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.

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.

Low doses of ethanol markedly potentiate the anti-seizure effect of diazepam in a mouse model of difficult-to-treat focal seizures.

Ethanol is commonly used as a solvent in injectable formulations of poorly water-soluble drugs. The concentrations of ethanol in such formulations are generally considered reasonably safe. It is long known that ethanol can potentiate central effects of sedatives and tranquillizers, particularly the benzodiazepines, most likely as a result of a synergistic interaction at the GABAA receptor. However, whether this occurs at the low systemic doses of ethanol resulting from its use as solvent in parenteral formulations of benzodiazepines is not known. In the present study we evaluated whether a commercial ethanol-containing aqueous solution of diazepam exerts more potent anti-seizure effects than an aqueous solution of diazepam hydrochloride or an aqueous emulsion of this drug in the intrahippocampal kainate model of temporal lobe epilepsy in mice. Spontaneous epileptic seizures in this model are known to be resistant to major antiepileptic drugs. Administration of the ethanol-containing formulation of diazepam caused an almost complete suppression of seizures. This was not seen when the same dose (5 mg/kg) of diazepam was administered as aqueous solution or emulsion, although all three diazepam formulations resulted in similar drug and metabolite concentrations in plasma. Our data demonstrate that ethanol-containing solutions of diazepam are superior to block difficult-to-treat seizures to other formulations of diazepam. To our knowledge, this has not been demonstrated before and, if this finding can be translated to humans, may have important consequences for emergency treatment of acute seizures, series of seizures, and initial treatment of status epilepticus in patients.

Long-term storage of authentic postmortem forensic blood samples at -20°C: measured concentrations of benzodiazepines, central stimulants, opioids and certain medicinal drugs before and after storage for 16-18 years.

The long-term stability of benzodiazepines, opioids, central stimulants and medicinal drugs in authentic postmortem blood samples was studied. All together, 73 samples were reanalyzed after storage at -20°C for 16-18 years. At reanalysis samples containing diazepam, nordiazepam and flunitrazepam demonstrated only small changes during long-term storage when mean and median drug concentrations were compared, while clonazepam concentrations tended to decrease. Samples containing amphetamine, morphine, codeine and 'acidic' medicinal drugs as paracetamol and meprobamate also showed small changes over 16-18 years in mean and median drug concentrations at a group level. For many drugs, however, single samples could demonstrate marked concentration changes, both increases and decreases during storage. For 'alkaline' medicinal drugs, concentration losses were observed in most cases.

Direct determination of diazepam and its glucuronide metabolites in human whole blood by µElution solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A µElution solid-phase extraction (SPE) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of diazepam, nordiazepam, oxazepam, oxazepam glucuronide, temazepam and temazepam glucuronide in human whole blood is presented. 200 µL of whole blood samples were loaded onto a Waters Oasis HLB 96-well µElution SPE plate using 75 µL of methanol as the elution solvent, and the eluents were injected into an Eclipse XDB C18 column. No hydrolysis, solvent transfer, evaporation or reconstitution was involved in the sample preparation procedures. Tandem mass spectrometric detection with Turbo Ion Spray was conducted via multiple reaction monitoring (MRM) under positive ionization mode. The method was validated and proved to be accurate (accuracy within 93-108%), precise (intra-day RSD<9.9% and inter-day RSD<7.2%) and sensitive with limits of detection (LOD) in the range of 0.05-0.25 ng/mL for all the compounds. Extraction recoveries were in the range of 31-80% for all the analytes. This method demonstrated to be reproducible and reliable. The applicability of the method was demonstrated by analysis of several forensic cases involving diazepam and its metabolites.

Concentrations of drugs determined in blood samples collected from suspected drugged drivers in England and Wales.

This communication reports the blood concentrations of alcohol and drugs from 376 cases of alleged driving under the influence of drugs analysed at the Forensic Science Service Chorley and London laboratories between February 2010 and March 2011. The samples were analysed for alcohol, amphetamine, benzodiazepines, cocaine, MDMA, opiates, γ-hydroxybutyrate (GHB), ketamine, methadone and methylmethcathinone (the 4-isomer of which is known as mephedrone). The results were interpreted with respect to the number and type of drugs of abuse detected and the concentrations measured. Alcohol was quantified in 113 cases (30%), and of these a level in excess of the prescribed UK limit for driving of 80 mg% was present in 90 cases. In 80 cases, only the concentration of alcohol was measured, the concentrations of both drugs and alcohol were measured in 33 cases. In the remaining 263 cases, only the concentrations of relevant drugs of abuse were measured. The most common drug of abuse quantified was cocaine which was detected in 92 cases, either as the active drug or as its major metabolite benzoylecgonine, followed by diazepam which was quantified in 76 cases. Concentrations of some new drugs, and drugs rarely reported in driving under the influence cases are also presented.

Evaluation of plasma diazepam and nordiazepam concentrations following administration of diazepam intravenously or via suppository per rectum in dogs.

