Quantitative analysis of quazepam and its metabolites in human blood, urine, and bile by liquid chromatography-tandem mass spectrometry.

Quazepam (QZP), which is a long-acting benzodiazepine-type hypnotic, and its 4 metabolites, 2-oxoquazepam, N-desalkyl-2-oxoquazepam (DOQ), 3-hydroxy-2-oxoquazepam (HOQ), and 3-hydroxy-N-desalkyl-2-oxoquazepam, in human blood, urine, and bile were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. The analytes were extracted from blood by protein precipitation followed by solid phase extraction, and from urine and bile by liquid-liquid extraction and cleanup using a PSA solid phase extraction cartridge. This method was applied to a medico-legal autopsy case, in which the deceased had been prescribed QZP approximately 3 weeks before his death. In blood, the concentrations of free DOQ (160±7 ng/mL for heart blood and 181±12 ng/mL for femoral blood) were the highest of all the analytes and in agreement with the concentration at a steady state. This indicates that the deceased consecutively received QZP for at least several days until the concentrations reached approximately the same level as that in the steady state. An extremely high concentration of total HOQ (the sum of conjugated and free HOQ) in bile was also found (56,200±1900 ng/mL). This accumulation of HOQ in bile is probably due to enterohepatic circulation. This study demonstrates that the combination of the concentrations of QZP and its metabolites in biological matrices can provide more information about the amount and frequency of QZP administration.

Gas chromatography-tandem mass spectrometry assay for the quantification of four benzodiazepines and citalopram in eleven postmortem rabbit fluids and tissues, with application to animal and human samples.

Pharmacokinetic studies and postmortem toxicological investigations require a validated analytical technique to quantify drugs on a large number of matrices. Three-step liquid/liquid extraction with online derivatization (silylation) ahead of analysis by gas chromatography-tandem mass spectrometry was developed and validated on rabbit specimens in order to quantify citalopram and 4 benzodiazepines (diazepam, nordazepam, oxazepam and temazepam) in 11 biological matrices (blood, urine, bile, vitreous humor, liver, kidney, skeletal muscle, brain, adipose tissue, bone marrow (BM) and lung). Since the 11 biological matrices came from the same animal species, full validation was performed on 1 matrix, bone marrow (considered the most complex), while the other 10 underwent partial validation. Due to non-negligible matrix effects, calibration curves were performed on each matrix. Within-day and between-day precision (less than 12.0% and 12.6%, respectively) and accuracy (from 88.9% to 106.4%) were acceptable on BM at both low and high concentrations. Assessment on the other matrices confirmed accuracy and within-day precision (less than 12%, and generally between 85.1% and 114.5%, respectively). The lower limit of quantification of the method was 1ng/g for nordazepam, 5ng/g for citalopram and 10ng/g for oxazepam, diazepam and temazepam. The combination of 3-step extraction and MS/MS detection provided good selectivity in all matrices, including the most lipid-rich. Application to real-case samples showed that the method was sensitive enough to describe distribution patterns in an animal experiment, and specific enough to detect molecules in highly putrefied samples from human postmortem cases.

Enantioselective recognition of 2,3-benzodiazepin-4-one derivatives with anticonvulsant activity on several polysaccharide chiral stationary phases.

The retention behaviour of racemic 1-(4-aminophenyl)-1,2,3,5-tetrahydro-7,8-methylendioxy-4H-2,3-benzodiazepin-4-one derivatives with anticonvulsant activity on several chiral stationary phases was investigated. The selective performances of six polysaccharide phases, namely, Chiralcel OA, OD, OF, OG, OJ and Chiralpak AD were studied and normal phase HPLC methods were optimized to separate the enantiomeric forms of this class of compounds. The chiral recognition mechanism between the analytes and the chiral selectors was discussed. A molecular modeling study was carried out with the aim to explore the enantioselective molecular recognition process with the Chiralcel OG stationary phase.

Direct liquid chromatography determination of the reactive imine SJG-136 (NSC 694501).

