Preparation of a novel sorptive stir bar based on vinylpyrrolidone-ethylene glycol dimethacrylate monolithic polymer for the simultaneous extraction of diazepam and nordazepam from human plasma.

A new monolithic coating based on vinylpyrrolidone-ethylene glycol dimethacrylate polymer was introduced for stir bar sorptive extraction. The polymerization step was performed using different contents of monomer, cross-linker and porogenic solvent, and the best formulation was selected. The quality of the prepared vinylpyrrolidone-ethylene glycol dimethacrylate stir bars was satisfactory, demonstrating good repeatability within batch (relative standard deviation < 3.5%) and acceptable reproducibility between batches (relative standard deviation < 6.0%). The prepared stir bar was utilized in combination with ultrasound-assisted liquid desorption, followed by high-performance liquid chromatography with ultraviolet detection for the simultaneous determination of diazepam and nordazepam in human plasma samples. To optimize the extraction step, a three-level, four-factor, three-block Box-Behnken design was applied. Under the optimum conditions, the analytical performance of the proposed method displayed excellent linear dynamic ranges for diazepam (36-1200 ng/mL) and nordazepam (25-1200 ng/mL), with correlation coefficients of 0.9986 and 0.9968 and detection limits of 12 and 10 ng/mL, respectively. The intra- and interday recovery ranged from 93 to 106%, and the relative standard deviations were less than 6%. Finally, the proposed method was successfully applied to the analysis of diazepam and nordazepam at their therapeutic levels in human plasma. The novelty of this study is the improved polarity of the stir bar coating and its application for the simultaneous extraction of diazepam and its active metabolite, nordazepam in human plasma sample. The method was more rapid than previously reported stir bar sorptive extraction techniques based on monolithic coatings, and exhibited lower detection limits in comparison with similar methods for the determination of diazepam and nordazepam in biological fluids.

Pharmacokinetics of the cytochrome P-450 substrates phenytoin, theophylline, and diazepam in healthy Greyhound dogs.

The purpose of this study was to determine the pharmacokinetics of phenytoin, theophylline, and diazepam in six healthy Greyhound dogs. Additionally, the pharmacokinetics of the diazepam metabolites, oxazepam and nordiazepam, after diazepam administration was determined. Phenytoin sodium (12 mg/kg), aminophylline (10 mg/kg), and diazepam (0.5 mg/kg) were administered IV on separate occasions, and blood was collected at predetermined time points for the quantification of plasma drug concentrations by fluorescence polarization immunoassay (phenytoin, theophylline) or mass spectrometry (diazepam, oxazepam, and nordiazepam). The terminal half-life was 4.9, 9.2, and 1.0 h, respectively, for phenytoin, theophylline, and diazepam, and 6.2 and 2.4 h for oxazepam and nordiazepam after IV diazepam. The clearance was of 2.37, 0.935, and 27.9 mL · min/kg, respectively, for phenytoin, theophylline, and diazepam. The C(MAX) was 44.7 and 305.2 ng/mL for oxazepam and nordiazepam, respectively, after diazepam administration. Temazepam was not detected above 5 ng/mL in any sample after IV diazepam.

Diazepam pharmacokinetics after nasal drop and atomized nasal administration in dogs.

The standard of care for emergency therapy of seizures in veterinary patients is intravenous (i.v.) administration of benzodiazepines, although rectal administration of diazepam is often recommended for out-of-hospital situations, or when i.v. access has not been established. However, both of these routes have potential limitations. This study investigated the pharmacokinetics of diazepam following i.v., intranasal (i.n.) drop and atomized nasal administration in dogs. Six dogs were administered diazepam (0.5 mg/kg) via all three routes following a randomized block design. Plasma samples were collected and concentrations of diazepam and its active metabolites, oxazepam and desmethyldiazepam were quantified with high-performance liquid chromatography (HPLC). Mean diazepam concentrations >300 ng/mL were reached within 5 min in both i.n. groups. Diazepam was converted into its metabolites within 5 and 10 min, respectively, after i.v. and i.n. administration. The half lives of the metabolites were longer than that of the parent drug after both routes of administration. The bioavailability of diazepam after i.n. drop and atomized nasal administration was 42% and 41%, respectively. These values exceed previously published bioavailability data for rectal administration of diazepam in dogs. This study confirms that i.n. administration of diazepam yields rapid anticonvulsant concentrations of diazepam in the dog before a hepatic first-pass effect.

