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Chromatograms of Bromazepam, Midazolam and Medazepam using the mixture of acetonitrile, methanol and 0.05 M ammonium acetate (10:57:33, v/v/v) as mobile phase with a flow rate of 1 mL/min at 25 1C column temperature.
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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...
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... with 3 mL of water, withdraw. -Apply the serum solution. -Benzodiazepines were eluted with 10 mL of a mixture of methanol and acetonitrile (1:1). The eluates were left to dry for 15 min. Dry residues were reconstituted with mobile phase. The solutions were filtered through 0.45 mm HPLC filters before injected directly to HPLC. Fig. 3) with retention times at 5.12, 13.61 and 26.65 min, respectively. For confirmation, a solution of each compound was injected ...
Citations
... Many analytical techniques have been reported in the literature for the assay of BRZ either alone or in conjunction with other drugs in dosage form and/or body fluids. These techniques include ultraviolet (UV)-visible spectroscopy [8][9][10][11][12], fluorimetry [8,13], liquid chromatography (LC) [14][15][16][17][18][19][20][21][22][23][24][25][26], gas chromatography connected to mass spectrometry (GS/MS) [27][28][29], and capillary electrophoresis (CE) [30,31]. Potentiometric membrane sensors, either inner liquid [32][33][34] or solid-contact electrodes [35], were used for the determination of BRZ in its tablet form, biological fluids, and wastewater effluents. ...
Stability-indicating methods are awesome tools to ensure the safety and efficacy of active pharmaceutical ingredients (APIs). An accurate comparative study involving the use of potentiometric sensors for the determination of bromazepam (BRZ) in the presence of its main product of degradation and impurity was performed by the fabrication of two membrane electrodes. A screen-printed electrode (SPE) and a solid-contact glassy carbon electrode (SCE) were fabricated and their performance optimized. The fabricated sensors showed a linear electrochemical response in the concentration range 1.0 × 10−6 M to 1.0 × 10−2 M. The electrodes exhibited Nernstian slopes of 59.70 mV/decade and 58.10 mV/decade for the BRZ-SPE and BRZ-SCE membrane electrodes, respectively. The electrochemical performance was greatly affected by the medium pH. They showed an almost ideal electrochemical performance between pH 3.0 and pH 6.0. The fabricated membranes were applied successfully for the quantification of BRZ in the presence of up to 90% of its degradation product. Moreover, a successful application of the fabricated electrodes was performed for the sensitive and selective quantification of BRZ in its tablet form without any pretreatment procedure.
... These methods comprise spectrophotometric and spectrofluorimetric methods [10][11][12]. Also, high performance liquid chromatography (HPLC) [13][14][15][16][17] and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS) [18][19][20] were applied for the quantification of the studied drug in bulk, dosage forms, and biological fluids. At the same time, many electrochemical methods were applied for the determination of BRZ, which itself was determined by the application of different polarographic and voltammetric techniques [21][22][23][24]. ...
The application of ion-selective electrodes (ISEs) in the detection and determination of environmental pollutants has become a very important mission in the last few years. Two selective and sensitive membrane electrodes were fabricated in the laboratory and intended to evaluate the electrochemical response of bromazepam (BRZ) using phosphotungstic acid (PTA) and sodium tetraphenylborate (TPB) as ion pairing agents. The linearity range of the fabricated electrodes was between 1 × 10−6 M to 1 × 10−3 M. Nernstian slopes of 54 mV/decade and 57 mV/decade were obtained for the BRZ-PTA and BRZ-TPB membrane electrodes, respectively. The performance of the fabricated membranes was optimum in the pH range of 3–6. Optimum electrochemical response was attained through the careful adjustment of all assay settings. The cited method was successfully applied for the selective determination of BRZ in either its pure form or real wastewater samples obtained from a pharmaceutical industrial plant. The main core of novelty in the suggested method lies in the application of the membranes for the sensitive, selective, and economic determination of BRZ in real wastewater effluents without the tedious sample pretreatment procedures. This can make the suggested method considered an eco-friendly method, as it minimizes the use of organic solvents and chemicals used in the pretreatment process.
