Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864).

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC MS/MS), liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4'-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.

Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography-tandem mass spectrometry.

Benzodiazepines and zolpidem are generally prescribed as sedative, hypnotics, anxiolytics or anticonvulsants. These drugs, however, are frequently misused in drug-facilitated crime. Therefore, a rapid and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for identification and quantification of benzodiazepines, zolpidem and their metabolites in urine using deuterium labeled internal standards (IS). Urine samples (120 µL) mixed with 80 µL of the IS solution were centrifuged. An aliquot (5 µL) of the sample solution was directly injected into the LC-MS/MS system for analysis. The mobile phases consisted of water and acetonitrile containing 2mM ammonium trifluoroacetate and 0.2% acetic acid. The analytical column was a Zorbax SB-C18 (100 mm × 2.1 mm i.d., 3.5 µm, Agilent). The separation and detection of 18 analytes were achieved within 10 min. Calibration curves were linear over the concentration ranges of 0.5-20 ng/mL (zolpidem), 1.0-40 ng/mL (flurazepam and temazepam), 2.5-100 ng/mL (7-aminoclonazepam, 1-hydroxymidazolam, midazolam, flunitrazepam and alprazolam), 5.0-200 ng/mL (zolpidem phenyl-4-carboxylic acid, α-hydroxyalprazolam, oxazepam, nordiazepam, triazolam, diazepam and α-hydroxytriazolam), 10-400 ng/mL (lorazepam and desalkylflurazepam) and 10-100 ng/mL (N-desmethylflunitrazepam) with the coefficients of determination (r(2)) above 0.9971. The dilution integrity of the analytes was examined for supplementation of short linear range. Dilution precision and accuracy were tested using two, four and ten-folds dilutions and they ranged from 3.7 to 14.4% and -12.8 to 12.5%, respectively. The process efficiency for this method was 63.0-104.6%. Intra- and inter-day precisions were less than 11.8% and 9.1%, while intra- and inter-day accuracies were less than -10.0 to 8.2%, respectively. The lower limits of quantification were lower than 10 ng/mL for each analyte. The applicability of the developed method was successfully verified with human urine samples from drug users (n=21). Direct urine sample injection and optimized mobile phases were introduced for simple sample preparation and high-sensitivity with the desired separation.

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.

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.

Ignition of a human body by a modest external source: a case report.

A case of sustained combustion of a human body that occurred in 2006 in Geneva, Switzerland, is presented. The body of a man was discovered at home and found to have been almost completely incinerated between the knees and the mid-chest, with less damage to the head, arms, lower legs and feet. His dog was also found dead just behind the house door. The external source of ignition was most likely a cigarette or a cigar. The chair in which the man had been sitting was largely consumed while other objects in the room exhibited only a brown oily or greasy coating and were virtually undamaged. Toxicological analyses carried out on the blood from the lower legs showed low levels of desalkylflurazepam. Alcohol concentration was 1.10 per thousand, carboxyhaemoglobin levels were 12% and cyanide concentration was 0.05 mg/L. Toxicological analyses carried out on the dog's blood showed carboxyhaemoglobin levels at 65%.

Benzodiazepine-mediated structural changes in the multidrug transporter P-glycoprotein: an intrinsic fluorescence quenching analysis.

P-glycoprotein expressed in Pichia pastoris was used to study the drug binding sites of different benzodiazepines. The effect of bromazepam, chlordiazepoxide, diazepam and flurazepam on P-glycoprotein structure was investigated by measuring the intrinsic fluorescence of the transporter tryptophan residues. Purified mouse mdr1a transporter in mixed micelles of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid and 1,2-dimiristoyl-sn-glycerol-3-phosphocholine emitted fluorescence at 340 nm indicative of the fluorophores in a relatively apolar environment. Acrylamide and iodide ion were used as collisional quenchers toward distinct regions of the transporter, the protein and the interface protein-surface, respectively. Binding of ATP induced conformational changes at the protein surface level in accordance with the location of the nucleotide binding sites. Bromazepam interaction with the transporter was located at the protein-surface interface, diazepam at the membrane region and chlordiazepoxide at the protein surface. Only the flurazepam interaction site was not detected by the quenchers used. All benzodiazepines were able to elicit reorientation of the protein fluorophores on the P-glycoprotein-ATP complex.