Quantification of chlordesmethyldiazepam by liquid chromatography-tandem mass spectrometry: application to a cloxazolam bioequivalence study.

A rapid, sensitive and specific LC-MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid-liquid extraction (diethyl-ether/hexane, 80/20, v/v) procedure. The LC-MS/MS method on a RP-C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5-50 ng/mL (R > 0.999). The between-run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification-LLOQ (0.500 ng/mL). The between-run accuracies were 0.1, -1.5, -2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam -- test, Eurofarma Lab. Ltda and Olcadil -- reference, Novartis Biociências S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUC0-inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA.

Bioavailability and dose proportionality of intramuscular diazepam administered by autoinjector.

A diazepam 10-mg autoinjector was evaluated in bioequivalence and dose proportionality studies; both involved 24 young, healthy subjects and used randomized, open-label, 2-treatment, 2-period crossover designs with a 3-week washout period between treatments. The bioequivalence study compared a single diazepam 10-mg autoinjector with a conventional needle and syringe containing 10 mg of diazepam injectable. The dose proportionality study compared the pharmacokinetics of a single diazepam 10-mg autoinjector with that of 2 diazepam 10-mg autoinjectors given simultaneously (20 mg). Injections were intramuscular in the midanterolateral thigh in both studies. The studies showed that the diazepam autoinjector produced consistent plasma diazepam levels, with a rapid onset of absorption. The diazepam 10-mg autoinjector given intramuscularly was bioequivalent to a conventional syringe containing diazepam 10 mg. A single (10-mg) autoinjector and 2 (20-mg) diazepam autoinjectors administered simultaneously produced plasma diazepam concentrations that were essentially dose proportional.

Cardiac and peripheral blood similarities in the comparison of nordiazepam and bromazepam blood concentrations.

Concomitant heart and peripheral blood determinations were performed on 40 fatal cases involving nordiazepam (20 cases) and bromazepam (20 cases). The heart blood concentration for the two drugs (588 ng/mL for nordiazepam and 802 ng/mL for bromazepam) does not differ from the corresponding peripheral blood concentration (587 ng/mL for nordiazepam and 883 ng/mL for bromazepam). The mean ratios for the heart and peripheral blood concentrations were 0.95 for nordiazepam and 0.86 for bromazepam. No postmortem redistribution was observed for these two benzodiazepines. The authors thus suggest that corresponding heart blood can be proposed in the quantitative analysis of these drugs when peripheral blood is unavailable. The present study also shows the stability of the two drugs after a year of storage.

Voriconazole and fluconazole increase the exposure to oral diazepam.

OBJECTIVE: We assessed the effect of voriconazole and fluconazole on the pharmacokinetics and pharmacodynamics of diazepam. METHODS: Twelve healthy volunteers took 5 mg of oral diazepam in a randomised order on three study sessions: without pretreatment, after oral voriconazole 400 mg twice daily on the first day and 200 mg twice daily on the second day, or after oral fluconazole 400 mg on the first day and 200 mg on the second day. Plasma concentrations of diazepam and N-desmethyldiazepam were determined for up to 48 h. Pharmacodynamic variables were measured for 12 h. RESULTS: In the voriconazole phase, the area under the plasma concentration time curve (AUC 0-infinity) of diazepam was increased (geometric mean ratio) 2.2-fold (p < 0.05; 90% confidence interval [CI] 1.56 to 2.82). This was associated with the prolongation of the mean elimination half-life (t(1/2)) from 31 h to 61 h (p < 0.01) after voriconazole. In the fluconazole phase, the AUC 0-infinity of diazepam was increased 2.5-fold (p < 0.01; 90% CI 1.94 to 3.40), and the t(1/2) was prolonged from 31 h to 73 h (p < 0.001). The peak plasma concentration of diazepam was practically unchanged by voriconazole and fluconazole. The pharmacodynamics of diazepam were changed only modestly. CONCLUSION: Both voriconazole and fluconazole considerably increase the exposure to diazepam. Recurrent administration of diazepam increases the risk of clinically significant interactions during voriconazole or fluconazole treatment, because the elimination of diazepam is impaired significantly.

