Simultaneous determination of chlordiazepoxide and selected antidepressants using CZE.

A simple CE method was developed and validated for the simultaneous determination of chlordiazepoxide (CHL), amitriptyline, and nortriptyline (mixture I) or the determination of CHL and imipramine (mixture II) using the same BGE. Sertraline and amitriptyline were used as internal standards for the first and second mixtures, respectively. The method allows amitriptyline to be completely separated from its impurity and main metabolite nortriptyline, which can be quantified from 0.2 µg/mL. The separation was achieved using 20 mM potassium phosphate buffer pH 5 containing 12 mM ß-cyclodextrin and 1 mM carboxymethyl-ß-cyclodextrin. UV detection was performed at 200 nm and a voltage of 15 kV was applied on an uncoated fused-silica capillary at 25°C. These experimental conditions allowed separation of the compounds to be obtained in 7 min. Calibration graphs proved the linearity up to 40 µg/mL for CHL, up to 100 µg/mL for amitriptyline and imipramine, and up to 5 µg/mL for nortriptyline. The accuracy and precision of the method have been determined by analyzing synthetic mixtures and pharmaceutical formulations. The analytical results were quite good in all cases indicating that the method was linear, sensitive, precise, accurate, and selective for both mixtures.

Optimization of dispersive liquid-liquid microextraction with central composite design for preconcentration of chlordiazepoxide drug and its determination by HPLC-UV.

A simple, rapid, and sensitive method based on dispersive liquid-liquid microextraction combined with HPLC-UV detection applied for the quantification of chlordiazepoxide in some real samples. The effect of different extraction conditions on the extraction efficiency of the chlordiazepoxide drug was investigated and optimized using central composite design as a conventional efficient tool. Optimum extraction condition values of variables were set as 210 µL chloroform, 1.8 mL methanol, 1.0 min extraction time, 5.0 min centrifugation at 5000 rpm min(-1), neutral pH, 7.0% w/v NaCl. The separation was reached in less than 8.0 min using a C18 column using isocratic binary mobile phase (acetonitrile/water (60:40, v/v)) with flow rate of 1.0 mL min(-1) The linear response (r(2) > 0.998) was achieved in the range of 0.005-10 µg mL(-1) with detection limit 0.0005 µg mL(-1) The applicability of this method for simultaneous extraction and determination of chlordiazepoxide in four different matrices (water, urine, plasma, and chlordiazepoxide tablet) were investigated using standard addition method. Average recoveries at two spiking levels were over the range of 91.3-102.5% with RSD < 5.0% (n = 3). The obtained results show that dispersive liquid-liquid microextraction combined with HPLC-UV is a fast and simple method for the determination of chlordiazepoxide in real samples.

Validated HPLC determination of the two fixed dose combinations (chlordiazepoxide hydrochloride and mebeverine hydrochloride; carvedilol and hydrochlorothiazide) in their tablets.

Simple, rapid, and selective RP-HPLC methods with UV detection were developed for simultaneous determination of chlordiazepoxide hydrochloride and mebeverine hydrochloride (Mixture I) and carvedilol and hydrochlorothiazide (Mixture II). The chromatographic separation in both mixtures was achieved by using an RP-C8 (octylsilyl) analytical column. For Mixture I, a mobile phase composed of acetonitrile-0.05 M disodium hydrogen phosphate-triethylamine (50 + 50 + 0.2, v/v/v), pH 2.5, was used; the detector wavelength was 247 nm. For Mixture II, the mobile phase consisted of acetonitrile-0.05 M disodium hydrogen phosphate (50 + 50, v/v), pH 4.0, and the detector was set at 220 nm. Quantification of the analytes was based on measuring their peak areas. Both mixtures were resolved in less than 6 min. The reliability and analytical performance of the proposed HPLC procedures were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. The linear dynamic ranges were 2.5-150 and 2.5-500 microg/mL for chlordiazepoxide HCI and mebeverine HCI, respectively, and 0.25-200 and 0.25-150 microg/mL for carvedilol and hydrochlorothiazide, respectively. The validated HPLC methods were successfully applied to the analysis of their commercial tablet dosage forms, for which no interfering peaks were encountered from common pharmaceutical adjuvants.