Suicide by multidrug ingestion: hypothesis on the role played by the self-administration of activated charcoal.

A fatal suicidal ingestion of drugs, together with activated charcoal, is reported. The death occurred 31 hours after the self-administration. The autopsy revealed a large amount of gastric content that appeared to be a compact mass of black color. Toxicologic analyses showed the presence of toxic levels of desalkylflurazepam and trazodone; metamizole and pridinol were also detected. The obtained results supported the hypothesis of a death due to acute intoxication delayed by the self-administration of activated charcoal, which elimination was probably hindered by the action of pridinol.

Quantification of benzodiazepines in whole blood and serum.

A high-performance liquid chromatography method for the determination of benzodiazepines and their metabolites in whole blood and serum using mass spectrometry (MS) and photodiode array (PDA) detection is presented. The combination of both detection types can complement each other and provides extensive case relevant data. The limits of quantification (LOQ) with the MS detection lie between 2 and 3 microg/l for the following benzodiazepines or metabolites: 7-amino-flunitrazepam, alprazolam, desalkyl-flurazepam, desmethyl-flunitrazepam, diazepam, flunitrazepam, flurazepam, alpha-hydroxy-midazolam, lorazepam, midazolam, nitrazepam, nordazepam and oxazepam, respectively 5 microg/l for lormetazepam and 6 microg/l for bromazepam. The LOQ of clobazam determined with the PDA detector is 10 microg/l. A convenient approach for determining the measurement uncertainty of the presented method--applicable also for other methods in an accreditation process--is presented. At low concentrations (<10 microg/l), measurement uncertainty was estimated to be about 50%, and at concentrations >180 microg/l, it was estimated to be about 15%. One hundred and twenty-eight case data acquired over 1 year are summarised.

Effects of itraconazole on the plasma kinetics of quazepam and its two active metabolites after a single oral dose of the drug.

The effects of itraconazole, a potent inhibitor of cytochrome P450 (CYP) 3A4, on the plasma kinetics of quazepam and its two active metabolites after a single oral dose of the drug were studied. Ten healthy male volunteers received itraconazole 100 mg/d or placebo for 14 days in a double-blind randomized crossover manner, and on the fourth day of the treatment they received a single oral 20-mg dose of quazepam. Blood samplings and evaluation of psychomotor function by the Digit Symbol Substitution Test and Stanford Sleepiness Scale were conducted up to 240 h after quazepam dosing. Itraconazole treatment did not change the plasma kinetics of quazepam but significantly decreased the peak plasma concentration and area under the plasma concentration-time curve of 2-oxoquazepam and N-desalkyl-2-oxoquazepam. Itraconazole treatment did not affect either of the psychomotor function parameters. The present study thus suggests that CYP 3A4 is partly involved in the metabolism of quazepam.

Benzodiazepine binding studies on living cells: application of small ligands for fluorescence correlation spectroscopy.

We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.

Rapid down-regulation of [3H]zolpidem binding to rat brain benzodiazepine receptors during flurazepam treatment.

