Optimization of modified dispersive liquid-liquid microextraction coupled with high-performance liquid chromatography for the simultaneous preconcentration and determination of nitrazepam and midazolam drugs: An experimental design.

A simple, sensitive, and rapid microextraction method, namely, ultrasound-assisted surfactant-enhanced emulsification microextraction based on the solidification of floating organic droplet method coupled with high-performance liquid chromatography was developed for the simultaneous preconcentration and determination of nitrazepam and midazolam. The significant parameters affecting the extraction efficiency were considered using Plackett-Burman design as a screening method. To obtain the optimum conditions with consideration of the selected significant variables, a Box-Behnken design was used. The microextraction procedure was performed using 29.1 µL of 1-undecanol, 1.36% (w/v) of NaCl, 10.0 µL of sodium dodecyl sulfate (25.0 µg mL(-1)), and 1.0 µL of Tween80 (25.0 µg mL(-1)) as an emulsifier in an extraction time of 20.0 min at pH 7.88. In order to investigate the validation of the developed method, some validation parameters including the linear dynamic range, repeatability, limit of detection, and recoveries were studied under the optimum conditions. The detection limits of the method were 0.017 and 0.086 ng mL(-1) for nitrazepam and midazolam, respectively. The extraction recovery percentages for the drugs studied were above 91.0 with acceptable relative standard deviation. The proposed methodology was successfully applied for the determination of these drugs in a number of human serum samples.

Silver nanoparticle enhanced chemiluminescence method for the determination of nitrazepam.

We report on a simple and sensitive chemiluminescence (CL) method to determine nitrazepam. This method is based on the fact that rhodamine 6G (Rh6G) enhanced the weak CL emission of the reaction of hexacyanoferrate with nitrazepam, and that it was further enhanced by silver nanoparticles (AgNPs). The effects of the concentrations of K3Fe(CN)6, Rh6G, AgNPs and NaOH on the CL reaction were investigated. Under the optimum conditions, the CL intensity was proportional to the concentration of nitrazepam in the range from 1.0 nM to 10.0 µM. The detection limit (3σ) was at 0.1 nM. The relative standard deviation was 2.1% (at a 0.1 µM concentration and for n = 11). The method was successfully applied to the determination of nitrazepam in Coca-Cola beverage, urine and plasma, and the recovery was 98 - 103%. We also considered the possible CL reaction mechanism.

[Simultaneous determination of 5 sedative hypnotics in human plasma by reversed phase high-performance liquid chromatography].

OBJECTIVE: To determine diazepam, nitrazepam, oxazepam, estazolam, and alprazolam simultaneously in human plasma by reversed phase high-performance liquid chromatography (RP-HPLC). METHODS: Ten microliter carbamazepine (50 mg/L)as the internal standard was added into 1 mL sample, which contained the 5 mixed sedative hypnotics as standard substance and human plasma as ground substance. They were extracted with acetoacetate from plasma samples, and then were dissolved by 100 microL mobile phase. The blood drug levels were analyzed by high performance liquid chromatograph with 20 microL sample injection on a chromatographic column C18 (4.6 mm x 250 mm) at 30 degree. The mobile phase consisted of methanol and water (65:35),and the flow rate was 1.0 mL/min.The ultraviolet detection wavelength was 230 nm. RESULTS: The linearity range of the 5 drugs was 5-1,200 microg/L (r> or =0.9966, P<0.05). The recovery rate was 95.5%-105.6%. The extraction recovery rate was more than 75%. The relative standard deviation (RSD) of intra-day and inter-day was less than 10% (n=5). CONCLUSION: RP-HPLC method is convenient, accurate and sensitive for simultaneous determination of the concentration of diazepam, nitrazepam, oxazepam, estazolam, and alprazolam in human plasma.

Extraction and analysis of clonazepam and 7-aminoclonazepam in whole blood using a dual internal standard methodology.