Massive intoxication involving unusual high concentration of amitriptyline.

Amitriptyline is a tricyclic antidepressant widely used in the treatment of depression. Antidepressant drugs are among the most commonly encountered causes of self-poisoning, as illustrated by several published cases in the literature. This investigation reports a case of massive amitriptyline intoxication, involving a 44-year old female found dead in bed. The presence of this tricyclic antidepressant was revealed by a routine screening procedure. The concentration was calculated by gas chromatography/ electron ionization-mass spectrometry in the selected ion monitoring mode after solid-phase extraction using proadifen as internal standard and was in the post-mortem whole blood sample 85.9 mug/mL. This value was much higher than the reported toxic values ever found in the literature, and may therefore have caused the victim's death. Nortriptyline was also detected in the toxic concentration range, as well as therapeutic levels of diazepam and nor-diazepam. Taking into account both the available circumstantial information and toxicological results, it is very likely that death was caused by self-poisoning. Human & Experimental Toxicology (2007) 26, 667-670.

Site-, Technique-, and Time-Related Aspects of the Postmortem Redistribution of Diazepam, Methadone, Morphine, and their Metabolites: Interest of Popliteal Vein Blood Sampling.

Sampling site, technique, and time influence postmortem drug concentrations. In 57 cases, we studied drug concentration differences as follows: subclavian vein-dissection/clamping versus blind stick, femoral vein-dissection/clamping versus blind stick, right cardiac chamber, and popliteal vein-dissection and clamping only. Cases were distributed in group #1 (all cases with both techniques), group #2 (dissection/clamping), and group #3 (blind stick). Sampled drugs were diazepam, methadone, morphine, and their metabolites. To assess PMR, mean concentrations and ratios were calculated for each group. Time-dependent variations of blood concentrations and ratios were also assessed. Results indicate that site, method, and time may influence postmortem distribution interpretation in different ways. Popliteal blood seems less subject to PMR. In conclusion, our study is the first to evaluate concurrently three main aspects of PMR and confirms that the popliteal vein may represent a site that is more resistant to the changes seen as a result of PMR.

Liquid chromatographic-electrospray ionization mass spectrometric quantitative analysis of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in rat plasma.

A liquid chromatographic-mass spectrometric method with electrospray ionization is presented for the simultaneous determination of buprenorphine, nordiazepam and their pharmacologically active metabolites, norbuprenorphine and oxazepam, in rat plasma. The drugs were extracted from plasma by liquid-liquid extraction and chromatographically separated using a gradient elution of aqueous ammonium formate and acetonitrile. Following electrospray ionization, the analytes were quantified in the single ion storage mode. The assay was validated according to current acceptance criteria for bioanalytical method validation. It was proved to be linear from 0.7 to 200 ng/ml plasma for buprenorphine, 1.0 to 200 ng/ml for norbuprenorphine, 2.0 to 200 ng/ml for nordiazepam, and from 5.0 to 200 ng/ml for oxazepam. The average recoveries of buprenorphine, norbuprenorphine, nordiazepam and oxazepam were 89, 39, 88 and 82%, respectively, with average coefficients of variation ranging from 1.8 to 14.3%. The limits of quantitation for these drugs were 0.7, 1.0, 2.0 and 5.0 ng/ml, respectively, with associated precisions within 17% and accuracies within +/-18% of the nominal values. Both the intra- and inter-assay precision values did not exceed 11.3% for the four analytes. Intra- and inter-assay accuracies lay within +/-15% of the nominal values. The validated method was applied to the determination of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in plasma samples collected from rats at various times after intravenous administration of buprenorphine and nordiazepam.