In a previous study, it was found that down-regulation of benzodiazepine (BZ) binding in rats treated 4 weeks with flurazepam was relatively greater and more widespread when measured with [3H]zolpidem, a selective 'BZ1 receptor' ligand, than that measured with the non-selective ligand, [3H]flunitrazepam. In the present study, the time course for down-regulation of [3H]zolpidem binding was studied in rats treated with flurazepam. [3H]Zolpidem binding was also studied in rats given a midazolam treatment shown to cause tolerance. Rats were chronically treated with flurazepam for 1 or 2 weeks, or with midazolam for 3 weeks, then killed immediately after the treatment. Another group of rats was acutely treated with desalkyl-flurazepam and killed 30 min later. After 2 weeks of flurazepam treatment, the Bmax of [3H]zolpidem binding was decreased by 22% in cerebral cortex, 26% in cerebellum and 33% in hippocampus, with no change in the Kd in any region. After 1 week of flurazepam treatment, the Bmax was decreased by 23% in cerebellum and 14% in hippocampus, but not changed in cerebral cortex. The Kd was increased in cerebral cortex, but not in cerebellum or hippocampus. Neither the Bmax nor the Kd of [3H]zolpidem binding was affected by acute desalkyl-flurazepam treatment, or by 3 weeks of midazolam treatment. These results, in combination with previous findings, which showed no change in [3H]flunitrazepam binding after 1 or 2 week flurazepam treatment, and no change in cerebellum even after the 4 week treatment, may indicate a shift in BZ receptor subtypes in flurazepam-tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Gas chromatographic-mass spectrometric method for the determination of flurazepam and its major metabolites in mouse and rat plasma.

A capillary gas chromatographic-negative chemical ionization (NCI) mass spectrometric method for the determination of flurazepam and its metabolites N-1-hydroxyethyl-flurazepam and N-1-desalkyl-flurazepam in mouse and rat plasma was described. Derivatization of the metabolites of flurazepam with BSTFA allowed a highly stable, accurate, and sensitive GC-MS analysis. The use of a single internal standard (halazepam) for the quantification of all compounds saved cost and time. The detection limits were 0.1 ng/ml for N-hydroxyethyl-flurazepam-TMS (M(r) = 404), 0.5 ng/ml for desalkyl-flurazepam-TMS (M(r) = 360), and 0.5 ng/ml for flurazepam (M(r) = 387) with an injection volume of 1 microliter at a signal-to-noise ratio greater than 5. The quantitation limit was set to 10 ng/ml for all compounds.

Quazepam and flurazepam: differential pharmacokinetic and pharmacodynamic characteristics.

Quazepam and flurazepam share pharmacokinetic properties that result in prevention of early-morning insomnia, daytime rebound anxiety, and withdrawal rebound insomnia. Yet sleep laboratory and performance studies demonstrated that during a 1- to 4-week administration period quazepam had a low potential for causing daytime drowsiness or impairment. This profile may be related to several factors, such as differences in quazepam's metabolic pathways; plasma pharmacokinetics; rate of brain uptake, redistribution, and clearance; as well as differences in receptor binding and kinetics.

Nonisotopic receptor-binding assay for benzodiazepine receptors utilizing a fluorophore labeled ligand.

A nonisotopic receptor-binding assay method provides a new approach for the study of receptor-ligand interactions and a possible receptor assay for benzodiazepine drugs. The proposed method is based upon the use of fluorescence-labeled drugs and a chromatographic system which accepts samples without deproteinization. The effectiveness of the technique is illustrated in a study of benzodiazepine receptor-drug-binding interactions.

Propranolol does not alter flutoprazepam kinetics and metabolism in the rat.

The influence of propranolol on the disposition of flutoprazepam, a benzodiazepine derivative extensively biotransformed by hepatic microsomal oxidation, was evaluated in the rat. Propranolol was infused subcutaneously with osmotic minipumps (5 mg/day) to obtain steady-state concentrations of about 200 ng/ml. Flutoprazepam (5 mg/kg) was given intraperitoneally on the third day of propranolol infusion. There was some variability in flutoprazepam disposition, consistent with the concept of an extensive first-pass metabolism of high-extraction drugs. Propranolol had no significant effects on the kinetics of flutoprazepam or norflutoprazepam, an active metabolite possibly accounting for a substantial part of the parent compound's pharmacological and clinical effects. It was concluded that there is no evidence of any pharmacokinetic interaction between this beta-adrenoceptor blocker and flutoprazepam in the rat.

Plasma concentrations of flurazepam and midazolam in chronic insomniacs during 14-day use and their relationship to therapeutic effects and next-day performance and mood.

