ABSTRACT
A simultaneous analytical method for etizolam and its main metabolites (alpha-hydroxyetizolam and 8-hydroxyetizolam) in whole blood was developed using solid-phase extraction, TMS derivatization and ion trap gas chromatography tandem mass spectrometry (GC-MS/MS). Separation of etizolam, TMS derivatives of alpha-hydroxyetizolam and 8-hydroxyetizolam and fludiazepam as internal standard was performed within about 17 min. The inter-day precision evaluated at the concentration of 50 ng/mL etizolam, alpha-hydroxyetizolam and 8-hydroxyetizolam was evaluated 8.6, 6.4 and 8.0% respectively. Linearity occurred over the range in 5-50 ng/mL. This method is satisfactory for clinical and forensic purposes. This method was applied to two unnatural death cases suspected to involve etizolam. Etizolam and its two metabolites were detected in these cases.
Subject(s)
Diazepam/analogs & derivatives , Tranquilizing Agents/blood , Diazepam/blood , Diazepam/poisoning , Female , Forensic Toxicology , Gas Chromatography-Mass Spectrometry , Humans , Male , Middle Aged , Solid Phase Extraction , Suicide , Tandem Mass Spectrometry , Tranquilizing Agents/poisoningABSTRACT
Bone morphogenetic proteins (BMPs), which have been shown to be heparin-binding proteins, induce osteoblast differentiation in mesenchymal cells. In the present study, we examined the effects of heparin on the BMP activities in C2C12 myoblasts. Heparin dose dependently enhanced the osteoblast differentiation induced by not only homodimers of BMP-2 or BMP-4 but also heterodimers of BMP-2/6 or BMP-2/7. However, the osteoblast differentiation induced by the constitutively active BMPR-IA, a functional BMP type I receptor, was not affected by heparin. Heparan sulfate and dextran sulfate also enhanced the BMP-2 activity, although the chemically desulfated heparin-derivatives have lost this stimulatory capacity. Heparin dose-dependently suppressed the accumulation of BMP-2 from the culture media into the cell layer or BMPR-IA, and retained a large amount of BMP-2 in the culture media. The biological activity of BMP-2, which was evaluated using a BMP-responsive reporter gene expression, was prolonged in the presence of heparin. Taken together, these results suggest that sulfated polysaccharides enhance the biological activity of both homodimers and heterodimers of BMPs by continuously serving the ligands to their signaling receptors expressed on cell membranes.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Polysaccharides/metabolism , Transforming Growth Factor beta , Blotting, Western , Bone Morphogenetic Protein 2 , Bone Morphogenetic Protein 4 , Bone Morphogenetic Protein 6 , Bone Morphogenetic Protein 7 , Cell Differentiation , Cell Lineage , Cell Membrane/metabolism , Cells, Cultured , Culture Media, Conditioned/pharmacology , Dextran Sulfate/pharmacology , Dimerization , Dose-Response Relationship, Drug , Genes, Reporter , Heparin/metabolism , Heparitin Sulfate/pharmacology , Humans , Osteoblasts/metabolism , Plasmids/metabolism , Precipitin Tests , Signal Transduction , Sulfur/metabolismABSTRACT
A novel detection method for 21 different anions by thin-layer chromatography is presented. Anions on the target plate form salts with amine in a developing solvent and are visualized after staining with citric acid-acetic anhydride reagent as white spots contrasting against a pale red-pink background. This method has particularly high sensitivity for anions of chlorate, sulfate, phosphate, chromate and dichromate (0.02-0.05 microg). The method is demonstrated to efficiently detect toxic arsenite in curry sauce as an example application. The proposed method offers highly efficient indirect detection for a wide range of anions, and serves as a purification procedure for the preparation of anionic sample solutions for other analytical methods.