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma.

A method was developed and fully validated for the quantitation of prazepam and its major metabolites, oxazepam and nordiazepam, in human plasma. Sample pretreatment was achieved by solid-phase extraction using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled with single-quadrupole mass spectrometry (MS) with an electrospray ionization interface. The MS system was operated in the selected ion monitoring mode. HPLC was performed isocratically on a reversed-phase XTerra MS C18 analytical column (150 x 3.0 mm i.d., particle size 5 microm). Diazepam was used as the internal standard for quantitation. The assay was linear over a concentration range of 5.0-1000 ng ml(-1) for all compounds analyzed. The limit of quantitation was 5 ng ml(-1) for all compounds. Quality control samples (5, 10, 300 and 1000 ng ml(-1)) in five replicates from three different runs of analysis demonstrated an intra-assay precision (CV) of < or = 9.1%, an inter-assay precision of < or = 6.0% and an overall accuracy (relative error) of < 4.6%. The method can be used to quantify prazepam and its metabolites in human plasma covering a variety of pharmacokinetic or bioequivalence studies.

Withdrawal reaction from long-term, low-dosage administration of diazepam. A double-blind, placebo-controlled case study.

Symptoms of diazepam withdrawal developed in a young man who had been taking diazepam in dosages of 15 to 25 mg/day during a six-year period. This was verified in a study conducted under placebo-controlled, double blind conditions, with plasma levels of diazepam and its major metabolite, desmethyldiazepam, monitored throughout the course of the study. Severe symptoms of physiological withdrawal were observed within two days of replacement of diazepam with placebo capsules. The patient recovered promptly on reinstitution of diazepam administration, and relapsed during a second withdrawal phase. During an additional two week-period of placebo administration, the patient's condition first worsened, then gradually improved. Examination of plasma levels of diazepam and desmethyldiazepam indicated no obvious pharmacokinetic abnormalities. Thus, with long-term administration of diszepam, even in therapeutically accepted doses, withdrawal reactions can be encountered on abrupt termination.

Inhibition of diazepam metabolism by fluvoxamine: a pharmacokinetic study in normal volunteers.

The effect of fluvoxamine on the pharmacokinetics of diazepam and metabolically derived N-desmethyl-diazepam was investigated in eight healthy volunteers. Each subject received a single oral dose of diazepam (10 mg) in a control session and on the fourth day of a 16-day treatment with fluvoxamine maleate (100 to 150 mg daily). Compared with the control session, concurrent fluvoxamine intake was associated with increased mean peak plasma diazepam concentrations (from 108 to 143 ng/ml, geometric means, difference not significant), with a marked reduction in apparent oral diazepam clearance (from 0.40 to 0.14 ml/min/kg; p < 0.01) and with a prolongation in diazepam half-life (from 51 to 118 hours; p < 0.01). Although peak plasma N-desmethyldiazepam levels were similar in the two sessions, the time required for the metabolite to reach a peak was longer during fluvoxamine intake than in the control session (206 versus 62 hours; p < 0.01). N-Desmethyldiazepam area under the plasma concentration-time curve values were also significantly increased during fluvoxamine treatment. These data suggest that fluvoxamine inhibits the biotransformation of diazepam and its active N-demethylated metabolite. The magnitude of this interaction is likely to have considerable clinical significance.

Diazepam tapering in detoxification for high-dose benzodiazepine abuse.