SJG-136 (NSC 694501), 8,8'-[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one], which is being developed as a DNA-interactive antitumor agent, contains highly reactive imines in the diazepinone portions of the molecule. Water or alcohol adds readily to the imino moiety to form the corresponding carbinolamine or its alkyl ether, respectively. This sensitivity to protic substances poses a formidable challenge to the formulation and HPLC assay development for the compound. After studying the solution chemistry of SJG-136 and its potential interaction with various stationary phases, two reversed-phase liquid chromatographic assays for the compound have been developed. A direct assay that separates SJG-136 from its water or methanol adducts and an indirect assay that quantifies SJG-136 as its dihydrate adduct are reported. The latter method, which is more practical for drug development, has been validated. It is reproducible (R.S.D.<2%), linear (r2=0.9999) and accurate (within 98-102% recovery), with a lower detection limit of 2.5 ng.

Separation of oxazepam, lorazepam, and temazepam enantiomers by HPLC on a derivatized cyclodextrin-bonded phase: application to the determination of oxazepam in plasma.

The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3-hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 degrees C, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and (-) enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 degrees C and centrifuged at 0 degrees C for the collection of plasma. The results showed a difference in plasma S(+)- and R(-)-oxazepam concentrations in rabbits. No racemization of S(+)- or R(-)-Oxa enantiomers, added alone to blank plasma, was observed after extraction and enantioselective HPLC analysis.

Solid-phase extraction and liquid chromatographic quantitation of the antiarrhythmic drug L-768673 in a microemulsion formulation.

A sensitive and specific method based in solid-phase extraction and reverse-phase liquid chromatography was developed and validated for the quantitation of L-768673 in a microemulsion formulation. Following a water wash, the drug was eluted from the extraction column with acetonitrile and was analyzed on a reverse-phase C18 column with UV detection at 245 nm. The mobile phase consisted of acetonitrile-0.2% trifluoroacetic acid, 0.1% triethylamine (53:47 v/v). The retention time L-768673 was approximately 28 min with a flow rate of 1.5 ml min-1.

Two cellular and subcellular locations for the peripheral-type benzodiazepine receptor in rat liver.

Determination of ligand binding properties of the peripheral benzodiazepine receptor (PBBS) in liver, in hepatocytes, and in nonparenchymal cells demonstrated the presence of receptor-specific high-affinity binding in both hepatocyte and nonhepatocyte cells. Density gradient centrifugation showed that the high-affinity receptor in hepatocytes was localised to mitochondria, whereas in nonhepatocytes it was not mitochondrial, but with a possible biliary epithelial cell plasma-membrane location. Both receptors showed the peripheral-type specific high-affinity binding of PK 11195 and Ro5 4864 and could be photolabelled as 18 kDa proteins with [3H]PK 14105. Immunocytochemistry showed the presence of acyl-CoA binding protein, a putative endogenous ligand for the receptor, in both cell locations. Some other properties of the PBBS were investigated in liver. Diphosphatidyl glycerol had a strong inhibitory effect on receptor binding in both liver and adrenal, with Ro5 4864 more sensitive to inhibition than PK 11195. However, whereas soybean lipid and phosphatidyl serine increased the binding of both ligands to adrenal receptor, these lipids had no effect on liver, suggesting that liver PBBS may differ from the well-characterised adrenal PBBS in some of its protein conformation. Modulators of mitochondrial respiration that also influence intermembrane contact site formation were found to elicit no marked stimulatory or inhibitory effects on PBBS ligand binding in liver, a result also found for adrenal mitochondria, suggesting that the extent of contact site formation does not influence ligand binding and that the hepatocyte receptor may not play a role in regulating mitochondrial respiration. These two cellular and subcellular locations of the PBBS in liver and the different effect of phospholipids compared to other peripheral tissues may be important for the role(s) of PBBS in liver and also for the multiple roles ascribed to the receptor and to peripheral-type benzodiazepine ligands.

Autoxidation of drugs: prediction of degradation impurities from results of reaction with radical chain initiators.

