Development of a validated HPLC method for the separation and analysis of a Bromazepam, Medazepam and Midazolam mixture

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 °C using a mobile phase containing 25% acetonitrile, 45% methanol and 30% ammonium acetate (0.05 M). The pH was adjusted to pH=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%±1.06. 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.

Development and validation of a stability indicating RP-HPLC-DAD method for the determination of bromazepam

“…The obtained results by the established HPLC method were compared statistically to the reference HPLC method [15] for Lexotanil1 and Calmepam1 tablets, where both the Student's t-…”

“…These reported methods also reveal many analytical techniques for BMZ determination either alone or together with other benzodiazepines, in both pharmaceutical or biological matrices. These techniques include HPLC [13][14][15][16][17][18][19][20][21][22][23][24], LC-MS [25,26], GC-MS [27], TLC [28], capillary electrophoresis [29], spectrophotometry [30][31][32], ion-selective electrode [33][34][35], potentiometric [36] and voltammetric [37,38] methods.…”

Optimization of modified dispersive liquid–liquid microextraction coupled with high‐performance liquid chromatography for the simultaneous preconcentration and determination of nitrazepam and midazolam drugs: An experimental design

“…Numerous analytical procedures including HPLC, LC–MS/MS, flow injection fluorimetry, and TLC have been developed for the analysis of these drugs in various matrices . Generally, the concentration of these drugs in complex biological matrices is very low and so application of separation and preconcentration techniques is necessary.…”

Development and optimization of a RP-HPLC method to Quantify Midazolam in Rat Plasma after Transdermal Administration: Validation and Application in Pharmacokinetic Study

“…Although these methods utilizing MS detectors are more specific and sensitive than HPLC-UV assays, and provide very low limits of detection, the essential equipment may not be available in many laboratories. Most of the HPLC-UV methods suffer from various limitations, including inadequate sensitivity; use of expensive solid phase extraction cartridges, long run times, or rigor-ous operating requirement of mobile phase [11][12][13][14]19].…”