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%.

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.

Application of capillary electrophoresis to the simultaneous screening and quantitation of benzodiazepines.

Capillary electrophoresis (CE) is an attractive approach for the analysis of drugs in body fluids. We made a simultaneous analysis of nitrazepam, diazepam, estazolam, bromazepam, triazolam and flurazepam using CE with on-column detection at 200 nm. We obtained the best electropherograms under a condition of 5 mM phosphate-borate (pH 8.5) containing 50 mM SDS and 15% methanol. We examined the effect of the sample solvent matrix on the electropherograms obtained, indicating that increasing the methanol content in the sample solvent or the injection volume above a certain threshold limit decreased the resolution. We then focused on application of the CE to the analysis of the drugs in spiked serum, being appropriate for an analysis within 25 min. Linearity, the detection limit, accuracy and reproducibility were established using this method. The calibration curve was linear up to 1 mg/l of serum concentration. The lower limit of detection was 5 pg per injection and 0.025 mg/l of the serum concentration for all the compounds except for flurazepam, for which they were 40 pg/injection and 0.2 mg/l. The detection limits obtained allowed toxicological and pharmacological determinations for nitrazepam, diazepam, estazolam and bromazepam, but not for triazolam and flurazepam. Only toxic blood levels for the latter two benzodiazepines could be quantified by this method. We concluded that the CE could at least be applicable to simultaneous screening for toxic levels of benzodiazepines. We suggest that this technique may offer criminal toxicologists a rapid, simple and adaptable approach for the estimation of many other drugs in body fluids.

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.

Intranasal absorption of flurazepam, midazolam, and triazolam in dogs.

Intranasal delivery of flurazepam, midazolam, and triazolam was studied in a dog model as a possible alternate route of drug administration for treatment of insomnia. Four beagles received each hypnotic by both intranasal and oral routes on two separate occasions. Plasma concentrations for each hypnotic after dosing were measured by electron-capture gas-liquid chromatography. The mean intranasal absorption rates (tmax) of flurazepam, midazolam, and triazolam were 1.7, 2.0, and 2.6 times faster, respectively, compared with oral dosing. The mean dose-normalized peak concentrations (Cmax) after intranasal delivery were 16.4, 2.9, and 3.4 times higher, respectively, versus oral administration. The mean dose-normalized AUCs estimated for these compounds after nasal administration were 2.4-, 2.5-, and at least 2-fold larger than after oral administration for midazolam, triazolam, and flurazepam, respectively. If these observations can be extrapolated to humans, the faster absorption achieved by the intranasal route would appear to benefit insomniacs characterized by difficulty in falling asleep because of an anticipated faster sedative effect onset. The higher peak concentrations and larger amounts absorbed in the case of intranasal midazolam and triazolam delivery may lead to dose reduction.

Behavioral tolerance to flurazepam.

Rats were trained to earn 180 food pellets in daily, fixed-interval 1-min sessions. When performance had stabilized, a Before group was given a weekly 16 mg/kg flurazepam injection IP for 3 weeks immediately before the sessions, while an After group received their weekly injections immediately after the sessions. Then, the After group received 3 such weekly injections before the sessions. Behavioral tolerance developed by the 2nd flurazepam injection for the Before group, but for the After group, the 3 postsession flurazepam injections resulted in subsequent tolerance to presession flurazepam injection for session lever presses, but not for the time taken to earn 180 pellets. Dispositional tolerance to the serum elimination rate of flurazepam did not develop over the course of 3 injections. Behavioral suppression still evident in the initial portion of sessions with the 2nd and 3rd presession injection coincided with the duration of rising and high levels of serum flurazepam.

Determination of diazepam and nordazepam in milk and plasma in the presence of oxazepam and temazepam.

For studies on the excretion of drugs into milk a sensitive high-performance liquid chromatographic assay was developed to quantitate diazepam and nordazepam in the milk and plasma of humans and rabbits in the presence of their major metabolites, oxazepam and temazepam. Flurazepam was used as an internal standard. The assay involves extractions with diethyl ether and an additional acid clean-up step. Chromatographic separation was achieved by a LiChrospher 60 RP-select B (5 microns) column and KH2PO4- acetonitrile (69:31, v/v) adjusted to pH 2.80 as a mobile phase. The same extraction and chromatographic conditions were suited to both types of samples, milk and plasma. The limits of determination using ultraviolet detection at 241 nm was for diazepam 20 ng/ml and for nordazepam 15 ng/ml. The absolute recoveries of diazepam, nordazepam and flurazepam in human milk were 84, 86 and 92% and in human plasma 97, 89 and 94%, respectively. The within- and between-day accuracy and precision for diazepam and nordazepam in milk and plasma at all concentrations tested (20-1500 ng/ml) were better than 8%. The high fat content which occurs in rabbit milk presented no limitation for the extraction of lipophilic diazepam: the method was successfully used to monitor milk and plasma concentrations of diazepam and nordazepam in lactating New Zealand White rabbits during 26-h infusions of diazepam (1.4 mg/h).