Simultaneous determination of imipramine hydrochloride and chlordiazepoxide in pharmaceutical preparations by spectrophotometric, RP-HPLC, and HPTLC methods.

A binary mixture of imipramine HCl and chlordiazepoxide was determined by three different methods. The first involved determination of imipramine HCl and chlordiazepoxide using the first derivative spectrophotometric technique at 219 and 231.5 nm over the concentration ranges of 1-20 and 2-24 microg/mL with mean accuracies of 99.47 +/- 0.78 and 101.43 +/- 1.20%, respectively. The second method utilized RP-HPLC with methanol-acetonitrile-0.065 M ammonium acetate buffer (45 + 25 + 30, v/v/v, pH adjusted to 5.6 +/- 0.02 with phosphoric acid) as the mobile phase pumped at a flow rate of 1.0 mL/min. Quantification was achieved using UV detection at 240 nm over concentration ranges of 0.25-4.0 and 0.1-1.6 microg/mL, with mean accuracies of 101.17 +/- 0.56 and 100.67 +/- 0.40% for imipramine HCl and chlordiazepoxide, respectively. The third method was HPTLC with carbon tetrachloride-acetone-triethylamine (pH 8.3; 6 + 3 + 0.3, v/v/v) as the mobile phase. Quantification was achieved with UV detection at 240 nm over concentration ranges of 50-600 and 20-240 ng/spot with mean accuracies of 99.51 +/- 0.59 and 100.59 +/- 0.84% for imipramine HCl and chlordiazepoxide, respectively. The suggested procedures were checked using prepared mixtures, and were successfully applied for the analysis of pharmaceutical preparations. The accuracy and precision of the methods were confirmed when the standard addition technique was applied. The results obtained by applying the proposed methods were statistically analyzed.

Demoxepam Derivatization and GC-MS Analysis Produces Erroneous Nordiazepam and Oxazepam Results.

Demoxepam, when derivatized by silylation and analyzed using gas chromatography-mass spectrometry (GC-MS), produces artifacts which are falsely identified as nordiazepam and oxazepam. Demoxepam was analyzed unextracted at various concentrations, using different derivatization procedures, and on different GC-MS systems. Oxazepam and nordiazepam were consistently identified in neat demoxepam samples, despite the changing variables. Under certain conditions, oxazepam was identified as low as 50 ng/mL derivatized demoxepam, and nordiazepam identified as low as 500 ng/mL derivatized demoxepam. The analysis of underivatized demoxepam resulted in nordiazepam detection at levels ≥2,500 ng/mL, whereas oxazepam was not detectable at or below 10,000 ng/mL demoxepam. Isolating the derivatization procedures and GC-MS analyses demonstrates that these processes are responsible for any degradation or rearrangement reactions which are taking place. Laboratories which follow similar procedures for benzodiazepine confirmations should consider these findings when interpreting analytical data from chlordiazepoxide cases.

Novel Stability-Indicating Chemometric-Assisted Spectrophotometric Methods for the Determination of Chlordiazepoxide and Clidinium Bromide in the Presence of Clidinium Bromide's Alkali-Induced Degradation Product.

Two simple and accurate chemometric-assisted spectrophotometric models were developed and validated for the simultaneous determination of chlordiazepoxide (CDZ) and clidinium bromide (CDB) in the presence of an alkali-induced degradation product of CDB in their pure and pharmaceutical formulation. Resolution was accomplished by using two multivariate calibration models, including principal component regression (PCR) and partial least-squares (PLS), applied to the UV spectra of the mixtures. Great improvement in the predictive abilities of these multivariate calibrations was observed. A calibration set was constructed and the best model used to predict the concentrations of the studied drugs. CDZ and CDB were analyzed with mean accuracies of 99.84 ± 1.41 and 99.81 ± 0.89% for CDZ and 99.56 ± 1.43 and 99.44 ± 1.41% for CDB using PLS and PCR models, respectively. The proposed models were validated and applied for the analysis of a commercial formulation and laboratory-prepared mixtures. The developed models were statistically compared with those of the official and reported methods with no significant differences observed. The models can be used for the routine analysis of both drugs in QC laboratories.