... Because of the therapeutic importance of Bromazepam, many methods have been developed for its determination in pharmaceutical dosage forms and / or biological fluids. These methods include potentiometric titrations [7,8], high performance liquid chromatography (HPLC) [9][10][11][12], liquid chromatography-mass spectroscopy (LC-MS) [13], gas chromatography-mass spectroscopy (GC-MS) [14]. Howev--er, most of these methods are tedious and involve expensive and sophisticated experimental set up which many ordinary quality control laboratories cannot afford. ...
... The color development was monitored spectrophometrically at the maximum absorption, λmax = 498 nm. The analytical results obtained from the proposed spectrophotometric method were statistically compared with those obtained from a reference HPLC method [12], the most commonly used analytical method for determination and quantification of Bromazepam. The application of proposed method to the assay of Bromazepam in commercial samples indicated no significant difference in precision and accuracy, and was in a satisfactory agreement with a reference HPLC method. ...
... To demonstrate the potential of the proposed spectrophotometric determination of Bromazepam based on the formation of ion-association complex, the results obtained from the quantitative analysis of Bromazepam in its standard solutions and pharmaceutical products by the proposed method were compared with a reference HPLC method that exhibited appreciably sensitive and accurate quantitative determination of Bromazepam and some other benzodiazepines [9,12]. In this study, idealized HPLC analysis was performed on Phenomenex C18 analytical column (50 mm ×4.6 mm, 5 μm), Fig.s 6 and 7 show the calibration curves for the proposed spectrophotometric and HPLC reference method, both constructed in the same concentration range. ...
A rapid and validated spectrophotometric method has been developed for determination of Bromazepam in pure and pharmaceutical dosage forms. The method was based on ion-pair association reaction of Bromazepam with iron (III) thiocyanate complex in acidic medium. The color development was monitored spectrophometrically at the maximum absorption, λ max = 498 nm. The stoichiometry of the ion-pair associate was determined, and the reaction path way was postulated. The proposed method was successfully applied to the determination of Bromazepam in five marketed brands with three labeled dosages (1.5, 3, 6 mg per tablet/ capsule). The analytical results of the proposed spectrophotometric method were statistically compared with those obtained from standard HPLC procedures used as a reference method. Application of the proposed procedure shows acceptable linearity, precision, repeatability, and reproducibility. The analytical results were in good agreement with the label claims. F-and Student's t-tests proved that no significant difference regarding both accuracy and precision between the proposed and reference method, and that this spectrophotometric method can be employed for the routine analysis of Bromazepam in bulks as well as in the commercial formulations.
... � The values between parenthesis are corresponding to the theoretical values of t and F (p = 0.05). �� HPLC method using C 18 column, 25% acetonitrile, 45% methanol and 30% ammonium acetate (0.05M) (pH 9) as a mobile phase with UV detection at 240 nm [15]. ...
... 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. ...
... 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- test and the variance ratio F-test were used at a 95% confidence level. It was clear from Table 6 that calculated t-test and F-test values are less than that of the tabulated ones. ...
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 C 18 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.
... Clonazepam (CZ), 7-nitro-5-(2-chlorophenyl)-3H-1,4-benzodiazepine-2(1H)-one as internal standard (I.S.) and bromazepam are considered the most important 1,4-benzodiazepines ( Fig. 1) enhances the activity of gamma-aminobutyric acid, the most common inhibitory neurotransmitter in the central nervous system which an abused drug in which sudden withdrawal, particularly from high dosage, carries the risk of epileptic seizures that used in the management of alcohol withdrawal syndrome (15)(16)(17)(18). Several analytical methods have been reported for bromazepam determination in pharmaceutical and biological fluids including spectrophotometry (19)(20)(21)(22)(23)(24)(25)(26), spectrofluorimetry (19)(20)(21)(22)(23)(24)(25)(26)(27), spectroscopy (28,29), HPLC (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46), GC-MS (47)(48)(49)(50), TLC (51), capillary electrophoresis (52,53), ion-selective electrode (54,55), potentiometric (56) and voltammetric (21,57) methods. ...
