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.

[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.

Heterogeneity of cerebral capillary flow in man and its consequences for estimation of blood-brain barrier permeability.

Blood-brain barrier permeability studies made in man using the indicator dilution method revealed that the extraction of the test substance increases during the upslope of the venous (outflow) dilution curve. The present study aimed to obviate the possibility that this could result from intravascular phenomena, such as interlaminar diffusion (the result of differences in molecular size) and erythrocyte carriage. Several reference substances were employed for the determination of the extraction in order that careful correction could be made for differences in intravascular behavior of the test and reference substance. The test substances studied were D-glucose, L-phenylalanine, water, propranolol, and benzodiazepines, representing both carrier-transported and lipophilic substances. In-diethylenetriamine pentaacetic acid, Na+, Cl-, L-glucose, and L-lysine were employed as reference substances. For all the substances tested, and after correction for intravascular phenomena, the extractions were found to increase during the initial part of the dilution curve. This increasing extraction can be ascribed to heterogeneity of the cerebral circulation; the higher extraction corresponds to longer contact with the blood-brain barrier and indicates a longer transit time. Signs of heterogeneity were also present when blood flow was elevated above normal. Any influence that heterogeneity might have on the mean extraction value can be minimized by using an appropriate calculation of the extraction of the test substance.

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.

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.

Cimetidine impairs nitrazepam clearance.

The effect of cimetidine on hepatic clearance of the benzodiazepine derivative nitrazepam was evaluated in healthy subjects. Six received a single 5- or 10-mg oral nitrazepam dose in the drug-free state and again with therapeutic cimetidine doses. Nitrazepam kinetics were determined from multiple serum concentrations measured during the 72 hr after each dose. Cimetidine had no effect on nitrazepam absorption kinetics, since peak serum nitrazepam concentration and time of peak concentration were not altered. Cimetidine did not alter nitrazepam volume of distribution, but cimetidine consistently reduced nitrazepam clearance, from a mean of 1.41 ml/min/kg in the control state to 1.17 ml/min/kg during cimetidine treatment. This resulted in prolongation of nitrazepam elimination t1/2 from 22.2 to 27.8 hr. Thus the ability of cimetidine to impair drug oxidation in man extends to the capacity for clearance of nitrazepam, a compound biotransformed mainly by nitroreduction.

Age, sex, and nitrazepam kinetics: relation to antipyrine disposition.

Forty healthy men and women 19 to 80 years old received a single 10 mg oral dose of the 7-nitro benzodiazepine nitrazepam. Nitrazepam plasma concentrations were measured during the next 72 hours. Among men, the elderly had a larger volume of distribution (Varea) than did younger subjects (1.96 vs. 1.63 L/kg; P less than 0.05); because clearance did not change with age (0.84 vs. 0.95 ml/min/kg), the prolonged t1/2 in elderly men (28 vs. 20 hours; P less than 0.01) was a result of the larger Varea. Elderly and young women did not differ in nitrazepam Varea (2.58 vs. 2.55 L/kg), t1/2 (26 vs. 27 hours), or total clearance (1.19 vs. 1.09 ml/min/kg). The nitrazepam free fraction in plasma (18% to 19% unbound) was not related to age or sex. Among 18 subjects who also received antipyrine, the clearance of nitrazepam and antipyrine were not correlated (r = 0.23). Thus age minimally influences nitrazepam clearance (accomplished mainly by nitroreduction), which in turn is not significantly related to antipyrine oxidizing capacity.

Effects of butoctamide hydrogen succinate and nitrazepam on psychomotor function and EEG in healthy volunteers.

We studied the effects of butoctamide hydrogen succinate and nitrazepam on psychomotor function and EEG in eight male volunteers aged 19-32. The hypnotic effects, effects on psychomotor performance, EEG activity and standing steadiness between BAHS 1000 mg and nitrazepam 5 mg were compared at regular intervals for 10 h. The serum levels of both drugs were also assayed. The hypnotic effects of BAHS were very weak compared to those of nitrazepam. BAHS did not exert any effects on psychomotor performance and standing steadiness during the test period. In contrast, nitrazepam impaired psychomotor performance and standing steadiness as the serum drug levels increased. Nitrazepam decreased the alpha activity and increased the beta activity in a concentration-dependent manner. BAHS did not change the alpha activity but increased beta-2 activity at Fz and Cz at 10 h of the post-drug period. BAHS was eliminated more rapidly than nitrazepam. These results indicated that BAHS, at the dose used, was less potent than nitrazepam and the effects on psychomotor performance and standing steadiness were minimal.