In the study of the degradation of drug substances by molecular oxygen, their specific reaction mechanisms must be taken into account. The rate-determining step is usually the reaction of the substrate with a radical chain initiator, which is often an unknown impurity. The reactivity and selectivity of autoxidation can be controlled better by using a radical chain initiator, such as AIBN, than by changing the temperature or the oxygen pressure. In this paper the products profiles of four pharmaceutical substances in a simple oxidation test with AIBN are compared with the results of long term natural stability tests or with already established stabilities.

[An experimental study of the pharmacokinetic mechanisms for the development of gidazepam tolerance]. / Eksperimental'noe izuchenie farmakokineticheskikh mekhanizmov razvitiia tolerantnosti k gidazepamu.

It has been shown that the basis for the development of tolerance to the myorelaxant effect of gidazepam given long to rats is biotransformational changes followed by varying pharmacokinetic parameters: decreases in the AUC of gidazepam and its desalkyl metabolite in plasma, in brain in particular, redistribution of these substances between the plasma and brain of tolerant rats. Changes in pharmacokinetic parameters have no influence on their anxiolytic effects.

[The effect of pharmaceutic aids on gidazepam biotransformation and bioavailability]. / Vliianie vspomogatel'nykh veshchestv na biotransformatsiiu i biodostupnost' gidazepama.

When gidazepam was administered to rats in combination with 4% solutions of Tween 80 or polyvinylpyrrolidone, its bioavailability was higher than its combination with 4% polyethylene glycol 400 solution, as shown by high performance liquid chromatography. Increasing the quantities of these high-molecular compounds in gidazepam solutions was demonstrated to enhance gidazepam dealkylation in the animals after oral administration.

Determination of midazolam and its alpha-hydroxy metabolite in human plasma and urine by high-performance liquid chromatography.

We describe a new high-performance liquid chromatography (HPLC) method for measurement of midazolam and its major metabolite, alpha-hydroxymidazolam, in clinical samples. Plasma or urine was mixed with 100 ng internal standard Ro 05-6669 and borate buffer, 0.1 M, pH 9. Midazolam and its related compounds were extracted into diethylether. The organic phase was evaporated to dryness. The residue was dissolved in HPLC mobile phase [methanol-isopropyl alcohol-perchloric acid, 0.5 microM (57:25:18)] and injected into the chromatograph. The separation of substances was performed on an Spherisorb S5CN 250 x 4.6 mm HPLC column maintained at 45 degrees C. The detection was performed by absorption measurement at 245 nm. At a flow rate of 1.7 ml/min, the retention times of Ro 05-6669, 1,4 dihydroxymidazolam, alpha-hydroxymidazolam, 4-hydroxymidazolam and midazolam were 4.0, 6.7, 7.8, 9.6, and 10.8 min, respectively. In the concentration range of 5-1,000 ng/ml, the calibration graphs for both compounds were linear. The coefficients of variation of the between-day and within-day assay were < 14% for the concentration range 5-10 and < 7% for the range 10-600 ng/ml. The limits of detection for midazolam and alpha-hydroxymidazolam were 2 and 4 ng/ml, respectively. This assay is more sensitive than earlier methods; it is simple and rapid, and it enables the quantification of midazolam and its alpha-hydroxy metabolite with very good precision and accuracy in human plasma and urine.

Overpressured layer chromatographic study of retention behaviour of various benzodiazepine derivatives on layers impregnated with tricaprylmethylammonium chloride.

The retention behaviour of various benzodiazepine derivatives was investigated on silica gel layers impregnated with tricaprylmethylammonium chloride (TCMA). The chromatograms were developed by means of overpressured layer chromatography (OPLC). As for the case of amino- and nitrosalicylic acids, pyrimidine derivatives, barbiturates, penicillins, cephalosporins and tetracyclines, the retention of benzodiazepine derivatives increased with increasing layer TCMA concentration with eluents containing methanol and water, but not TCMA. On increase of the methanol content of the eluent, a retention-decreasing effect was observed. On layers impregnated with TCMA, a linear relationship existed between the RM values of the benzodiazepines and the methanol content of the eluent. A similar relationship held for silica gel layers impregnated with paraffin oil (traditional reversed-phase). There was no correlation between the results obtained on layers treated with TCMA or with paraffin oil. On TCMA-impregnated layers, the retention of compounds having different chemical structures showed no dependence on the pH of the eluent. There were two reasons for this. Firstly, as it was established, above a certain RF value, the pH of the layer in the presence of TCMA was almost identical irrespective of the original pH of the buffer. Secondly, below this RF value, the actual pH of the layer did not have a strong enough effect to cause appreciable differences between the retentions of the dissociated and undissociated species of the analytes. The conditions for optimum separation are given.

