HPLC Separation of Diazepam Conformers Coupled with Off-Line NMR Experiment

The HPLC analysis of diazepam conformers was carried out in the temperature range 273-313 K, increment 10 K. The pH of the mobile phase was 3.3; 5.5; 5.5 (I c =const; 6.5; 6.5 I c =const.). The various flow rates of 0.2; 0.5; 1.0; mL/min were used. Chiral stationary phase based on β-cyclodextrin (Ch...

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Bibliographic Details
Published in:Journal of liquid chromatography & related technologies 2006-06, Vol.29 (9), p.1351-1368
Main Authors: Fedurcová, Andrea, Lehotay, Jozef, Liptaj, Tibor, Prónayová, Nad'a, Čižmárik, Jozef
Format: Article
Language:English
Subjects:
Analysis
Biological and medical sciences
Chiral additive in mobile phase
Diazepam
Dynamic HPLC
General pharmacology
Interconversion
Medical sciences
NMR
Pharmacology. Drug treatments
Plateau
β-Cyclodextrin
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Summary:The HPLC analysis of diazepam conformers was carried out in the temperature range 273-313 K, increment 10 K. The pH of the mobile phase was 3.3; 5.5; 5.5 (I c =const; 6.5; 6.5 I c =const.). The various flow rates of 0.2; 0.5; 1.0; mL/min were used. Chiral stationary phase based on β-cyclodextrin (ChiraDex) was used for separation of diazepam. The influence of temperature, flow rate, pH of mobile phase, and ionic strength on the retention and elution profile on two peaks of diazepam conformers were studied. After that, the chiral β-cyclodextrin (0.001 mol/L; 0.003 mol/L; 0.005 mol/L) was added into the mobile phase. The increasing amount of β-cyclodextrin in the mobile phase caused the disappearance of the first peak. The complete chiral separation of diazepam was not possible because of simultaneous interconversion. The plateau between two peaks of diazepam appeared in the whole range studied, any inhibitory effects of chromatographic conditions (pH of mobile phase, temperature, flow rate, ionic strength) on interconversion was not observed, only the change of the area ratio between two peaks appeared. Off-line standard 1 H and COSY NMR experiments were used to study the diazepam structure in the achiral mobile phase. The results of 1 H and COSY NMR experiments confirmed that there is no pH dependent open-ring reaction of 1,4-benzodiazepine ring that could occur during the residence time of diazepam in the achiral mobile phase.
ISSN:1082-6076
1520-572X
DOI:10.1080/10826070600598852