Blood samples were drawn from each of 99 chronic insomniacs twice during washout (days -20 and -6) and six times during the study (mornings after study nights -1, 1, 2, 7, 13, and 14) to examine the relationship between morning-after drug plasma levels, sleep efficiency, next-day mood, and performance. Patients in the four treatment groups received either flurazepam 30 mg, flurazepam 15 mg, midazolam 15 mg, or placebo. Plasma drug concentrations of N-desalkylflurazepam and midazolam were measured by electron-capture gas chromatography. Values of midazolam during the 14-day study were at or near the sensitivity limit of the assay and were not used in the calculations. Levels of N-desalkylflurazepam increased as expected during the 14 days. Mean level for the high-dose flurazepam group was approximately twice that of the low-dose group. The main consistency in the correlations, which were found on days 13 and 14, was that the high-dose desalkylflurazepam concentrations had a negative correlation with two independent measures of sleep latency. However, otherwise there was little or no relationship between N-desalkylflurazepam levels and sleep efficiency or next-day behavior. Issues of tolerance, individual variability in baseline and response, and their contribution to the findings are discussed.

Carcinogenicity study of doxefazepam administered in the diet to Sprague-Dawley rats.

Groups of 50 male and 50 female Sprague-Dawley rats were given food containing sufficient doxefazepam, a benzodiazepine derivative, to ensure intakes of 0, 3, 10, or 30 mg/kg/day. These dosages respectively correspond to 2, 20, and 60 times the mean daily hypnotic dose level of an adult man. Rats were treated for 104 weeks and then euthanized. An extensive autopsy was performed on those animals that died intercurrently and on euthanized animals. The chronic administration of doxefazepam did not influence the survival of the rats. No treatment-related changes in clinical signs and body weight gains occurred and malignant tumor rates were similar in controls and treated animals. A significant linear trend in the incidence of hepatocellular neoplasms, primarily benign, was observed in the female treated groups. This higher incidence was not associated to a higher occurrence of focal hyperplasia or other preneoplastic lesions in treated rats. The brain, a target organ for the pharmacological activity of doxefazepam, was carefully examined to search for microscopic foci of proliferative cells. A total of 12 and 6 malignant gliomas were observed in male and female rats, respectively; only two were noticed at autopsy. These tumors were mainly of the oligodendroglioma type commonly found in aged rats. Their incidence was slightly higher in treated rats, but results were not of statistical significance. The overall evaluation of the present study indicates that doxefazepam is noncarcinogenic in rats. However, the increase in liver adenomas found here as well as in previous bioassays with similar drugs and the lack of reliable historical data on the incidence of brain tumors in benzodiazepine-treated rodents suggest that additional experimental and epidemiological studies should be undertaken to exhaustively assess the toxic potential of this widely used class of drugs.

Pharmacokinetics of flutoprazepam, a novel benzodiazepine drug, in normal subjects.

The single dose pharmacokinetics of flutoprazepam and its active N-desalkyl metabolite were determined in 8 normal subjects by using newly developed, highly sensitive, GC-MS and HPLC techniques. Following a 2 mg dose of the drug, the concentrations of unchanged flutoprazepam in serum were extremely low (below 5 ng/ml at 2 h) and declined rapidly to undetectable levels within 6-9 h after dosing. At all sampling times, the serum concentration of the N-dealkylated metabolite (N-desalkylflurazepam) was much greater than that of the parent compound. This metabolite appeared in serum rapidly (within 2 h), reached a peak between 2 and 12 h and declined slowly, with an elimination half-life of about 90 h on average. The serum concentration of two additional putative metabolites (3-hydroxy-flutoprazepam and N-desalkyl-3-hydroxy-flutoprazepam) was below the limit of detection (2 ng/ml) in all samples. Mild CNS effects (documented by prolonged choice reaction time) were present at 2 and 4 h but were no longer detectable at 9 h. It is suggested that unchanged flutoprazepam is unlikely to contribute significantly to clinical effects and that the drug exerts its therapeutic activity through conversion to the slowly eliminated N-desalkyl metabolite.