Sensitive, simple, rapid, selective, reliable and expeditious GC-FID method without derivatization for the quantitative analysis of bromazepam (BZ) drug has been developed for different analytical and forensic toxicology laboratories in pure form, pharmaceutical preparations, urine, blood, biscuits and beverages. The chromatographic system was carried out using a GC-17AGas Chromatograph SHIMDZU in conjunction with a column (DP-5, length: 30 meter and ID: 0.25 ml) was validated. The validation guidelines in terms of linearity, accuracy, precision, matrix effects, stability, selectivity, and recovery have been validated as per US-FDA bioanalytical guidelines. The method was linear over the concentration range of 0.6-800 µg ml −1 with limits of detection ad quantification of 0.0253 and 0.108 µg ml −1 , respectively. The intraday and interday precisions and accuracy expressed by the relative standard deviation (RSD, %) and the relative standard error (RE, %) were both not more than 4.52 % and 2.5 %, respectively. The proposed method was successfully applied in pure form, pharmaceutical preparations, urine, blood, biscuits and beverages, 100 µg ml-1 clonazepam (CZ) as internal standard (I.S.) has been developed for different forensic toxicology laboratories. The stability of BZ in the presence of all endogenous applications components was studied and the new GC-FID method was successfully employed.
... There are number of publications for the analysis and determination of therapeutic and toxic blood concentration of BMZ [11,12]. Many publications can be found that have introduced various different analytical methods for the determination of BMZ either individually or in combination with other benzodiazepines in pharmaceutical and/or biological fluids including spectrophotometry [13][14][15][16][17][18][19], spectrofluorimetry [13,20], spectroscopy [21,22], HPLC [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39], GC-MS [40][41][42][43], TLC [44], capillary electrophoresis [45,46], ion-selective electrode [47,48], potentiometric [49] and voltammetric [15,50] BMZ, similar to most benzodiazepine derivatives, can undergo hydrolysis in acidic aqueous solutions, leading to the formation of degradation products. Because of the BMZ extensive use, a study on the mechanism and kinetics of its hydrolysis is a matter of great concern. ...
... For the developed spectrophotometric methods presented here, we can find that the results obtained here were statistically compared to those obtained by applying the reported HPLC method [25] for Lex-otanil® and Calmepam® tablets. No significant difference regarding accuracy and precision between the results was obtained. ...
... The values between parenthesis are corresponding to the theoretical values of t and F (P = 0.05). b HPLC method using C 18 column, 25% acetonitrile, 45% methanol and 30% ammonium acetate (0.05 M) (pH = 9) as a mobile phase with UV detection at 240 nm[25]. ...
Four simple, sensitive and selective stability indicating spectrophotometric methods are presented for quantitative determination of the benzodiazepine drug; bromazepam (BMZ) and one of its reported potential impurities and degradation product; 2-(2-amino-5-bromobenzoyl) pyridine (ABP) in methanol. Method A, is isoabsorptive point coupled with D⁰ method, where good linearity was obtained by measuring the absorbance of BMZ at 264 nm (Aiso) in the concentration range of 2–25 μg mL⁻¹, and the absorbance of ABP at its λmax 396 nm in concentration range of 0.5–24 μg mL⁻¹. Method B, is ratio subtraction; the absorbance was measured at 233 nm for BMZ using 20 μg mL⁻¹ of ABP, while ABP was determined directly at its λmax 396 nm using methanol as a solvent. Method C, was based on measuring the total peak amplitude of the first derivative of the ratio spectra (DD¹) of BMZ from 301 to 326 nm using 10 μg mL⁻¹ of ABP as a divisor and determination of ABP at peak amplitude of 293 nm using 5 μg mL⁻¹ of BMZ as a divisor. In method D, ratio difference method, good linearity was achieved for determination of BMZ and ABP by measuring the differences between the amplitudes of ratio spectra at 312 nm and 274 nm and differences between the amplitudes of ratio spectra at 274 nm and 312 nm, respectively. The stability of BMZ was investigated under different ICH recommended forced degradation conditions. The suggested methods were then successfully applied for determination of BMZ in its pharmaceutical formulations.