Simultaneous determination of mebeverine hydrochloride and chlordiazepoxide in their binary mixture using novel univariate spectrophotometric methods via different manipulation pathways.

Smart, sensitive, simple and accurate spectrophotometric methods were developed and validated for the quantitative determination of a binary mixture of mebeverine hydrochloride (MVH) and chlordiazepoxide (CDZ) without prior separation steps via different manipulating pathways. These pathways were applied either on zero order absorption spectra namely, absorbance subtraction (AS) or based on the recovered zero order absorption spectra via a decoding technique namely, derivative transformation (DT) or via ratio spectra namely, ratio subtraction (RS) coupled with extended ratio subtraction (EXRS), spectrum subtraction (SS), constant multiplication (CM) and constant value (CV) methods. The manipulation steps applied on the ratio spectra are namely, ratio difference (RD) and amplitude modulation (AM) methods or applying a derivative to these ratio spectra namely, derivative ratio (DD(1)) or second derivative (D(2)). Finally, the pathway based on the ratio spectra of derivative spectra is namely, derivative subtraction (DS). The specificity of the developed methods was investigated by analyzing the laboratory mixtures and was successfully applied for their combined dosage form. The proposed methods were validated according to ICH guidelines. These methods exhibited linearity in the range of 2-28µg/mL for mebeverine hydrochloride and 1-12µg/mL for chlordiazepoxide. The obtained results were statistically compared with those of the official methods using Student t-test, F-test, and one way ANOVA, showing no significant difference with respect to accuracy and precision.

Preconcentration and determination of chlordiazepoxide and diazepam drugs using dispersive nanomaterial-ultrasound assisted microextraction method followed by high performance liquid chromatography.

Benzodiazepines (BDs) are used widely in clinical practice, due to their multiple pharmacological functions. In this study a dispersive nanomaterial-ultrasound assisted- microextraction (DNUM) method followed by high performance liquid chromatography (HPLC) was used for the preconcentration and determination of chlordiazepoxide and diazepam drugs from urine and plasma samples. Various parameters such as amount of adsorbent (mg: ZnS-AC), pH and ionic strength of sample solution, vortex and ultrasonic time (min), and desorption volume (mL) were investigated by fractional factorial design (FFD) and central composite design (CCD). Regression models and desirability functions (DF) were applied to find the best experimental conditions for providing the maximum extraction recovery (ER). Under the optimal conditions a linear calibration curve were obtained in the range of 0.005-10µgmL(-1) and 0.006-10µgmL(-1) for chlordiazepoxide and diazepam, respectively. To demonstrate the analytical performance, figures of merits of the proposed method in urine and plasma spiked with chlordiazepoxide and diazepam were investigated. The limits of detection of chlordiazepoxide and diazepam in urine and plasma were ranged from 0.0012 to 0.0015µgmL(-1), respectively.

New conventional coated-wire ion-selective electrodes for flow-injection potentiometric determination of chlordiazepoxide.

New chlordiazepoxide hydrochloride (Ch-Cl) ion-selective electrodes (conventional type) based on ion associates, chlordiazepoxidium-phosphomolybdate (I) and chlordiazepoxidium-phosphotungstate (II), were prepared. The electrodes exhibited mean slopes of calibration graphs of 59.4 mV and 60.8 mV per decade of (Ch-Cl) concentration at 25 degrees C for electrodes (I) and (II), respectively. Both electrodes could be used within the concentration range 3.16 x 10(-6)-1 x 10(-2) M (Ch-Cl) within the pH range 2.0-4.5. The standard electrode potentials were determined at different temperatures and used to calculate the isothermal coefficients of the electrodes, which were 0.00139 and 0.00093 V degrees C(-1) for electrodes (I) and (II), respectively. The electrodes showed a very good selectivity for Ch-Cl with respect to the number of inorganic cations, amino acids and sugars. The electrodes were applied to the potentiometric determination of the chlordiazepoxide ion and its pharmaceutical preparation under batch and flow injection conditions. Also, chlordiazepoxide was determined by conductimetric titrations. Graphite, copper and silver coated wires were prepared and characterized as sensors for the drug under investigation.

Clinical pharmacokinetics of chlordiazepoxide.