Introduction to the pharmacokinetics and pharmacodynamics of benzodiazepines.

The contributions of pharmacokinetics and pharmacodynamics to the generation and maintenance of drug responses are reviewed from the literature. Potential pharmacokinetic determinants of duration of drug action are dose, lipid solubility and elimination half-life. Certain paradoxes are inherent in the view that clinical differences among benzodiazepines are due primarily to their elimination half-lives. It is concluded that the respective roles of pharmacokinetics and pharmacodynamics in the generation and maintenance of clinical responses to benzodiazepines require clarification.

Influence of cystamine on pharmacokinetics and pharmacodynamics of nitrazepam.

The influence of cystamine on pharmacokinetics of nitrazepam was studied. Experiments were performed on the third day after the administration of cystamine, since the author's previous investigations showed that the protective action of cystamine in radiation sickness was strongest at this time. CA premedication resulted in more rapid resorption, lower level of nitrazepam in blood, and more rapid elimination of the drug.

Direct derivative spectrophotometric determination of nitrazepam and clonazepam in biological fluids.

The use of fifth order derivative spectra allows the direct determination of nitrazepam in urine at 388 nm with a limit of detection of 1 microgram ml-1. The determination of nitrazepam in blood plasma can be carried out directly by measurement at 402 nm in the fourth order derivative spectra with a limit of detection of 1.5 micrograms ml-1. Clonazepam can be determined directly in urine samples at 384 nm by using sixth order derivative spectra with a limit of detection of 1 microgram ml-1 and in blood plasma by using fourth order derivative spectra at 384 nm with a limit of detection of 0.5 ppm.

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.

Dialysis as an on-line sample-pretreatment technique for column liquid chromatography: influence of experimental variables upon the determination of benzodiazepines in human plasma.

An evaluation is provided of dialysis, coupled on-line to column liquid chromatography, as a sample pretreatment procedure for macromolecule-containing biological samples. The influence of parameters such as acceptor phase flow rate, temperature, hydrophobicity of the analytes, pH, ionic strength and viscosity of the sample on the recovery and rate of dialysis is studied. In addition, methods to reduce the degree of drug-protein binding and thereby improve the recovery are reported. Diazepam, nitrazepam and oxazepam are used as model compounds. A method is reported for the fully automated determination of these compounds in human plasma using only 100 microliters of sample. Data on repeatability, linearity and detectability are given.

A radioreceptor assay for benzodiazepines.

A simple, rapid and sensitive radioreceptor assay for determining benzodiazepines in serum is based on the displacement by the drug of specific [3H]diazepam binding to a membrane fraction from rat brain. The limit of detection of the more active benzodiazepines is about 0.5 ng. Diazepam, nitrazepam, clobazam and HR 458 have been assayed in human serum after a single oral clinical dose. The results can be used for determining pharmacokinetic parameters. The technique measures not only the parent benzodiazepine but also clinically active metabolites.

Simultaneous HPLC analysis of the hypnotic benzodiazepines nitrazepam, estazolam, flunitrazepam, and triazolam in plasma.

A new analytical methodology was developed for simultaneous high-performance liquid chromatographic quantitation of nitrazepam, estazolam, flunitrazepam, and triazolam in plasma. After addition of the internal standard nor-prazepam, biological samples and calibration standards were extracted at basic pH into diethyl ether-methylene chloride (2:1, v/v). The reconstituted extract was separated on a reversed-phase Nova Pak C18 column with acidic buffer (6mM)-acetonitrile-methanol (64:23:13, v/v) as mobile phase. The detection was performed at 242 nm. Within-run and day-to-day precision values were about 2-8%. The method is thus sensitive and reproducible for use in clinical and experimental toxicokinetic studies.

Complications in correlation studies between serum, free serum and saliva concentrations of nitrazepam.

The relation between free serum and saliva concentrations of nitrazepam in healthy male volunteers has been studied. It was found that the average value of free serum concentrations from all volunteers was twice the average value of corresponding saliva concentrations. Sometimes mean values of serum, free serum and saliva concentrations are used in correlation studies. However, the data from individual volunteers showed that there was no correlation between them. Statistics can easily introduce false pictures for correlations between free serum and saliva concentrations of nitrazepam, whereas no correlation could be found on the basis of results from individuals. In response studies such as the effects of drugs on driving performance, the free serum and saliva concentrations of a drug from individual volunteers should therefore be considered. This will complicate the use of saliva in epidemiological studies on drugs and driving.

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.

Polarographic determinations of nimetrazepam in serum and whole blood.

In the elaborated polarograhic technique, serum has been shown the best biological material for the direct determination of nimetazepam.]

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.