... 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 rigorous operating requirement of mobile phase [11][12][13][14]19]. ...
... All benzodiazepines share the same mechanism of action, which is the modulation of γ-aminobutyric acid (GABA) uptake; the principle inhibitory neurotransmitter in the brain [2,[5][6][7]. Once bound, the benzodiazepine locks the GABA receptor into a conformation with much higher affinity for the neurotransmitter and enhances ion conduction, resulting in the reduction of neural excitability [2]. ...
Two common analytical chemical problems often encountered when using chromatographic techniques in drug analysis are matrix interferences and ion suppression. Common sample preparation often involves the dilution of the sample prior to injection onto an instrument, especially for liquid chromatography–mass spectrometry (LC–MS) analyses. This practice frequently does not minimize or eliminate conditions that may cause ion-suppression and therefore, suffer more from reduced method robustness. In order to achieve higher quality results and minimize possible interferences, various sample preparation techniques may be considered. Through the use of QuEChERS (“catchers”), a novel sample preparation technique used for high aqueous content samples, benzodiazepines can be extracted from biological fluids, such as blood and urine. This approach has shown increased recoveries of target compounds when using quantification by both external and internal standard. This increase in the recoveries has been attributed to a matrix enhancement and was determined through the use of the method of standard addition. While improving the overall analytical method for gas chromatography–mass spectrometry (GC–MS) analysis, it is not clear if this approach represents an overall benefit for laboratories that have both GC–MS and high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) capability. Demonstrating evidence of variable ionization (enhancement, ion source inertness, etc.), the method of quantification should be focused on in future studies.
In the present study, comprehensive experimental and quantum chemical calculations on 7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one (Bromazepam) were carried out using X-ray single crystal analysis, FT IR, ¹H NMR and UV/vis. Bromazepam crystallizes in monoclinic crystal system of P21/a group with a = 10.3224(5) Å, b = 15.8882(5) Å, c = 8.1627(3) Å and β =106.848 (12)° and forms an infinite chain structure with a trans-zigzag type in the solid state along the crystallographic axis ''a'' through N6...H-C13 intermolecular hydrogen bond with a distance of 2.584 Å. Optimized geometrical structures, and harmonic vibrational frequencies were investigated by DFT(B3LYP) and HF methods. TD-DFT calculations to assign the experimental electronic transitions. The chemical shift of the ionizable NH should be described by the cluster model rather than using the default polarizable continuum model (PCM). The bond lengths calculated by B3LYP functional agree with the crystal data specifically by using combined basis sets in the optimization process.
Abstract- Polyvinyl chloride (PVC) membrane sensor is described and characterized for the determination of a Benzodiazepine drug; bromazepam (BMZ). The sensor based on the use of the ion association complex of BMZ cation with tetraphenyl borate (BMZ-TPB) counter anion as ion exchange sites in the PVC matrix plasticized with dibutylsebathete (DBS) as a solvent mediator. The performance characteristics of thissensor were evaluated according to IUPAC recommendations; achieve a fast, stable and linear response for BMZ over the concentration range 10–6 to 10–2 M with slope of 44.13 mV per concentration decade. The direct potentiometric determination of BMZ using the proposed sensor gave average recovery of 100.05±0.66. The sensor is used for determination of BMZ in pharmaceutical formulations and in plasma. Validation of the method shows suitability of the proposed sensor for use in the quality control assessment of BMZ. The developed method was proved to be simple, accurate and precise when statistically compared with a reference HPLC method.