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.

Repeated dose effects of lormetazepam and flurazepam upon driving performance.

The residual effects of lormetazepam 1 mg and 2 mg in soft gelatine capsules on driving performance were assessed and compared to those of flurazepam 30 mg, which is also a powerful hypnotic, but possesses a far less favourable pharmacokinetic profile with a long-acting sedative metabolite. Driving performance was tested 10 to 11 h and 16 to 17 h post administration, after 2 days on placebo (baseline), and 2, 4 and 7 days of drug treatment (active), and after 1 and 3 days following the resumption of placebo (washout). The driving test consisted of operating an instrumented motor-vehicle over a 72 km highway circuit in light traffic. Flurazepam 30 mg significantly impaired the ability to control the lateral position of the vehicle compared to placebo baseline measurements. The degree of impairment was substantial in the female subjects and was greater in the morning than in the afternoon. Lormetazepam 1 mg showed no residual effect on driving performance. Lormetazepam 2 mg impaired driving performance to some extent on the following morning, 10 to 11 h post administration, but no residual effect was found in the afternoon. All drugs improved sleep quality and prolonged sleep duration to more or less the same extent.

Simultaneous determination of flurazepam and its metabolites in human plasma by high-performance liquid chromatography.

A sensitive isocratic high-performance liquid chromatographic method is described, which allows the precise and accurate quantification of flurazepam and four metabolites with a single determination. A pharmacokinetic study was performed on nine volunteers and the main pharmacokinetic data are reported. The method was used to demonstrate that monodesethylflurazepam and didesethylflurazepam are major metabolites in men. One more unidentified flurazepam metabolite was detected.

Pharmacokinetic determinants of dynamic differences among three benzodiazepine hypnotics. Flurazepam, temazepam, and triazolam.

Healthy adult volunteers (n = 52) received single oral doses of flurazepam hydrochloride (15 mg), temazepam (15 mg), triazolam (0.25 mg), or placebo in a parallel, double-blind study. Sedative effects were greatest with triazolam, followed next by temazepam; peak effects closely coincided with peak plasma concentrations. Differential recovery from sedation corresponded in part to differences in mean elimination halflife, although sedative effects returned to baseline before plasma drug concentrations became undetectable. Sedation following flurazepam administration was less intense than with triazolam and temazepam. When tested at three hours after dosing, none of the active treatments impaired learning of a 16-item word list. However, at 24 hours, triazolam recipients could not recall a significant fraction of what was learned. Thus, dynamic differences among three benzodiazepine hypnotics may be partly explained by kinetic differences, as well as, we should caution, by possible "clinical inequivalence" in dosage.

Liquid chromatographic assay and pharmacokinetics of quazepam and its metabolites following sublingual administration of quazepam.

A reverse-phase liquid chromatographic method is described for simultaneous quantification of quazepam, and two of its metabolites, 2-oxoquazepam and N-desaklyl-2-oxoquazepam. The method uses a solid-phase extraction procedure to prepare plasma samples. After extraction, the methanolic extract is evaporated; the residue is then reconstituted in a small volume of mobile phase and chromatographed. The total chromatography time for a single sample is about 20 min. A sensitivity of 1 ng/ml for quazepam and its metabolites is attained when 1 ml of plasma is extracted. Analytical recovery of quazepam and its metabolites added to plasma ranged from 87 to 96%. The maximum within-day and day-to-day coefficients of variation for each compound at concentrations of 20 and 60 ng/ml were 7.6 and 11.2%, respectively. The method was applied to sublingual pharmacokinetic studies of quazepam in healthy volunteers.

Determination of flurazepam and its major metabolites N-1-hydroxyethyl- and N-1-desalkylflurazepam in plasma by capillary gas chromatography.

