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.

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.

Benzodiazepine stability in postmortem samples stored at different temperatures.

Benzodiazepine (lorazepam, estazolam, chlordiazepoxide, and ketazolam) stability was studied in postmortem blood, bile, and vitreous humor stored at different temperatures over six months. The influence of NaF, in blood and bile samples, was also investigated. A solid-phase extraction technique was used on all the studied samples, and benzodiazepine quantification was performed by high-performance liquid chromatography-diode-array detection. Benzodiazepine concentration remained almost stable in all samples stored at -20°C and -80°C. Estazolam appeared to be a stable benzodiazepine during the six-month study, and ketazolam proved to be the most unstable benzodiazepine. A 100% loss of ketazolam occurred in all samples stored over 1 or 2 weeks at room temperature and over 8 or 12 weeks at 4°C, with the simultaneous detection of diazepam. Chlordiazepoxide suffered complete degradation in all samples, except preserved bile samples, stored at room temperature. Samples stored at 4°C for 6 months had a 29-100% decrease in chlordiazepoxide concentration. The data obtained suggest that results from samples with these benzodiazepines stored long-term should be cautiously interpreted. Bile and vitreous humor proved to be the most advantageous samples in cases where degradation of benzodiazepines by microorganisms may occur.

Use of a Sonogel-Carbon electrode modified with bentonite for the determination of diazepam and chlordiazepoxide hydrochloride in tablets and their metabolite oxazepam in urine.

Sonogel-Carbon electrode (SngCE) modified with bentonite (BENT) shows an interesting alternative electrode to be used in the determination of 1,4-benzodiazepines by square wave adsorptive cathodic stripping voltammetry (SWAdCSV). Diazepam (DZ) and chlordiazepoxide hydrochloride (CPZ), were determined using SngCE modified by 5% BENT. An electrochemical study of different parameters (such as pH, buffer type, ionic strength, accumulation potential, scan rate, and accumulation time) which affect the determination of DZ and CPZ is reported. Linear concentration ranges of 0.028-0.256 µg mL(-1) DZ (r=0.9997) and 0.034-0.302 µg mL(-1) CPZ (r=0.9997) are successfully obtained after an accumulation time of 60s. The quantification and detection limits were calculated to be 14.0 and 4.0 ng mL(-1) for DZ, and 16.0 and 5.0 ng mL(-1) for CPZ, respectively. The surface of the proposed electrode was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX). The developed method was applied to the analysis of commercially available tablets and human urine real samples. Analysis was performed with better precision, very low detection limits, and faster than previously reported voltammetric techniques.

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 spectrophotometric determination of chlordiazepoxide and clidinium using multivariate calibration techniques.

Three multivariate modelling approaches including partial least squares regression (PLS), genetic algorithm-partial least squares regression (GA-PLS), and principal components-artificial neural network (PC-ANN) analysis were investigated for their application to the simultaneous determination of chlordiazepoxide and clidinium levels in pharmaceuticals. A set of synthetic mixtures of drugs in ethanol and 0.1 M HCL was made, and the prediction abilities of the aforementioned methods were examined using RSE% (relative standard error of the prediction). The PLS and PC-ANN methods were found to be comparable, and GA-PLS produced slightly better results. The predictive models that we built were successfully applied to simultaneously determine the levels of chlordiazepoxide and clidinium in coated tablets.

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.

Stability-indicating HPLC method for simultaneous determination of clidinium bromide and chlordiazepoxide in combined dosage forms.

The study describes development and subsequent validation of a stability indicating reverse-phase high-performance liquid chromatography method for the simultaneous estimation of clidinium bromide (CLI) and chlordiazepoxide (CHLOR) from their combination drug product. Chromatographic separations are performed at ambient temperature on a Phenomenex Luna C(18) (250 mm x 4.6 mm, i.d., 5 microm) column using a mobile phase consisting of potassium dihydrogen phosphate buffer (0.05 M, pH 4.0 adjusted with 0.5% orthophosphoric acid)-methanol- acetonitrile (40:40:20, v/v/v). The flow rate is 1.0 mL/min, and the detection wavelength is 220 nm. The method is validated with respect to linearity, precision, accuracy, system suitability, and robustness. The utility of the procedure is verified by its application to marketed formulations that were subjected to accelerated degradation studies. The method distinctly separated the drug and degradation products even in actual samples. The products formed in marketed tablet dosage forms are similar to those formed during stress studies.

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.

[Determination of five components in compound hypotensive tablet by HPLC].

AIM: To establish a method for the determination of the five components (reserpine, chlordiazepoxide, hydrochlorothiazide, dihydralazine sulfate, triamterene) in compound hypotensive tablet. METHODS: The chromatography was performed using a CN column with acetontrile-0.1 mol L(-1) sodium heptasulfonate solution (7:3) and (5:5) as the mobile phases. The detection wavelength was 267 nm for reserpine, chlordiazepoxide and hydrochlorothiazide, 310 nm for dihydralazine sulfate, 360 nm for triamterene. RESULTS: The linear range of each component was tested, and the recovery and stability of each component was satisfactory, three lots of samples were determined using the method. CONCLUSION: This is an accurate and credible quality control method for compound hypotensive tablet.

[Determination of diazepine derivatives: alprazolam, medazepam, chlordiazepoxid mixture by high performance liquid chromatography]. / Diazepino dariniu: alprazolamo, medazepamo, chlordiazepoksido misinio tyrimas didelio slegio skysciu chromatografijos metodu.

The aim of this research was to determine possibility of qualitative analysis of diazepine derivatives: alprazolam, medazepam, chlordiazepoxid in the mixture. The high performance liquid chromatography was used for the investigation, the reagents--tablets of alprazolam, medazepam, chlordiazepoxid. It was found out that the most acceptable eluent for the analysis of the mixture by high pressure liquid chromatography is phosphate buffer, containing 0.02 M penthanesulfonic acid:etannitrile (55:45) (pH=3.5). The optimal wavelength for identification of the drugs in the mixture is 254 nm.

Simultaneous determination of chlordiazepoxide and clidinium bromide in pharmaceutical formulations by derivative spectrophotometry.

A direct and simple first derivative spectrophotometric method has been developed for the simultaneous determination of clidinium bromide and chlordiazepoxide in pharmaceutical formulations. Acetonitrile was used as solvent for extracting the drugs from the formulations and subsequently the samples were evaluated directly by derivative spectrophotometry. Simultaneous determination of the drugs can be carried out using the zero-crossing method for clidinium bromide at 220.8 nm and the graphical method for chlordiazepoxide at 283.6 nm. The calibration graphs were linear in the ranges from 0.983 to 21.62 mg/l of clidinium bromide and from 0. 740 to 12.0 mg/l of chlordiazepoxide. The ingredients commonly found in commercial pharmaceutical formulations do not interfere. The proposed method was applied to the determination of these drugs in tablets.

[The preservability of sibazon breakdown products in the cadavers of experimental animals]. / Sokhraniaemost' produktov raspada sibazona v trupakh éksperimental'nykh zhivotnykh.

Cibasone degradation products were measured in corpses of 15 cats poisoned with cibasone in a dose of 750 mg/kg. Cibasone was assessed by semiquantitative method on Silufol UV-254 plates from the content of the main degradation product 3-methylamino-5-chloro-benzophenone (MCB) in parallel with label in the benzene system. MCB was detectable in cadaveric material and in the container bottom panel for up to 7 years 8 months and in soft tissue until their complete putrefactive destruction. MCB concentrations were the highest in the stomach, thin intestine, and liver and the lowest in the muscles and bones. The main product of elenium (chlozepide) degradation product 2-amino-chloro-benzophenone (ACB) was detected along with MCB in a year and up to 4 years 7 months after burial, although elenium was not injected. Degradation products were identified by electron spectroscopy in UV and visible bands of the spectrum: lambda max = 238 and 410 nm for MCB and 238 and 390 nm for ACB. ACB is the product of MCB degradation-dimethylation in putrefactive degradation of tissues. Ethanol extraction of degradation products detects MCB and ACB 7 years 8 months postmortem.

Use of direct-probe mass spectrometry as a toxicology confirmation method for demoxepam in urine following high-performance liquid chromatography.

The identification of the metabolite demoxepam in human urine establishes that chlordiazepoxide, a common benzodiazepine, has been administered. Like N-oxide metabolites of other drugs, demoxepam cannot be detected by gas chromatography-mass spectrometry (GC-MS), due to thermal decomposition, and the product, nordiazepam, is a metabolite common to many benzodiazepines. Demoxepam can be readily screened using a high-performance liquid chromatography (HPLC) system such as REMEDi HS; at 35 degrees C, no thermal decomposition will occur. Currently, there is no confirmation method available for the detection of demoxepam in urine samples. In this study, we demonstrated that following collection of the HPLC fraction, demoxepam can be confirmed using the technique of direct-probe MS. The mass spectra of demoxepam and nordiazepam differ and are easily distinguishable from each other. Ten urine samples that were analyzed by HPLC and determined to contain demoxepam were evaluated; demoxepam was confirmed in each case by direct-probe MS.

Development of tolerance to anxiolytic effects of chlordiazepoxide in elevated plus-maze test and decrease of GABAA receptors.

Repeated administration of benzodiazepines has been reported to produce tolerance in animals and humans. Using an elevated plus-maze test and an autoradiographic technique, we investigated whether repeated administration of chlordiazepoxide produced tolerance to its anxiolytic effects, and whether such repeated administration altered benzodiazepine and GABAA receptors. Tolerance to the anxiolytic effect of chlordiazepoxide was produced when it was administered at a dose of 30 mg/kg (i.p.) once a day for 10 and 14 days. In the quantitative autoradiographical study, although repeated chlordiazepoxide treatment had no effect on [3H]flunitrazepam and [3H]Ro 15-4513 binding to benzodiazepine receptors, such treatment reduced [3H]muscimol binding to GABAA receptors in the cortex, caudate putamen, and hippocampus. These results suggest firstly, the production of tolerance to the anxiolytic effects of chlordiazepoxide, and, secondly, that this tolerance may be due to the down-regulation of GABAA receptors, but not of benzodiazepine receptors.

Fourier transform infrared analyses of some particulate drug mixtures using a diamond anvil cell with a beam condenser and an infrared microscope.

Although the diamond anvil cell (DAC) has been used in many forensic science laboratories for the analysis of trace evidence, few applications of this technique for the analysis of controlled substances have been reported. This may be due to both an unfamiliarity on the part of forensic drug chemists with this accessory and the nature and quality of spectra that result from use of a DAC on a dispersive instrument. Along with low energy throughput, which results in relatively high noise levels, strong broad diamond absorptions occur. With the use of a Fourier transform infrared instrument, these do not present a problem and nanogram quantities of materials can be analyzed when the DAC is used with an infrared microscope. Since single crystals can be sampled with the DAC, simple physical separations (involving particle-picking) can be used in certain cases to isolate drugs from particulate mixtures for infrared analysis. This method is especially useful for some "difficult" mixtures and residues, and several examples of such analyses involving samples of forensic science interest are presented.