In this paper, a simple and robust method for the determination of clonazepam and its primary metabolite (7-aminoclonazepam) in whole blood is described. Clonazepam (klonopin) is a popular prescription drug that has been implicated in the field of drug facilitated sexual assaults (DFSA). Clonazepam, 7-aminoclonazepam and the internal standards (deuterated analogues for GC-MS analysis and nitrazepam for analysis by LC-PDA/GC-MS) were spiked into blood samples. The samples were buffered with a pH 6-phosphate solution (5 mL) and extracted from phenyl spe columns. The columns were washed with 5% acetonitrile in pH 6-phosphate buffer (3 mL) and eluted with ethyl acetate (2 x 3 mL). The eluents were evaporated for further chromatographic analysis. For GC-MS, the samples were derivatized prior to analysis. When performed with LC-PDA the samples were reconstituted in distilled water. From this method LOQs of 5 ng/mL of sample is easily achievable by either chromatographic system. By using GC-MS in EI mode, 1 ng/mL of sample can be detected. Data is presented here to show the simplicity and efficiency of the extraction scheme. By employing the properties of GC-MS and LC-PDA, this extraction and analysis procedure further extends the number of tools open to the forensic toxicologist for the analysis of this drug.

Voltammetric behavior of nitrazepam and its determination in serum using liquid chromatography with redox mode dual-electrode detection.

A method involving high-performance liquid chromatography with dual-electrode electrochemical detection in the redox mode (LC-DED) has been successfully developed for the determination of the benzodiazepine tranquilizer, nitrazepam, in serum. To elucidate the electrochemical mechanism occurring at a glassy carbon electrode, cyclic voltammetry was preformed with 1 mM solutions of nitrazepam at pH values between 2 and 12, using a potential range from -1.5 to +1.5 V. Two reduction peaks were observed over the whole pH range; the first, designated R1, was consistent with the 4e-, 4H+ reduction of the 7-nitro group to a hydroxylamine species; the second more negative peak, designated R2, was shown to be the result of a 2e-, 2H+ reduction of the 4-5 azomethine group. On the reverse anodic scan, an oxidation signal was observed, designated O1, which was considered to result from a 2e-, 2H+ oxidation of the hydroxylamine to a nitroso group. On the second forward scan, a new reduction peak, designated R3, was observed, which was considered to result from reduction of the nitroso species back to the hydroxylamine species. Studies were then undertaken to exploit the hydroxylamine/nitroso redox couple using LC-DED detection for the measurement of nitrazepam in serum. The optimal chromatographic conditions were found to comprise a mobile phase containing 60% methanol, 40% 50 mM pH 4.1 acetate buffer, in conjunction with a Hypersil C18 250 mm x 4.6 mm column. Hydrodynamic voltammetric studies were undertaken to optimize the operating potentials required for dual-electrode detection. It was found that an applied potential of -2.4 V was optimum for the "generator" cell and +0.5 V for the "detector" cell. The proposed method was evaluated by carrying out replicate nitrazepam determinations on spiked bovine and human serum samples. The former evaluation was preformed at a concentration of 11.2 microg mL(-1), and the latter at 1670 ng mL(-1). For bovine serum, the recovery of nitrazepam was found to be 75.8% and the associated coefficient of variation was 6.1% (n = 6). For human serum, the recovery was 74.1% with a coefficient of variation of 7.8% (n = 7). These data suggest that the method holds promise for applications in toxicology and where an alternative reliable method to confirm drug abuse may be required.

Extraction and analysis of flunitrazepam/7-aminoflunitrazepam in blood and urine by LC-PDA and GC-MS using butyl SPE columns.

The forensic toxicology community has recognized flunitrazepam and its metabolite (7-aminoflunitrazepam) as compounds of concern for several years. In this procedure, the analytes were extracted from whole blood and urine onto single mode solid phase cartridges (butyl) using nitrazepam as an internal standard. The columns were washed with distilled water and hexane. All three compounds were eluted from the sorbent using an ethyl acetate-methanol solvent mixture. After collection and evaporation of the solvent, the residue was dissolved in A, 0.1% (v/v) aqueous trifluoroacetic acid for HPLC-PDA analysis or B, ethyl acetate for derivatization with pentafluoropropionic anhydride (PFPA) for analysis by gas chromatography-mass spectrometry (selected ion monitoring, SIM). A limit of quantitation for this method using HPLC-PDA was found to be 5 and 1.0 ng mL(-1) by SIM.

[Analysis of benzodiazepine derivative mixture by gas-liquid chromatography]. / Benzodiazepino dariniu misinio analize duju ir skysciu chromatografijos metodu.

