Multinuclear NMR (1H, 13C and 19F) spectroscopic re-examination of the solvolytic behaviour of flurazepam dihydrochloride.

The dissolution of reference and archival samples of flurazepam dihydrochloride (2) was studied in DMSO-d6 and in D2O by 1H-, 13C- and 19F-NMR spectroscopy to identify and distinguish solvated species of the parent drug (2), the "benzophenone" (4) and glycine (5) hydrochloride degradation products. In DMSO-d6, for most samples, only the ring intact form (2) could be detected by 13C-NMR whereas the inherently greater sensitivity of 19F-NMR allowed detection of initial trace amounts (< 1%) of the open-ring form (3). 19F-NMR spectroscopy also afforded the best means of quantifying the various entities in solution, including the increase towards equilibrium levels of the open-ring entity and detection/quantitation of a new equilibrium species, possibly the cis/trans rotamer of the open-chain entity (3). Various chemical shifts for flurazepam dihydrochloride and USP flurazepam related reference standards C and F are reported for DMSO-d6 solutions. The bases for 1H- and 19F-NMR assay of DMSO-d6 solutions of (2) for (4) are discussed with comparative data. The solvation characteristics of (2) in D2O at 0 and 27 degrees C were found to be too complex to follow by 13C-NMR; however, 19F-NMR studies at these temperatures permitted one to clearly discern that no additional formation of entity (4) occurred beyond whatever initial levels were present in degraded samples while the open-ring entity (3) was observed to increase to an equilibrium level of 56% over 24 h at 27 degrees C. Dissolution in D2O at either 0 or 27 degrees C does not contribute to solvolytic degradation of (2) to (4) over 24 h.

Determination of droperidol in plasma by liquid chromatography.

A reversed-phase high performance liquid chromatographic method is described for the determination of droperidol concentrations in plasma. Following extraction, separation of droperidol and the internal standard flurazepam was achieved with a Spherisorb Nitrile, 5 microns, S5CN 250 mm x 4.6 mm column at 200 nm. The mobile phase was phosphate buffer (0.05 M, pH 2.4), acetonitrile and ethanol (65:20:15, v/v/v). The assay was rapid, sensitive and linear over the range 2-4000 ng ml-1. Precision of the assay expressed as the intra- and inter-day relative standard deviations (%RSD) did not exceed 10%. Flunitrazepam, midazolam and nitrazepam were also resolved with this technique and did not interfere with droperidol or flurazepam. Resolution of all five compounds was complete in less than 6 min. The assay was used to study the pharmacokinetics of high dose droperidol infusions during and after cardiac surgery.

Biopharmacological data and high-performance liquid chromatographic analysis of 1,4-benzodiazepines in biological fluids: a review.

A review with 123 references on the analysis of 1,4-benzodiazepines in biological samples using HPLC is presented. Some important physico-chemical and biopharmacological data for the development of analytical methods are collected. Different methods of sample pretreatment, chromatographic conditions and detection systems are discussed.

Quantitation of flurazepam and three metabolites by electron capture gas liquid chromatography.

Flurazepam and three of its metabolic products (desalkyl, hydroxyethyl, and aldehyde metabolites) can be simultaneously quantitated without derivatization by gas chromatography with electron capture detection. After addition of a suitable internal standard, unknown biological samples and calibration standards are extracted at neutral pH into benzene/isoamyl alcohol. The reconstituted extract is chromatographed at 275 degrees C with a 10% OV-101 liquid phase, which allows resolution of all 5 compounds. In some cases a 1% OV-225 liquid phase is used for quantitation of hydroxyethylflurazepam. The method is sufficiently sensitive and reproducible for use in clinical and experimental pharmacokinetic studies.

A fatality due to flurazepam.

A fatality attributed to suicidal ingestion of up to 2.2 grams of flurazepam is described. The deceased was a 52-year old female with a history of depression and suicidal attempts. No significant pathology was found at autopsy. Full toxicological analyses detected only flurazepam and metabolites in her tissues. The concentrations of flurazepam in femoral blood, liver, bile, vitreous humor and urine were 5.5 mg/L, 130 mg/kg, 33 mg/L, 1.3 mg/L and 3.3 mg/L, respectively. Analysis of gastric contents showed 600 mg of flurazepam. Desalkylflurazepam was also detected in blood, liver, bile and vitreous, but at much lower concentrations than the parent compound.

Concentrations of phenobarbital, flurazepam, and flurazepam metabolites in autopsy cases.

