified by external standard method. The pre-treatment method of the samples and the chromatographic conditions of the analysis were critically examined. The linear ranges of 10 sulfonylureas were 0.1 - 10.0 mg/L, and the correlation coefficients were 0.999 6 - 0.999 7. The average recoveries of ten sulfonylurea herbicides in spiked soybeans ranged from 69.8% to 100.7%, and the relative standard deviations were between 1.89% and 10.43%. The limit of detection was 20 microg/kg. The results have indicated that the method developed is easier, faster, and has better purification effect. It has also demonstrated that this multiresidue analytical method can meet the requirements for the simultaneous determination of many sulfonylurea herbicides in import and export inspection for soybeans.