Chlordiazepoxide was the first benzodiazepine derivative made available for clinical use. The metabolic pathway of chlordiazepoxide is complex, since the drug is biotransformed into a succession of pharmacologically active products: desmethylchlordiazepoxide, demoxepam, desmethyldiazepam, and oxazepam. The elimination half-life (tl/2beta) of chlordiazepoxide following single doses, in healthy individuals generally ranges from 5 to 30 hours, and the volume of distribution from 0.25 to 0.50 liters/kg. The hepatic extraction ratio is well under 5%. Elimination of the parent compound is mirrored by formation of the first active metabolite. Clearance of chlordiazepoxide is reduced and tl/2beta is prolonged in the elderly, in those with cirrhosis, and in those receiving concurrent disulfiram therapy. Oral chlordiazepoxide is rapidly and completely absorbed, but intramuscular injection is painful and results in slow and erratic absorption. Multiple-dose therapy with chlordiazepoxide results in accumulation of the parent compound, as well as two or more of its active metabolites. The rate and extent of accumulation varies considerably between individuals. A relation between plasma concentrations of chlordiazepoxide and its metabolites to clinical effects has been suggested in some studies and is currently under further investigation.

Pharmacokinetic studies on Ro 15-1788, a benzodiazepine receptor ligand, in the brain of the rat.

Methods for determining Ro 15-1788 in brain tissue were developed using gas chromatography with nitrogen-phosphorus detection, and using reverse-phase high performance liquid chromatography. Application of the methods to pharmacokinetic studies in the rat found the elimination half-life of Ro 15-1788 from rat brain to be 16 min. Ro 15-1788 was undetectable in rat plasma at the time points studied. Concentrations of Ro 15-1788 in the brain were reduced if chlordiazepoxide was coadministered.

Simultaneous high-performance liquid chromatographic determination of chlordiazepoxide and amitriptyline hydrochloride in two-component tablet formulations.

A rapid, precise, and accurate high-performance liquid chromatographic procedure is presented for the stimultaneous determination of amitriptyline hydrochloride and chlordiazepoxide in two-component tablet formulations. The impurities and decomposition products of both components were separated, making the determination specific for amitriptyline hydrochloride and chlordiazepoxide. The method was used for the assay, content uniformity, and dissolution testing of dosage forms containing 5--30 mg of chlordiazepoxide and 12.5--75 mg of amitriptyline.

High-pressure liquid chromatographic determination of chloridazepoxide and its major metabolites in biological fluids.

A simple, isocratic, reversed-phase, high-pressure liquid chromatographic procedure was developed for the determination of chlordiazepoxide and its major metabolites in plasma and urine. The within-run coefficient of variation was 3.4-8.0%, and the day-to-day variation was 4.0-8.0%. Recoveries of 80-91% with sensitivity limits of 50 ng/ml were obtained for the parent drug and its metabolites. Plasma and urine samples collected after single intravenous and single oral doses were analyzed using this procedure.

Formation of thermally stable derivatives of chlordiazepoxide and its desmethyl metabolite for GLC.

A derivatization procedure is described for the GLC determination of subnanogram amounts of chlordiazepoxide and nanogram amounts of N-desmethylchlordiazepoxide. Treatment with acetic anhydride at elevated temperature eliminates the highly polar and unstable nitrone group of these compounds by rearrangement and acetylation. The mass spectrometric fragmentation pattern of the acetyl derivatives is recorded and interpreted.

Effect of amitriptyline on polarography of chlordiazepoxide.

With increasing amounts of electroinactive amitriptyline, each of the three chlordiazepoxide reduction waves shifted to more cathodic half-wave potentials and decreased in limiting current. The shift was most pronounced up the 1:1 mole ratio but continued up to ratios of 200:1. This behavior was observed in several supporting electrolytes and was not due to change in pH since this factor was maintained constant as the amitriptyline concentration was increased. Shifts in E1/2 and reductions in limiting current may arise in several ways, such as complex formation between the two drugs or adsorption of the amitriptyline onto the surface of the dropping mercury electrode hindering chlordiazepoxide reduction. Most data point to adsorption as the cause.

Kinetics and mechanisms of hydrolysis of 1,4-benzodiazepines I: chlordiazepoxide and demoxepam.

