Abstract
The purpose of this work was to develop a rapid, sensitive and validated HPLC method for
the separation and analysis of a Bromazepam, Medazepam and Midazolam mixture. The three
benzodiazepine compounds were separated on a reversed-phase C18 column at 50 1C using a mobile
phase containing 25% acetonitrile, 45% methanol and 30% ammonium acetate (0.05 M). The pH was
adjusted to pH¼ 9 by the addition of ammonia solution (35%, w/w). The samples were detected using a
UV detector at 240 nm. The validation study of the method included the effect of temperature, flow
rate, ratio of the components of the mobile phase and the pH of the mobile phase on the efficiency
of separation. The linear range of Bromazepam and Midazolam was between 0.12 and 0.18 mg/mL, while
that of Medazepam was between 0.08 and 0.12 mg/mL. The relative standard deviation for precision
was less than 2%. The linearity, selectivity, accuracy and robustness of the developed method
showed acceptable values. The method was applied to the analysis of the samples of raw material of
the three compounds under study, and the percentage of recoveries was 99.89%71.06. It was also
applied to the analysis of samples of pharmaceutical preparations of those compounds and spiked
serum samples. Recoveries from serum samples ranged between 91.5% and 99.0%. The developed method
is suitable for quality control of Bromazepam, Medazepam and Midazolam in their mixtures and in
pharmaceutical preparations (tablets, capsules, ampoules). It can also be used to determine their
concentrations in serum.
& 2012 Xi’an Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved.