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.

The rapid hydrolysis of chlordiazepoxide to demoxepam may affect the outcome of chronic osmotic minipump studies.

BACKGROUND: In chronic studies, the classical benzodiazepine chlordiazepoxide (CDP) is often the preferred drug because, unlike other benzodiazepines, it is soluble in water. However, rapid CDP hydrolysis in solution has been described. This would diminish plasma levels in chronic minipump studies and introduce the corelease of active compounds. METHODS: Therefore, the present study aimed to explore the putative hydrolysis of CDP in aqueous solution over time and to identify the hydrolysis products. Moreover, we aimed to characterize the hydrolysis products for their in vitro (3H-flunitrazepam binding and oocyte electrophysiology) and in vivo (stress-induced hyperthermia paradigm) GABAA receptor potency. RESULTS: CDP in solution hydrolyzed to the ketone structure demoxepam which was confirmed using mass spectrometry. The hydrolysis was concentration dependent (first-order kinetics) and temperature dependent. CDP exerted greater potency compared to demoxepam in vitro (increased activity at GABAA receptors containing α1 subunits) and in vivo (stress-induced hyperthermia), although 3H-flunitrazepam binding was comparable. CONCLUSIONS: The classical benzodiazepine CDP is rapidly hydrolyzed in solution to the active compound demoxepam which possesses a reduced activity at the GABAA receptor. Chronic studies that use CDP in aqueous solution should thus be interpreted with caution. It is therefore important to consider drug stability in chronic minipump applications.

Sensitivity of P-glycoprotein tryptophan residues to benzodiazepines and ATP interaction.

Plasma membrane P-glycoprotein is a member of the ATP-binding cassette family of membrane transporters. In the present study tryptophan intrinsic fluorescence was used to understand the P-glycoprotein response to three benzodiazepines (bromazepam, chlordiazepoxide and flurazepam) in the presence and absence of ATP. Fluorescence emission spectra showed a red shift on the maximal emission wavelength upon interaction of P-glycoprotein with all benzodiazepines. Benzodiazepine association with nucleotide-bound P-glycoprotein also showed this trend and the quenching profile was attributed to a sphere-of-action model, for static fluorescence. Furthermore, quenching data of benzodiazepine-bound P-glycoprotein with ATP were concentration dependent and saturable, indicating that nucleotide binds to P-glycoprotein whether drug is present or not. These results seems in agreement with the proposal of the ATP-switch model by Higgins and Linton, where substrate binding to the transporters initiates the transport cycle by increasing the ATP binding affinity.

Voltammetric behavior and quantification of the sedative-hypnotic drug chlordiazepoxide in bulk form, pharmaceutical formulation and human serum at a mercury electrode.

Chlordiazepoxide is a sedative-hypnotic drug widely employed as a transquilizer and anti-depressant. Its electrochemical behavior in Britton-Robinson (B-R) buffers of pH 2-11 at a mercury electrode has been investigated using dc-polarography, cyclic voltammetry and controlled-potential coulometry. Polarograms of the drug in B-R buffers of pH 2-10 exhibited three 2-electron waves, while at pH>10, only a single 4-electron wave was observed. The first, second, and third waves in buffers of pH10) may be due to the reduction of both the N-oxide and C=N centers in a one step. The shift of the E(1/2,) values to more negative potentials upon the increase of pH indicated the involvement of protons in the electrode reaction and that the proton-transfer reaction precedes the electrode process proper. The estimated data indicated that, one proton and two electrons are participated in the rate-determining step of each of the reduced centers. The general sequence of chlordiazepoxide reduction processes via each of its reactant centers may be expressed as: H(+), e, e, H(+)((fast)).Based on the interfacial adsorptive character of the drug onto the mercury electrode, a validated direct square-wave adsorptive cathodic stripping (SWAdCS) voltammetric procedure has been described for the trace determination of the drug in bulk form, tablets and human serum. The procedure did not require sample pretreatment or time-consuming extraction or evaporation steps prior to the assay of the drug. The optimized operational conditions of the proposed procedure have been found to be: accumulation potential E(acc.)=-0.9 V, accumulation time t(acc.)=30s, pulse-amplitude=50 mV, scan increment=10 mV and frequency=120 Hz. The proposed procedure is much more simple, fast, sensitive, costly low and achieved much more lower limits of detection (LOD) (4.4 x 10(-10)M and 6.6 x 10(-10)M) and limits of quantitation (LOQ) (1.5 x 10(-9)M and 2.2 x 10(-9)M), respectively in pharmaceutical formulation and spiked human serum, compared to previously reported methods.

Prediction of benzodiazepines solubility using different cosolvency models.

The solubility of four benzodiazepines (BZPs) including diazepam (DIZ), lorazepam (LRZ) clonazepam (CLZ), and chlordiazepoxide (CHZ) in water-cosolvent (ethanol propylene glycol and polyethylene glycol 200) binary systems were studied. In general, increasing the volume fraction of cosolvents resulted in an increase in the solubility of benzodiazepines. The mole fraction solubilities were fitted to the various cosolvency models, namely extended Hildebrand approach (EHA), excess free energy (EFE), combined nearly ideal binary solvent/Redlich-Kister (CNIBS/R-K), general single model (GSM), mixture response surface (MR-S). double log-log (DL-L), and linear double log-log (LDL-L). The results showed that DL-L model was the best model in predicting the solubility of all drugs in all the water-cosolvent mixtures (OAE% = 4.71). The minimum and maximum errors were observed for benzodiazepine's solubility in water-propylene glycol and water-ethanol mixtures which were 2.67 and 11.78%, respectively. Three models (EFE, CNIBS/R-K and LDL-L) were chosen as general models for solubility descriptions of these structurally similar drugs in each of the solvent systems. Among these models, the EFE model was the best in predicting the solubility of benzodiazepines in binary solvent mixtures (OAE% = 11.19).

Derivative spectrophotometry as a tool for the determination of drug partition coefficients in water/dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) liposomes.

The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.

Acid-base properties and solubility of pindolol, diazepam and chlordiazepoxide in SDS micelles.

The effect of sodium dodecyl sulphate (SDS) on the acid-base properties and on the solubility of a beta-blocker (pindolol) and of two benzodiazepines (diazepam and chlordiazepoxide) has been assessed. The study was performed by potentiometric and spectrophotometric determinations of the acidity constants and by spectrophotometric evaluation of the solubilities of the pharmaceutical drugs in aqueous solution and in solutions to which was added SDS with concentrations below and above the critical micelle concentration (cmc), at 25 degrees C and at an ionic strength 0.1 M (NaCl). The effect of the organized assemblies on the pKa values was quantified by the application of two theoretical models that differ in the inclusion of ionic exchange between positively charged species in solution. These models have allowed the determination of the binding constants for drug/micelle and yielded values in good agreement with those obtained by the solubility method, and in addition provide a more detailed picture of the effect of drug charge on its partition. The results can be taken to evidence different interaction modes of the drugs with the SDS micelles.

Solid-state characterization of chlordiazepoxide polymorphs.

A novel crystal form (form II) of the benzodiazepine chlordiazepoxide is reported. The new polymorphic phase was characterized and distinguished from the standard form (form I) by X-ray diffractometry, differential scanning calorimetry, infrared spectroscopy, microscopy, solution calorimetry, and solid-state nuclear magnetic resonance. The formation of form II was dependent on the crystallizing solvent, being the predominant form isolated from methanol. Recrystallization from other alcoholic solutions (ethanol, propanol, and butanol) and toluene yielded form I. Differential scanning calorimetry and powder X-ray diffraction indicated that the two forms were enantiotropically related with a transition of form II to form I occurring between 200 and 225 degreesC. Visual examination by hot stage microscopy in this temperature range revealed a dramatic solid-state transition. Single-crystal X-ray analysis was performed on form II which was found to crystallize in the triclinic space group P1 with a = 10.736(2) A, b = 16.921(4) A, c = 17.041(4) A, alpha = 100.76(1) degrees, beta = 95.27(1) degrees, gamma = 97.53(1) degrees, Z = 8, and dcal = 1.33 g/cm3. When compared with the published crystal structure of form I, the cell symmetry, volume, and density were similar. Both structures consisted of four crystallographically independent molecules linked in pairs through intermolecular hydrogen bonding. Differences were observed in the packing arrangement of the dimers in the polymorphs. The small heat of transition calculated from solution calorimetry (1.5 kJ mol-1) was sufficient to effect a crystallographic rearrangement of the dimers.

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.

Acid-base equilibria of 1,4-benzodiazepine 4-oxides by spectrophotometry.

Chlordiazepoxide (a 1,4-benzodiazepine 4-oxide) is an anxiolytic/hypnotic drug in clinical use. It was reported to be predominantly protonated at the N-oxide oxygen in acidic aqueous solutions at pH << 4.6 (pKa). We have studied the acid-base equilibria of three 1,4-benzodiazepine 4-oxides (chlordiazepoxide, diazepam 4-oxide, and nordiazepam 4-oxide) by ultraviolet-visible spectrophotometry. The results indicate that chlordiazepoxide is not protonated at the N-oxide oxygen, but rather at the nitrogen of an imine bond between C2 carbon and its nitrogen substituent in acidic media.

Determination of chlordiazepoxide in mouse plasma by gas chromatography-negative-ion chemical ionization mass spectrometry.

A gas chromatographic-negative-ion chemical ionization mass spectrometric (GC-NCIMS) method for the determination of chlordiazepoxide (Librium) in mouse plasma is described. Chlordiazepoxide was not suitable for GC analysis because of its thermal instability. The specific derivatization technique described permits the analysis using GC-MS with high sensitivity and selectivity. The use of halazepam as an internal standard instead of an isotopic internal standard decreases the cost and time of the analysis. The detection limit for chlordiazepoxide was 0.1 ng/ml with an injection volume of 1 microliter at a signal-to-noise ratio > 5. The limit of quantification was 5 ng/ml.

Dissociation constant of chlordiazepoxide in montmorillonite suspension and its pharmaceutical application for a controlled-release dosage form.

The changes in the distribution of the ionic species of a drug due to interactions with a clay surface may have important consequences in drug therapy. The binding of chlordiazepoxide (CDO) into montmorillonite clay, veegum: HV (V), surface was studied under several pH conditions. In vitro drug release behaviour from CDO-V products was investigated. Changes in the partial molar free energy of the ionic species of CDO, delta Gi (i = CDO, CDOH+) as a result of interaction with V were determined by using differential absorbance spectroscopic techniques at 260 and 310 nm. The stronger interaction between the negatively charged clay surface and the protonated species (CDOH+) relative to the parent compound is responsible for the apparent displacement of its pKa value in V suspensions from 4.64 to 5.67. In vitro drug release behaviour from CDO-V product to the simulated gastrointestinal fluids showed a controlled pattern. The in vivo results indicated a prolonged drug effect after the oral administration of drug-clay preparation.