Differential absorbance spectroscopy was successfully used to follow the hydrolysis kinetics of chlordiazepoxide and demoxepam from pH 1 to 11. Loss of the methylamino group from chlordiazepoxide produced demoxepam. Demoxepam degraded by a parallel consecutive reaction to 2-amino-5-chlorobenzophenone and a glycine derivative. Two intermediates were observed by TLC for demoxepam hydrolysis. One was assigned the open-ring structure resulting from amide hydrolysis, which kinetically appears to be the major mechanistic route leading to the benzophenone product. The other intermediate, representing an alternative but minor pathway, presumably results from initial scission of the azomethine linkage. Protonation of the N-oxide slightly alters the importance of these two pathways. Recyclization of the carboxylic acid intermediate was facile at pH values below the pKa of this intermediate. The stability parameters involving buffer catalysis, ionic strength effects, and temperature dependence of rate constants are reported.

Determination of major impurity in chlordiazepoxide formulations and drug substance.

Procedures for quantitating the lactam impurity, 7-chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one 4-oxide, which can be present in chlordiazepoxide formulations, is presented. The method consists of trapping chlordiazepoxide in sulfuric acid in kieselguhr, eluting the impurity with ether, and quantitating by UV spectrophotometry in absolute alcohol at 312 nm.

Assay of chlordiazepoxide and demoxepam in chlordiazepoxide formulations by difference spectrophotometry.

Rapid difference spectrophotometric methods for chlordiazepoxide and demoxepam in chlordiazepoxide formulations are described which overcome the nonspecificity of the official spectrophotometric assays. The procedures are based on the measurement of the difference absorbance at 269 nm of equimolar solutions of chlordiazepoxide at pH 8 and pH 3 and the difference absorbance at 263 nm of equimolar solutions of demoxepam at pH 13 and pH 8. The methods are specific for chlordiazepoxide and demoxepam in the presence of both compounds, 2-amino-5-chlorobenzophenone, certain coformulated drugs, and formulation excipients. Analyses of commercial dosage forms of chlordiazepoxide have shown the presence of demoxepam at concentrations in excess of the pharmacopoeial specifications in some aged samples.

High-performance liquid chromatographic determination of chlordiazepoxide and major related impurities in pharmaceuticals.

A rapid, precise, forward-phase (adsorption) high-performance liquid chromatographic procedure is presented for the determination of chlordiazepoxide and two common impurities, 7-chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one 4-oxide and 2-amino-5-chlorobenzophenone, in commercial formulations and for the determination of the benzophenone in the chlordiazepoxide drug substance. The method involves simultaneous quantitation of chlordiazepoxide and the 1,3-dihhydro impurity, followed by quantitation of the benzophenone from a separate sample extract using a second mobile phase. A single microparticulate silica gel column is used throughout. Nitrazepam and o-dinitrobenzene are the internal standards, Quantitation is by peak area using a computing integrator, except that the peak due to the benzophenone is quantitated by peak height. The described procedure is of equivalent precision, but superior accuracy, to the BP 1973 spectrophotometric procedure for the analysis of chlordiazepoxide in chlordiazepoxide formulations. Quantitation of the 1,3-dihydro and the benzophenone impurities at levels as low as 6.3 and 0.9 ng, respectively, is demonstrated.

Liquid chromatography in pharmaceutical analysis IV: determination of antispasmodic mixtures.

Parameters associated with the separation of antianxiety-antispasmodic agents were investigated using high-pressure liquid chromatography. Eight widely prescribed drugs were studied. The compounds were chromatographed on reversed-phase octadecyltrichlorosilane (C18) or diphenyldichlorosilane (phenyl) columns, using mixtures of absolute methanol and distilled water buffered with ammonium dihydrogen phosphate, ammonium acid phosphate, or ammonium carbonate. A mixture of phenobarbital-propantheline bromide was selected to demonstrate the utility of the separation and quantification method. The mixture was chromatographed on a phenyl column, using absolute methanol-aqueous 1 percent ammonium dihydrogen phosphate (60:40) (pH 5.85) at a flow rate of 1.4 ml/min. Each determination can be achieved in approximately 15 min with an accuracy of 1-2 percent.