The analysis of mixture of benzodiazepine derivates (chlordiazepoxide, flunitrazepam, medazepam, nitrazepam, oxazepam and tetrazepam) by gas--liquid chromatography (GLC) in purpose to separate and identify these psychotropic drugs in mixture is presented in this article. The experiment was carried out in vitro, accommodating this method for identification and separation of drugs, isolated from biological objects (blood and urine). Referring to data of annual reports of chemical investigations (1) above-mentioned psychotropic drugs are very frequent among drug intoxication. In most cases they are detected in the mixture of the same or different pharmacological group, and this causes difficulty for separation and identification. The analysis of the mixture was carried out by GLC, which is widely used in practice of forensic-chemical examination. Adsorbents and stationery phases were changed; the conditions and parameters of chromatography were modified, in purpose totally separate preparations in the mixture. For the separation and identification of all three preparation the column packed with Inerton Super with stationary phase 3% OV-17 is suitable. The column temperature-290 degrees C. The mixture of these drugs was excreted from body fluids (blood and urine) in vitro and investigated by GLC under these conditions. The results of investigation were similar.

Sexual assault under benzodiazepine submission in a Paris suburb.

Sexual assaults under benzodiazepine submission have been described, since use of benzodiazepine enables non consensual sexual activity but rarely fully reported. An accurate evaluation of the phenomenon has seemed interesting. Files of 23 adult males and females examined at the Emergency Forensic Unit of an University Teaching Hospital near Paris were reviewed. All the victims had complained from sexual assault under drug submission, in the years 1996 and 1997. A complete examination for sexual assault was realised linked to clinical examination of drug intoxication. Every victim of rape under drug submission was sampled for urine screening (mean delay of 17.5 h after sexual assault) and blood alcohol level quantification. Urine was screened for benzodiazepines, cocaine, opiates and cannabinoids with qualitative immunochromatographic test. Traumatic lesions of sexual penetration were retrieved in 10 victims and sperm in 5. Clinical signs of benzodiazepine intoxication were retrieved in 12 out of 23 victims. Urine benzodiazepine screening was positive, over the cut-off values (300 ng/mL)when sampled less than 20 h after the facts. In 6 out of 23 victims, drugs of abuse and alcohol were associated to benzodiazepines. A reinforced attention can be brought to the rape under drug submission including the need of a proper examination and samplings shortly after the alleged facts to ascertain the diagnosis and to help the victim facing the Justice inquiry.

Stability of nitrobenzodiazepines in postmortem blood.

Studies were undertaken to determine the stability of nitrobenzodiazepines and their 7-amino metabolites in water and blood. At 22 degrees C nitrazepam and clonazepam were stable in sterile fresh blood containing preservative over 28 days, whereas 25% of flunitrazepam was degraded. At 37 degrees C all three drugs were substantially lost over 9 h (29-51%). There was only a small loss observed for the 7-amino metabolites and no substantial amounts of parent drug and 7-amino metabolite were degraded in water under these conditions. In the absence of preservative substantial amounts (25-50%) of parent drugs were lost in fresh blood over 10 days at 22 degrees C. In bacterially-contaminated postmortem blood all three drugs were completely degraded over 8 h at 22 degrees C with almost all drug completely converted to the respective 7-amino metabolite. These metabolites were also partially degraded (10-20%) over 45 h at 22 degrees C. All 3 nitrobenzodiazepines were stable in blood stored for up to 24 months at -20 degrees C, or 4 degrees C over 10 months. Their respective 7-amino metabolites were, however, relatively unstable at -20 degrees C with a significant loss (29%) after 2 months. At 4 degrees C a 21% loss occurred after 1 month. Freeze/thawing was found not to affect the concentration of nitrobenzodiazepine and 7-amino metabolites. These results show that the nitrobenzodiazepines and their metabolites are unstable chemically and metabolically in blood. We advise that blood collected for the purpose of nitrobenzodiazepine determinations should be preserved with sodium fluoride, stored at -20 degrees C and assayed as soon as practicable, preferably within a week of collection.

Postmortem distribution and redistribution of nitrobenzodiazepines in man.

The distribution of the nitrobenzodiazepines, flunitrazepam, clonazepam and nitrazepam, and their respective 7-amino metabolites were examined in blood, serum, vitreous humor, liver, bile and urine of decedents taking these drugs. Peripheral blood, serum and liver concentrations were not significantly different to each other. However, vitreous concentrations were one-third of blood, while bile concentrations were 5-12 fold higher. Blood, serum and vitreous contained predominantly the 7-amino metabolite, liver contained only the metabolite, while bile contained significant concentrations of both the parent drug and the 7-amino metabolite. Urine contained only small concentrations of parent drug, however, as expected a number of metabolites were detected. Redistribution studies compared the drug concentrations of femoral blood, taken at body admission to the mortuary, with femoral blood taken at autopsy approximately 39 h later in 48 cases. The concentrations of 7-amino metabolites were not significantly different, however the concentrations of parent nitrobenzodiazepines were significantly higher in the admission specimens. In 6 cases in which subclavian blood was taken, the concentrations were not significantly different to the concentrations in admission blood. Similar findings were observed when femoral and subclavian blood concentrations were compared in 6 cases. There was also no apparent difference in total blood concentrations of nitrobenzodiazepines when blood concentrations taken in hospital shortly prior to death were compared to postmortem blood. Postmortem diffusion into peripheral blood is therefore not a confounding factor in the interpretation of nitrobenzodiazepine concentrations.