Benzodiazepines inhibit temperature-dependent L-[125I]triiodothyronine accumulation into human liver, human neuroblast, and rat pituitary cell lines.

L-T3 (T3) accumulates into cells in a temperature-dependent saturable manner through a purported iodothyronine membrane carrier protein. We report energy-dependent uptake of picomolar [125I]T3 into differentiated cell lines derived from human liver, human neuroblast, and rat pituitary malignancies. Furthermore, this cellular uptake is inhibited by classical and nonclassical benzodiazepine-type drugs (BZs); the apparent half-maximal inhibitory concentrations range from 50 nM to 50 microM, varying with drug and cell type. The site of this T3-BZ interaction was explored with cross-competitive radioligand binding to rat liver cell fractions. No interaction was seen in experiments cross-competing unlabeled T3 (10(-9)-10(-5) M) against [3H]Ro5 4864, a peripheral BZ receptor ligand, for binding sites in a crude rat liver mitochondrial fraction. As well, lormetazepam and triazolam, BZs that potently inhibit cellular uptake of [125I]T3, have no effect on [125I]T3 binding to rat liver nuclear sites. Studies of [3H]diazepam and [3H]Ro5 4864 show very little temperature-dependent uptake into HepG2 cells (less than 0.5% over 90 min) and no effect from coincubation of unlabeled T3 (1 microM). Thus, the possibility that BZs are substrates for the T3 carrier protein and are causing the reduced cellular hormonal accumulation via competitive uptake and dilution of the radiolabeled cellular T3 is unlikely. In summary, 1) drugs from the BZ class inhibit high affinity temperature-dependent cellular accumulation of thyroid hormone into cell lines from rat and human species; 2) the site of action of BZ inhibition does not involve direct antagonism of the T3 nuclear receptor, nor is it likely that the peripheral BZ receptor is the iodothyronine carrier. BZs could be interacting with the purported iodothyronine carrier protein itself to block uptake.

Monitoring of concentrations of clobazam and norclobazam in serum and saliva of children with epilepsy.

Clobazam was added to the previous antiepileptic drug therapy of 90 children suffering from drug resistant epilepsy. Ten patients became seizure free, although four of these later developed tolerance. Thirty-three patients experienced a decrease in seizure frequency, and 24 of these, too, developed tolerance. Forty-four patients showed no change in seizure frequency, and three experienced an increase. The best results were experienced by patients with myoclonic seizures, whereas patients with complex partial seizures usually developed tolerance. The concentrations of clobazam and its active metabolite norclobazam were measured in 251 serum and 57 saliva samples. The group of seizure-free patients had the lowest clobazam and norclobazam concentrations; tolerance was associated with the highest concentrations. Beneficial side effects were associated with low, and adverse effects with high, concentrations of norclobazam. The concentrations of clobazam and norclobazam in saliva correlated with concentrations in serum. Monitoring of serum and salivary concentrations of clobazam and norclobazam is of limited value only, and no therapeutic target range can be given.

New pyrrolobenzodiazepine antibiotics, RK-1441A and B. II. Isolation and structure.

Antibacteriophage antibiotics, RK-1441A and B, related to neothramycin were isolated from the culture broth of Streptomyces sp. and their structures were deduced from spectroscopic analyses. The structure of RK-1441A was 8,11-dihydroxy-3,7-dimethoxy-5-oxo-1H-pyrrolo[2,1-c:1,4]benzodiazepine. RK-1441B is a tautomeric mixture at C-3 of the structure, 3,8,11-trihydroxy-7-methoxy-5-oxo-1H-pyrrolo[2,1-c:1,4]benzodiazepine.