OBJECTIVE: To evaluate the pharmacokinetics of diazepam administered per rectum via compounded (ie, not commercially available) suppositories and determine whether a dose of 2 mg/kg in this formulation would result in plasma concentrations shown to be effective for control of status epilepticus or cluster seizures (ie, 150 to 300 ng/mL) in dogs within a clinically useful interval (10 to 15 minutes). ANIMALS: 6 healthy mixed-breed dogs. PROCEDURES: Dogs were randomly assigned to 2 groups of 3 dogs each in a crossover-design study. Diazepam (2 mg/kg) was administered IV or via suppository per rectum, and blood samples were collected at predetermined time points. Following a 6- or 7-day washout period, each group received the alternate treatment. Plasma concentrations of diazepam and nordiazepam were analyzed via reversed phase high-performance liquid chromatography. RESULTS: Plasma concentrations of diazepam and nordiazepam exceeded the targeted range ≤ 3 minutes after IV administration in all dogs. After suppository administration, targeted concentrations of diazepam were not detected in any dogs, and targeted concentrations of nordiazepam were detected after 90 minutes (n = 2 dogs) or 120 minutes (3) or were not achieved (1). CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of these results, administration of 2 mg of diazepam/kg via the compounded suppositories used in the present study cannot be recommended for emergency treatment of seizures in dogs.

Blood drug concentrations of benzodiazepines correlate poorly with actual driving impairment.

BACKGROUND: The use of benzodiazepine receptor agonists can significantly impair driving performance. The aim of this review was to determine if there is a relation between blood concentrations of these drugs and the degree of driving impairment. METHODS: A literature search was conducted to identify driving studies that examined the effects of benzodiazepine receptor agonists. Studies were included if the on-the-road driving test was employed, using the standard deviation of lateral position (SDLP), i.e., the weaving of the car, as primary outcome measure. RESULTS: A total of 24 studies were identified that employed the on-the-road driving test to examine driving performance after administration of benzodiazepine receptor agonists. Eleven of these studies (45.8%) measured blood drug concentrations after the on-the-road driving test was performed. Technical reports of some of these studies provided individual data on blood drug concentrations and ΔSDLP (the ΔSDLP difference between drug and placebo). While group differences in concentrations were found as evidenced by significant effects of dose and time of driving since time of drug ingestion, no significant relationship between individual blood drug concentrations and ΔSDLP was found in any of the studies. CONCLUSION: While group mean average ΔSDLP and blood drug concentration sometimes correlate, individual differences in blood drug concentrations of benzodiazepine receptor agonists correlate poorly with driving impairment. From the currently available data, it must be concluded that there are no significant relationships between individual blood drug concentration and ΔSDLP. Future driving studies should assess blood drug levels as a standard procedure, to enable further research into the relationship between blood drug concentration and performance impairment.

Concentrations of diazepam and nordiazepam in 1,000 blood samples from apprehended drivers--therapeutic use or abuse of anxiolytics?

Using an in-house forensic toxicology database, we selected 1000 cases of driving under the influence of drugs (DUIDs) over a 12-month period if diazepam (D) and nordiazepam (ND) were both present in the blood samples. Quantitative analysis of D and ND in blood was done by solvent extraction (butyl acetate) and capillary column gas chromatography (GC) with a nitrogen-phosphorous (N-P) detector. The limits of quantitation of this analytical method for D and ND in blood were 0.05 mg/L. The correlation between D and ND concentrations in blood was statistically significant (r = .58, P < .001), as expected for a parent drug and its primary metabolite. However, the frequency distributions were markedly skewed to the right with mean (median) and highest concentrations of 0.37 (0.20) and 6.1 mg/L for D and 0.39 (0.20) and 5.6 mg/L for ND. The mean (median) total concentration (D + ND) was 0.76 mg/L (0.50 mg/L), and the concentration ratios D/ND and ND/D were 1.29 (median 0.95) and 1.41 (median 1.06), respectively. In 90 cases (9%), the concentration of D in blood exceeded 0.83 mg/L, which corresponds to an upper therapeutic limit in plasma (∼1.5 mg/L), assuming a plasma/blood distribution ratio of 1.8:1.

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.

Biotransformation of diazepam in a clinically relevant flat membrane bioreactor model using primary porcine hepatocytes.

In vitro biotransformation of drug using commercial culture medium with serum may not be the ideal culture medium for clinical application in extracorporeal bioartificial liver support (BAL) systems. In these systems, patient's blood or plasma is plumbed to primary hepatocytes within a seeded bioreactor, creating interaction between plasma and seeded hepatocytes. To address this situation, we investigated the biotransformation potential of diazepam in primary porcine hepatocytes with a flat membrane bioreactor (FMB); we used human plasma exposure and serum-free media in organotypical double gel culture model for long-term culture. We investigated diazepam clearance and all major metabolites of diazepam, such as oxazepam, temazepam, and desmethyldiazepam, in conventional single gel and organotypical sandwich models and compared them to the FMB model. Diazepam elimination was higher in double gel cultures with exposure to both SF 3 medium conditions and plasma, when compared to the single gel model in a Petri dish. It was observed that in the FMB, diazepam elimination was stable at about 3 pg/h/cell in plasma and SF 3 exposure. Oxazepam synthesis in the bioreactor was approximately one quarter less than in the Petri dish, but there were no differences between N-desmethyldiazepam and temazepam synthesis in double gel culture. In the flat membrane bioreactor, there was no decrease in the biotransformation of diazepam in plasma exposure compared with the control group. Our results suggest that this plasma exposure bioreactor may offer a useful approach in clinical use of extracorporeal BAL, as well as for drug metabolite investigation into toxicological research.

