PubMed · 10384785
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T A Brettell, K Inman, N Rudin, R Saferstein. 1999-06-15. Forensic science.. https://doi.org/10.1021/a1990009c
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A simple and sensitive high performance liquid chromatographic (HPLC) method using a semi-micro column, C8 reversed-phase column (3 mm i.d.) and a low flow rate 0.3 ml/min was developed and validated for the determination of five frequently prescribed benzodiazepines: clonazepam, diazepam, flunitrazepam, midazolam and oxazepam. Quantification was performed at 220 nm with methylclonazepam as internal standard. The method involved a simple extraction from alkalinized blood (1 ml) into 1-chlorobutane and provided excellent sensitivity, recovery, accuracy and reproducibility for benzodiazepines in therapeutic or toxic concentrations.
Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (P<0.05). Accordingly, BT-20 and MDA-MB-435-S had the highest capacity for binding [(3)H]-Ro5-4864, while the ER-positive cells exhibited only low binding of the benzodiazepine. PBR expression correlated inversely with cell doubling time (r = 0.78) and positively with Ki-67 expression (r = 0.77). The amount of mitochondria was significantly higher in cells with high PBR expression. As PBR could be demonstrated only after permeabilization of cells, PBR is suggested to be localized within the cytoplasm. Moreover, colocalization of PBR and mitochondria was shown by confocal microscopy analysis. The highest amounts of both PBR and mitochondria were found in cell lines with high mitotic activity. Therefore, it is concluded that the level of PBR is dependent on the number of mitochondria. PBR and its putative endogenous ligand diazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.
Packed column capillary electrochromatography (CEC), open-tubular CEC and microcolum liquid chromatography (LC) using a cholesteryl silica bonded phase have been studied to compare the retention behavior for benzodiazepines. It has been found that packed column CEC gives better resolution, faster analysis time than microcolumn LC for benzodiazepines maintaining similar selectivity except for some solutes which are charged species under the separation conditions. However, open-tubular CEC gave different selectivities to a larger extent for charged benzodiazepines from that which should be produced by the chromatographic properties of the cholesteryl silica phase. Charged species migration times are mainly influenced by electrophoretic mobility rather than the chromatographic interactions.