[Drug analysis with polarographic methods. 33. Electroanalytic studies of a new benzodiazepine: [6(o-chlorophenyl)-2,4-dihydro-2-[(4-methyl-1-piperazinyl) methylene]-8-nitro-1H-imidazol[1,2-a][1,4]benzodiazepine-1-one (WHO) (loprazolam,1)] and content determination its drug forms]. / Elektroanalytische Untersuchungen an einem neuen Benzodiazepin: [6-(2- Chlorphenyl)-2-(4-methyl-1-piperazinylmethylen)-8-nitro-2H-imidazol[1,2 -a] [1,4]benzodiazepin-1(4H)-on (VO)(Loprazolam,1)] und Gehaltsbestimmung seiner Arzneiformen.

Loprazolam (1) is a tricyclic benzodiazepine containing a new butazadiene moiety, which has not been investigated by polarography up to now. 1 is reduced in three waves at a DME over the whole pH-region. In BRP (pH 2-9) 10 electrons are consumed in this process. The first step is suitable for the determination of 1 in Loprazolam tablets containing 1 or 2 mg. These tablets are on the pharmaceutical market in several European countries. The mechanism of the electrode process will be reported in the communication XXXIV.

Study of the interaction of clobazam with cyclodextrins in solution and in the solid state.

Inclusion complexes of clobazam with alpha-, beta-, gamma-cyclodextrins (CyDs) and heptakis(2.6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD) in aqueous solution and in the solid phase were studied by the solubility method, infrared (IR) spectroscopy, differential scanning calorimetry (DSC), and X-ray diffractometry. In addition, inclusion complex of clobazam with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin and the solid dispersion of clobazam with methyl cellulose (MC) in a ground mixture were investigated by IR, DSC and X-ray diffractometry. It was observed that DM-beta-CyD had the highest stability constant among the four CyDs in solution. Thermal and X-ray diffraction analyses showed that clobazam molecules existed in a molecularly dispersed state in the ground mixture of CyDs. Infrared spectra showed lower frequency shifts in the case of the ground mixtures of clobazam with natural CyDs, which can be attributed to the formation of hydrogen bonds between the two carbonyl groups of clobazam and hydroxyl groups of natural CyDs. In contrast, higher frequency shifts were observed in the case of the ground mixtures of clobazam with methylated CyDs and MC and these were considered to be due to the monomolecular dispersion of clobazam in a hydrophobic environment. The mode of interaction of clobazam with DM-beta-CyD was different from that with natural CyDs in the ground mixtures. Furthermore, the crystalline inclusion complex of clobazam with DM-beta-CyD was obtained by heating of the coprecipitate in vacuo at 120 degrees C for 1 h.

Structure of 9-chloro-7-(2-chlorophenyl)-3,5-dihydro-[1,2,4] triazino[4,3-a][1,4]benzodiazepin-2(1H)-one.

C17H12Cl2N4O, Mr = 359.2, triclinic, P1-, a = 8.777 (8), b = 12.715 (4), c = 14.883 (4) A, alpha = 95.69 (2), beta = 83.62 (3), gamma = 93.46 (3) degrees, V = 1640.5 A3, Z = 4, Dx = 1.454 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 3.99 cm-1, F(000) = 736, T = 293 K, R = 0.039 for 2783 observed reflections. The seven-membered heterocyclic ring has a cycloheptatriene-like boat conformation with bow and stern angles 60.5 (8) and 33.8 (8) degrees, and 59.8 (8) and 35.9 (8) degrees, respectively, in the two independent molecules. The triazino ring is near planar in both molecules. The angles between the 7-phenyl ring and the fused benzo moiety are 88.9 (8) and 82.0 (8) degrees. Corresponding bond lengths and angles in the two molecules are generally similar and agree well with accepted values.

Pinazepam: analytical study of synthesis, degradation, potential impurities.

Pinazepam specifications are presented, synthesis method and quality control in the course of the synthesis on the starting materials, intermediate products and solvents are described. The used analytical methods of studied potential impurities, both from the synthesis and from degradation under exceptional conditions, are summarized and their validation is done. At last the Pinazepam satisfactory big stability is shown.