In five cases of death resulting from acute intoxication with phenobarbital and flurazepam, the blood, urine, brain, lung, liver, and kidney levels of these drugs as well as the levels of N-1 hydroxyethyl, N-1 desalkyl, and N-1 desalkyl-3-hydroxy flurazepam metabolites were determined. Concentration of flurazepam and its metabolites was determined by using new gas chromatographic conditions employing a selective detector for nitrogen-containing substances and a column of 1% SP-1000. In addition, the EMIT technique was also employed on blood and urine samples and the results compared with GLC data.

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.

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.

Acute flurazepam intoxication: a case report.

A fatality due to ingestion of flurazepam is reported. Flurazepam is a benzodiazepine, a widely prescribed hypnotic drug for use in sleep disorders. There are only few documented reports of the disposition of flurazepam in deaths due to overdose. A 68-year-old woman was found deceased at home with no evidence of trauma or asphyxia. Toxicologic analyses were performed and drug levels measured by means of gas chromatography coupled to mass spectrometry. The flurazepam concentration in each specimen was as follows: heart blood 2.8 microg/mL, bile 323 microg/mL, and urine 172 microg/mL. Presence of flurazepam into gastric content was observed too. Based on the autopsy findings, patient history, and toxicologic results, the cause of death was determined to be acute intoxication of flurazepam and the manner, suicide.

Simple thin-layer chromatographic method for the investigation of six related compounds in flurazepam hydrochloride and its capsules.

A simple and rapid thin-layer chromatographic procedure is presented for the separation and tentative identification of flurazepam hydrochloride and its six related impurities in bulk samples and capsules. A methanolic sample solution is applied to a plate of silica gel containing a fluorescent indicator, which is developed once with a basic quaternary solvent system. Spots are visible under a short wavelength UV lamp, with a limit of detectability ranging from about 62.5 ng (related compound B) to 500 ng (related compound C). The proposed procedure shows several advantages over the related compounds test of the United States Pharmacopeia.

Direct liquid inlet liquid chromatographic/mass spectrometric identification and high-performance liquid chromatographic analysis of a benzodiazepine glucuronide.

The glucuronide of N-1-hydroxy-ethyl flurazepam has been analysed by a direct liquid inlet liquid chromatographic/mass spectrometric system using MeOH/H2O (70:30 v/v) as mobile phase, at a flow rate of 0.7 ml min-1. Urine samples were purified by amberlite XAD-2 chromatography; the glucuronide was quantified by high-performance liquid chromatography using a counterion (tetrabutyl ammonium nitrate in methanol). Chromatographic results were validated by an enzymatic method: treatment of the samples with beta-glucuronidase and extraction of the parent drug with ethyl ether at pH 9. The biological application of this method was demonstrated by determination of this glucuronide in the urine of healthy human volunteers following a single intravenous administration of 50 mg of N-1-hydroxy-ethyl 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).

GLC-ECD determination of 1-(2-hydroxyethyl)-3-hydroxy-7-chloro-1,3-dihydro-5-(O-fluorophenyl)-2H-1,4-benzodiazepin-2-one (SAS 643) in plasma and urine and identification of its main biotransformation products.

A method using gas-liquid chromatography-electron-capture detection method is described for rapid, accurate determination of SAS 643 in plasma and urine. The drug is extracted from biological fluid with benzene and converted to the O, O'-bistrimethylsilyl derivative with bis(trimethylsilyl) trifluoroacetamide. The glucuronide form of the drug is extracted after hydrolysis with beta-glucuronidase. Nimetazepam is used as internal standard. Moreover, some metabolites such as glucuronide and the N-1-dealkylated and N-1-yl-acetic products are identified. All compounds were confirmed by thin-layer chromatography, mass spectroscopy, and gas-liquid chromatography-mass spectroscopy by comparison with reference products.

Large-volume sample stacking of selected drugs of forensic significance by capillary electrophoresis.

Large-volume sample stacking capillary electrophoresis (LVSS-CE) and conventional capillary electrophoresis (CE) are compared for the separation of drugs of significance to forensic and clinical analyses. LVSS-CE for cations requires the use of an electroosmotic flow (EOF) modifier in conjunction with polarity switching to effect on-column concentration of an analyte and its subsequent migration in the capillary. The run buffer consists of 0.05 mol dm(-3) disodium tetraborate adjusted to pH 2.2 with orthophosphoric acid, and the EOF modifier is 0.002 mol dm(-3) cetyltrimethylammonium bromide. CE investigations used an identical run buffer minus the EOF modifier. LVSS-CE and CE investigations used injection times of 30 s and 3 s, respectively. Both modes of capillary electrophoresis are compared in terms of their limits of detection, efficiency, resolution and reproducibility. LVSS-CE is also applied to the analysis of a spiked urine sample.