Abstract
A reliable, selective and sensitive stability-indicating RP-HPLC assay was established for the quantitation of bromazepam (BMZ) and one of the degradant and stated potential impurities; 2-(2-amino-5-bromobenzoyl) pyridine (ABP). The assay was accomplished on a C18 column (250 mm × 4.6 mm i.d., 5 μm particle size), and utilizing methanol-water (70: 30, v/v) as the mobile phase, at a flow rate of 1.0 ml min-1. HPLC detection of elute was obtained by a photodiode array detector (DAD) which was set at 230 nm. ICH guidelines were adhered for validation of proposed method regarding specificity, sensitivity, precision, linearity, accuracy, system suitability and robustness. Calibration curves of BMZ and ABP were created in the range of 1–16 μg mL-1 with mean recovery percentage of 100.02 ± 1.245 and 99.74 ± 1.124, and detection limit of 0.20 μg mL-1 and 0.24 μg mL-1 respectively. BMZ stability was inspected under various ICH forced degradation conditions and it was found to be easily degraded in acidic and alkaline conditions. The results revealed the suitability of the described methodology for the quantitation of the impurity (ABP) in a BMZ pure sample. The determination of BMZ in pharmaceutical dosage forms was conducted with the described method and showed mean percentage recovery of 99.39 ± 1.401 and 98.72 ± 1.795 (n = 6), respectively. When comparing the described procedure to a reference HPLC method statistically, no significant differences between the two methods in regard to both accuracy and precision were found.
Highlights
One of the extensively used derivatives of the 1,4-benzodiazepine series is the compound bromazepam (7-bromo-5-(pyridyl-2-yl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one) [1]
BMZ was hydrolised in acidic condition by refluxing with 1 N HCL solution for 3 h giving its degradant, due to cleavege of the 4,5-azomethine bond, followed by breakage of the 1,2-amide bond to give the benzophenone derivative; “2-(2-amino-5-bromobenzoyl) pyridine” (ABP) in addition to glycine Fig 1
On the other hand, when BMZ was exposed to hydrolysis under alkaline-stress condition, by mixing BMZ with 1N NaOH for 3 h under reflux, or by oxidative degradation of BMZ when added to an aqueous solution of 30% H2O2 for 3 h, this lead to the same degradation product (ABP) being produced
Summary
One of the extensively used derivatives of the 1,4-benzodiazepine series is the compound bromazepam (7-bromo-5-(pyridyl-2-yl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one) [1]. BMZ is reported to have similar properties to those of diazepam. Development and validation of a stability indicating HPLC determination of bromazepam
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