Quantitation of benzodiazepines in blood and urine using gas chromatography-mass spectrometry (GC-MS).

The benzodiazepine assay utilizes gas chromatography-mass spectrometry (GC-MS) for the analysis of diazepam, nordiazepam, oxazepam, temazepam, lorazepam, alpha-hydroxyalprazolam, and alpha-hydroxytriazolam in blood and urine. A separate assay is employed for the analysis of alprazolam. Prior to solid phase extraction, urine specimens are subjected to enzyme hydrolysis. The specimens are fortified with deuterated internal standard and a five-point calibration curve is constructed. Specimens are extracted by mixed-mode solid phase extraction. The benzodiazepine extracts are derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSFTA) producing tert-butyldimethyl silyl derivatives; the alprazolam extracts are reconstituted in methanol without derivatization. The final extracts are then analyzed using selected ion monitoring GC-MS.

LC-MS/MS analysis of 13 benzodiazepines and metabolites in urine, serum, plasma, and meconium.

We describe a single method for the detection and quantitation of 13 commonly prescribed benzodiazepines and metabolites: alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam and 7-aminoclonazepam in urine, serum, plasma, and meconium. The urine and meconium specimens undergo enzyme hydrolysis to convert the compounds of interest to their free form. All specimens are prepared for analysis using solid-phase extraction (SPE), analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and quantified using a three-point calibration curve. Deuterated analogs of all 13 analytes are included as internal standards. The instrument is operated in multiple reaction-monitoring (MRM) mode with an electrospray ionization (ESI) source in positive ionization mode. Urine and meconium specimens have matrix-matched calibrators and controls. The serum and plasma specimens are quantified using the urine calibrators but employing plasma-based controls. Oxazepam glucuronide is used as a hydrolysis control.

Simultaneous determination and quantification of 12 benzodiazepines in serum or whole blood using UPLC/MS/MS.

The benzodiazepines are a large, commonly prescribed family of psychoactive drugs. We describe a method permitting the simultaneous detection and quantification of 12 benzodiazepines in serum using ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS). Analytes included alprazolam, temazepam, oxazepam, nordiazepam, clonazepam, lorazepam, diazepam, chlordiazepoxide, midazolam, flunitrazepam, 7-aminoclonazepam, and 7-aminoflunitrazepam. Sample pretreatment is simple consisting of protein precipitation using cold acetonitrile (ACN) mixed with the deuterated internal standards. Samples were capped and vortexed for 5 min to ensure maximum precipitation. Following a 5-min centrifugation period, 400 microL of the supernatant was transferred to a clean tube and evaporated down under nitrogen. Samples were reconstituted in 200 microL of a deionized water:ACN (80:20) mixture and transferred to appropriate vials for analysis. Chromatographic run time was 7.5 min, and the 12 analytes were quantified using multiple reaction monitoring (MRM) and 6-point calibration curves constructed for each analyte at concentrations covering a clinically significant range.

Simultaneous analysis of diazepam and its metabolites in rat plasma and brain tissue by HPLC-UV and SPE.

Diazepam is frequently used as an adjuvant during antidepressant therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. To clarify the matter, a study is currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significant set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam. A feasible and reliable HPLC method has been developed for the simultaneous determination of these compounds in plasma and brain tissue of rats. The method has been applied to "normal" rats and to genetic rat models of depression in order to estimate drug metabolism in different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer/acetonitrile mixture as the mobile phase. The detection wavelength was 238 nm. An original sample pre-treatment, based on solid-phase extraction (SPE) was developed in order to eliminate endogenous interference, using only 250 microL of matrix (brain homogenate or plasma) for a complete analysis. The method has been validated with good results in terms of precision, extraction yield, sensitivity, selectivity and accuracy on both matrices and has been successfully applied to samples from some rats subjected to the preliminary study. The obtained data will hopefully contribute to the clarification of possible differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.

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.

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.

Fatal intoxication with milnacipran.

The antidepressant milnacipran is a double serotonin/noradrenalin reuptake inhibitor. The low reported incidence of intoxication indicates excellent tolerance in comparison with tricyclic and second generation antidepressants. We report a fatal intoxication associating milnacipran, at blood levels (femoral=21.5 mg/l, cardiac=20 mg/l) 40-fold higher than the usual treatment concentration, and six other molecules (fluoxetine, norfluoxetine, sertraline, cyamemazine, nordazepam and oxazepam) at therapeutic levels. To the best of our knowledge, this is the first reported fatal intoxication involving milnacipran.

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.