The clinical characteristics and management of patients who abuse high doses of benzodiazepines are not well described. In a prospective open study, 23 subjects who abused high doses of benzodiazepines were admitted for detoxification. Urine or blood test results confirmed benzodiazepine use in all but one subject and multiple drug use in eight (35%). Median benzodiazepine dose was 150 mg (range 40 to 500 mg) of diazepam equivalent. Initial plasma concentrations (diazepam: median = 1245 ng/ml; desmethyldiazepam: median = 2961 ng/ml) were 400% to 800% higher than usual therapeutic concentrations. For detoxification, subjects were given a loading dose of diazepam equal to approximately 40% their reported daily consumption. This was followed with daily tapering of diazepam by 10%. This regimen resulted in a slow and gradual decline in drug concentrations. Withdrawal symptoms were assessed daily. Sixteen subjects completed detoxification in the hospital without complications. One subject became paranoid and confused on day 7 of withdrawal. This was attributed to a too-low initial loading dose and too-rapid tapering, which resulted in rapid drug elimination. Gradual reduction of diazepam dose appears to be an effective and safe approach for detoxifying abusers of high doses of benzodiazepines.

Multiple-dose halazepam kinetics.

Halazepam is a benzodiazepine used in the management of anxiety disorders or short-term relief of anxiety. Our study was undertaken to evaluate its steady-state kinetics and those of its major active plasma metabolite N- desalkylhalazepam . Eleven healthy men aged 19 to 35 yr were given oral, 40-mg halazepam tablets every 8 hr for 14 days. Plasma samples were analyzed by gas chromatography to determine levels of halazepam and N- desalkylhalazepam . Halazepam kinetics can best be described by a two-compartment open model with first-order absorption kinetics. The elimination phase t1/2s of halazepam and N- desalkylhalazepam were 34.7 and 57.9 hr. Steady-state levels were predictable from kinetic data and were reached by the third day for halazepam and by the eleventh day for N- desalkylhalazepam .

Desmethyldiazepam kinetics in the elderly after oral prazepam.

Our subjects were 15 young (aged 22 to 42 yr) and 14 elderly (aged 62 to 85 yr) people who took single oral doses of 20 mg prazepam. Plasma desmethyldiazepam (DMDZ) concentrations were determined in venous blood samples drawn up to 9 days after the dose. Appearance in blood of DMDZ was slow, with peak plasma levels reached in an average of 10 to 20 hr. First-order DMDZ appearance was observed in only 17 subjects. Volume of distribution of total DMDZ (range, 1.33 to 6.30 l/kg) and of unbound DMDZ after correction for protein binding (range, 43 to 243 l/kg) was larger in women than in men of all ages, and in the elderly as opposed to the young. Elimination half-life (range, 29 to 224 hr) rose with age in men (r = 0.66, p < 0.01) but not in women (r = -0.02). Clearance of unbound DMDZ (range, 2.9 to 31.2 ml/min/kg) was greater in women than in men of all ages, and declined with age in men (r = -0.40) but not in women (r = -0.06). As in the case of diazepam, age can influence DMDZ kinetics, but changes in drug disposition with age may differ between sexes.

Diazepam and N-desmethyldiazepam redistribution after heparin.

The effects of heparin, 1,000 U intravenously, on the blood, plasma, and free concentrations of diazepam and its metabolite, N-desmethyldiazepam, have been investigated 3 hr after oral administration of 10 mg diazepam to 5 normal subjects. The percent free diazepam and N-desmethyldiazepam increased 15 min after heparin from 1.66 +/- 0.35 to 3.99 +/- 1.88 (mean +/- SD; p less than 0.05) in the case of diazepam anolite. The actual free concentration of diazepam rose from 3.6 +/- 1.04 to 6.9 +/- 1.33 ng/ml (p less than 0.05) 15 min after heparin while total blood concentration was unchanged (144 +/- 54 vs 130 +/- 57 ng/ml). Free concentrations of N-desmethyldiazepam rose from 0.62 +/- 0.17 to 1.01 +/- 0.34 but the effect, though consistent, was not statistically significant. Blood concentrations did not change (15 +/- 3.2 vs 14 +/- 3.9 ng/ml). That free drug level rose without a change in blood or total plasma levels suggests that factors other than simple plasma binding displacement are involved in this drug interaction.