In the present paper we describe a method for the quantitative determination of flurazepam (I) and two of its metabolites, N-1-desalkylflurazepam (II) and N-1-hydroxyethylflurazepam (III), in serum after therapeutic dosings is described. The method is sensitive (lower limit of quantification for I and III: 1 ng/ml, for II: 2 ng/ml), selective and--compared to the analytical approaches already published--simple to handle. Thus this assay is well suitable for determinations during clinical studies (e.g. evaluating the pharmacokinetics, bioavailability/bioequivalence). Following simple extraction- and derivatization steps (the latter being only required for III) the extract is injected directly onto a fused-silica, bonded-phase capillary column of a gas chromatograph and the compounds of interest detected by an electron-capture detector (ECD). The assay has been used successfully during several clinical studies, especially as very low dosages result in also very low blood concentrations.

Pharmacokinetics and CSF entry of flurazepam in dogs.

Anesthetized dogs received a single 1.0-mg/kg intravenous dose of flurazepam hydrochloride, following which multiple blood and cerebrospinal fluid (CSF) samples were taken over the next 8 h. Concentrations of flurazepam and its metabolite, desalkylflurazepam, were determined by gas chromatography with electron-capture detection. Mean kinetic variables for flurazepam were: volume of distribution 7.9 l/kg, elimination half-life 2.3 h, clearance 37 ml/min/kg, serum free fraction 25% unbound. The metabolic product desalkylflurazepam appeared in serum in low concentrations, and was eliminated with a half-life of 4.9 h. Flurazepam rapidly entered CSF, then was eliminated in parallel with flurazepam in serum. However, the extent of entry into CSF was limited, with the mean ratio of area under the curve for CSF versus serum (0.24) nearly identical to the serum free fraction. Thus, intravenous flurazepam in dogs is characterized by extensive distribution, high clearance, and short half-life. Entry into CSF is rapid, and appears governed by passive diffusion. The extent of CSF entry is limited by protein binding in serum.

Simultaneous determination of flurazepam and five metabolites in serum by high-performance liquid chromatography and its application to pharmacokinetic studies in rats.

A reversed-phase high-performance liquid chromatographic method is described which allows the quantification of flurazepam and five of its metabolites with a single, isocratic determination. In addition, it has the advantage of possessing a low detection limit and high precision. A 2 mm I.D. column was used to minimize sample size (50 microliter), increase sensitivity and reduce solvent consumption. The method was used to demonstrate that N-1-desalkylflurazepam, the major metabolite, has a short half-life in the rat in contrast to its prolonged life in humans.

The respiratory effects of oral ethyl loflazepate in volunteers.

A volunteer study was undertaken to assess the respiratory effects of ethyl loflazepate, a new benzodiazepine, and to correlate these with plasma concentrations of the active metabolites. Twelve volunteers were given placebo, 2 mg ethyl loflazepate, and 6 mg ethyl loflazepate on separate occasions. Respiration and plasma metabolite levels were assessed hourly for 8 h and at 24 h. The 6 mg ethyl loflazepate treatment produced a significant decrease (P less than 0.02) in the ventilatory response to carbon dioxide at 5 h. However this did not equate with a peak in plasma metabolite concentrations which were maintained at a plateau level from 4 to 24 h.

Brain uptake of flurazepam and of N1-desalkyl flurazepam after administration of flurazepam to the cat.

A gas chromatographic-mass spectrometric procedure was employed to identify flurazepam and several of its metabolites in the plasma, cerebrospinal fluid (CSF), and brain of cats after an iv injection of flurazepam. Following tissue redistribution, flurazepam was lost from plasma with a mean half-time of 1.4 hr; although usually not detectable in plasma by 24 hr, significant quantities were found in the brain at this time, particularly in the corpus callosum. N1-Hydroxyethyl flurazepam appeared rapidly in plasma following iv flurazepam, peaked at about 40 min, and then declined with a mean half-time of 2.1 hr. N1-Desalkyl flurazepam accumulated in plasma in the first 6 hr after flurazepam was injected, and then declined slowly with a mean half-time of about 50 hr. At 24 hr, corpus callosum concentrations of the N1-desalkyl flurazepam exceeded those of flurazepam by 16- to 63-fold and produced brain/plasma ratios of 6 to 62 in three cats. CSF concentrations of flurazepam and N1-desalkyl flurazepam did not reflect brain concentrations but only the estimated plasma fractions of the unbound drugs. The results suggest that long term central effects of iv flurazepam are mediated to a large extent by the N1-desalkyl flurazepam, in species in which that metabolite accumulates.