Determination of Bromazepam in Blood by Electron-Capture GLC and of its Major Urinary Metabolites by Differential Pulse Polarography

• Published in: Journal of pharmaceutical sciences 1974-09, Vol.63 (9), p.1440-1445
• Main Authors: De Silva, J.Arthur F., Bekersky, Ihor, Brooks, Marvin A., Weinfeld, Robert E., Glover, William, Puglisi, Carl V.
• Format: Article
• Language: English
• Subjects: Benzodiazepinones - blood; Benzodiazepinones - urine; bromazepam in blood; bromazepam urinary metabolites; Bromazepam-electron-capture GLC analysis in blood; Bromazepam—electron-capture GLC analysis in blood, differential pulse polarographic analysis of major urinary metabolites; Buffers; Chromatography, Gas - methods; differential pulse polarographic analysis of major urinary metabolites; Differential pulse polarography-analysis; Differential pulse polarography—analysis, bromazepam urinary metabolites; electron capture-analysis; GLC; GLC, electron capture—analysis, bromazepam in blood; Glucuronates - urine; Glucuronidase; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Polarography - instrumentation; Polarography - methods; Pyridines - blood; Pyridines - urine; Sulfatases; Time Factors; Tranquilizing Agents - blood; Tranquilizing Agents - urine
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/jps.2600630923

A sensitive and specific electron-capture GLC assay was developed for the determination of bromazepam (I), 7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one, in blood. The overall recovery of I from blood is 85 ± 5.0%, and the sensitivity limit of detection is 5–10ng I/ml blood. In the determination of the urinary excretion, the major urinary metabolites, 3-hydroxybromazepam (II) and 2-amino-3-hydroxy-5-bromobenzoylpyridine (V), are selectively extracted into ether from urine buffered to pH 9.0 after incubation with glucuronidase—sulfatase, whereas I and 2-amino-5-bromobenzoylpyridine (IV) are directly extractable from urine into ether at pH 9.0. The residues of the respective ether extracts are dissolved in 1.0 M phosphate buffer (pH 5.5) and analyzed by differential pulse polarography, which yields two distinct peaks for the benzodiazepin-2-one and the benzoylpyridine component in each fraction. The overall recovery of I and IV is 80 ± 5.0% with sensitivity limits of 100 and 50ng/5ml urine, respectively; the recovery of II and V is about 45 ± 5.0% with sensitivity limits of 100ng each/5ml urine analyzed. Blood levels of bromazepam and the urinary excretion of the unchanged drug and its major metabolites were determined in humans following the administration of single 12-mg oral doses.