Repeated diazepam dosing in cirrhotic patients: cumulation and sedation.

Five medically stable male patients with cirrhosis and four healthy age- and sex-matched controls received single 5-mg oral doses of diazepam (DZ) daily for 22 consecutive days. Plasma concentrations of DZ and its major metabolite desmethyldiazepam (DMDZ) were measured daily during the period of dosing and in the 7-day washout period that followed. Clinical self-ratings of sedation, fatigue, mood state, and sleep patterns were obtained daily during the period of dosage with the use of visual analogue scales. Steady-state plasma DZ concentrations were higher (165 and 98 ng/ml), and DMDZ concentrations tended to be higher (399 and 206 ng/ml), in cirrhotics than in controls. Increases in self-rated daytime sedation also were greater in cirrhotics than in controls and correlated strongly with total DZ and DMDZ plasma concentration during the first 2 wk of diazepam dosing. Thus, reduced clearance of DZ in cirrhotics leads to increased cumulation during long-term dosing. This in turn is associated with increased clinical sedation. Sedative effects are partly offset as treatment proceeds because of adaptation or tolerance. Based on kinetic findings, diazepam can be given safely to cirrhotic patients provided daily dosage is reduced by approximately 50%.

Differences in in vitro binding of diazepam and N-desmethyldiazepam to maternal and fetal plasma proteins at birth: relation to free fatty acid concentration and other parameters.

The in vitro protein binding of diazepam and its major plasma metabolite, N-desmethyldiazepam (DMD), was measured in women at full term of pregnancy, in mixed cord plasma (fetal plasma), and in control subjects. The free fraction of both drugs was determined by ultrafiltration at 37 degrees. The mean free fraction of diazepam was 0.023 +/- 0.0043 in control subjects and rose to 0.040 +/- 0.0071 in women at term, whereas in their fetuses the free fraction was 0.021 +/- 0.0057. The unbound fraction of DMD was 0.030 +/- 0.0084 in control subjects, 0.052 +/- 0.015 in women at term, and 0.035 +/- 0.010 in fetal plasma. The free fractions of both compounds differed in women at term and in their fetuses. The higher free fraction of the two drugs in maternal plasma than in cord plasma may be explained by elevated maternal concentrations of free fatty acids (and triglycerides), which may act as displacing agents in maternal plasma. The higher binding of these drugs in fetal plasma than in maternal plasma may explain their cumulation in vivo and may be responsible for the frequently observed adverse effects of maternal diazepam treatment on the newborn infant.

N-desmethyldiazepam: a new metabolite of chlordiazepoxide in man.

Following administration of chlordiazepoxide HCl to man, N-desmethyldiazepam, a known metabolite of diazepam (Valium), was identified in plasma. The metabolite was identified on the basis of its thin-layer chromatographic (TLC) mobility, electron-capture gas-chromatographic (EC-GC) retention time, and mass spectrum relative to authentic N-desmethyldiazepam. Plasma levels of N-desmethyldiazepam in subjects receiving both single and chronic doses of chlordiazepoxide were determined by an EC-GC method with a limit of sensitivity of 10 ng/ml using 2-ml samples and by a radioimmunoassay procedure which had a limit of sensitivity of 20 ng/ml using a 0.1-ml sample. Both assay methods gave good agreement for the levels of N-desmethyldiazepam. In subjects receiving a single 30-mg oral or intravenous dose of chlordiazepoxide, measurable levels of N-desmethyldiazepam in plasma (10 to 60 ng/ml) were obtained 24 to 72 hr after administration. In 5 subjects receiving 10 mg of chlordiazepoxide three times a day, steady-state levels of N-desmethyldiazepam in plasma were reached after about 1 wk of administration. The mean maximum and minimum steady-state levels of N-desmethyldiazepam were 260 and 220 ng/ml of plasma, respectively. Similar steady-state levels were observed on treatment with 30 mg of chlordiazepoxide over 24 hr.