Analysis of low-dose benzodiazepines by HPLC with automated solid-phase extraction.

We describe a simple, specific, and sensitive reversed-phase HPLC method with automated solid-phase extraction (SPE) for analyzing alprazolam, clonazepam, and nitrazepam concentrations in human serum or plasma. We prepare samples and calibrators with an automated sample preparer, using 100-mg Bond-Elut C18 SPE columns. The HPLC method uses isocratic elution with acetonitrile:methanol:dipotassium hydrogen phosphate, 10 nmol/L, pH 3.7 (30:2:100 by vol), at a flow rate of 1.5 mL/min to separate the drugs. We detect the benzodiazepines with diode-array detector at 240 nm; analyses of peak purity are performed at 210-365 nm. The recoveries were between 94% and 100%. The intraassay and interassay CVs were between 1.0% and 4.1%. The detection limit is 5 nmol/L. The antiepileptic and antidepressant drugs tested did not interfere with the assay. We developed the method for use in a clinical laboratory for therapeutic drug monitoring.

Automated on-line dialysis for sample preparation for gas chromatography: determination of benzodiazepines in human plasma.

An on-line dialysis-solid-phase extraction-gas chromatographic (GC) approach has been developed for the determination of drugs in plasma, using some benzodiazepines as model compounds. Clean-up is based on performing the dialysis of 100 microliters samples for 7 min using water as acceptor phase and trapping the diffused analytes on a PLRP-S copolymer precolumn. After drying of the precolumn with nitrogen for 15 min, the analytes are desorbed with ethyl acetate (275 microliters) and injected on-line into the GC system via a loop-type interface. The system provides a very efficient clean-up, and offers the possibility of adding chemical agents which can help to reduce drug-protein binding and, thus, increase sensitivity. To demonstrate the potential of the described approach, the determination of benzodiazepines in plasma at their therapeutical levels is used as an example with flame ionization, thermionic and mass-selective detection.

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-microns porous microspherical silica gel.

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commercially available 2 or 5 microns particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C18 reversed-phase column (condition 1: 100 x 4.6 mm I.D., particle size 2 microns, TSK gel Super-ODS: conditon 2: 100 x 4.6 mm I.D., particle size 5 microns, Hypersil ODS-C18). The mobile phase was composed of methanol-5 mM NaH2PO4 (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (condition 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 microns was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.

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.

Determination of benzodiazepin-1,4 derivatives in biological material. Part I. An attempt to apply high-performance liquid chromatography (HPLC) in the determination of diazepam and nitrazepam in human serum.

The application of the high performance liquid chromatography method for the determination of diazepam and nitrazepam in serum is described. This method is highly sensitive, and specific, and rapid. Calibration curves are linear in the concentration range 1-1000 ng/ml.

Two cases of lethal nitrazepam poisoning.

This case report describes two cases of lethal poisoning caused by a combination of advanced chronic disease and an overdose of nitrazepam. In both cases, a relatively small blood concentration of nitrazepam was found postmortem.

High-performance liquid chromatographic procedure for the measurement of nitrobenzodiazepines and their 7-amino metabolites in blood.

A simple and sensitive HPLC method is described for the determination of the nitrobenzodiazepines, nitrazepam, flunitrazepam and clonazepam and their respective 7-amino metabolites in post-mortem blood. Using a single-step extraction the nitrobenzodiazepines were recovered from 0.5 ml of blood using butyl chloride. The solvent was evaporated to dryness and the reconstituted residue injected onto an HPLC system. Separation was achieved using a phenyl-bonded reversed-phase column with an acetonitrile-phosphate buffer mobile phase. Ultraviolet detection (240 nm) was used for quantitation. A linear response was observed between 0.01 and 0.60 mg/l. Assay precision between and within assays was less than 20% for all analytes. The limits of detection ranged from 0.001 to 0.008 mg/l with a limit of quantitation of 0.01 mg/l for all analytes.