Nitrazepam Research Articles

A novel turn-off green carbon dot-based fluorescent probe for selective determination of nitrazepam in its pharmaceutical dosage form and milk samples.

In this study, a new, rapid, simple, and cost-effective fluorescent probe based on carbon dots (CDs) has been developed for the selective determination of nitrazepam (NZP). The CDs were synthesized with high production yield (71.43%) through one step carbonization of dried banana peels. The fluorescence intensity of the CDs is quenched by increasing the NZP concentration as a result of the inner-filter effect and dynamic quenching mechanism. This fluorescent sensor effectively quantified NZP in the linear range of 0.30-26 μg mL-1, with a limit of detection of 0.10 μg mL-1. The performance of the sensor was validated according to ICH guidelines and exhibited high precision and accuracy. The proposed method was also successfully extended for determining NZP in tablet dosage form and milk samples.

Elucidation of Degradation Behavior of Nitrazepam, a Benzodiazepine Drug, under Basic Conditions: Study on Degradability of Drugs in Stomach (IV).

This study investigates the stability of nitrazepam (NZP), a benzodiazepine drug, under basic conditions, since alkaline putrefactive amines and ammonia are produced once bodies are left to decompose for a long period postmortem after a murder involving NZP or an accidental overdose of NZP. The degradation of NZP in an aqueous alkaline solution was investigated by LC/photodiode array detector (PDA) where the NZP degradation product was isolated and purified by solid-phase extraction using Oasis® MCX, and its chemical structure was determined by LC/time-of-flight (TOF)-MS, NMR spectroscopy, and single-crystal X-ray crystallography. The results revealed that NZP was immediately degraded under basic conditions with 2-amino-5-nitrobenzophenone being an intermediate which further degraded to provide 2-hydroxy-5-nitrobenzophenone as the final degradation product. These results are expected to be useful in clinical chemistry and forensic science, such as the detection of drugs during postmortem examination and suspected addiction.

Insights from molecular dynamics and DFT calculations into the interaction of 1,4-benzodiazepines with 2-hydroxypropyl-βCD in a theoretical study

This study delves into the interaction between benzodiazepine (BZD) drugs and 2-hydroxypropyl-β-cyclodextrin (2HPβCD), a cyclodextrin (CD) known to improve drug delivery and enhance therapeutic outcomes. We find that the 2HPβCD’s atoms become more rigid in the presence of chlordiazepoxide (CDP), clonazepam (CLZ), and diazepam (DZM), whereas they become more flexible in the presence of nordazepam (NDM) and nitrazepam (NZP). We also investigated the structure of 2HPβCD and found that loading these drugs increases both the area and volume of the 2HPβCD cavity, making it more suitable for drug delivery. Moreover, this research found that all drugs exhibited negative values for the binding free energy, indicating thermodynamic favorability and improved solubility. The binding free energy order of the BZDs was consistent in both molecular dynamics and Monte Carlo methods, with CDP and DZM having the highest affinity for binding. We also analyzed the contribution of different interaction energies in binding between the carrier and the drugs and found that Van der Waals energy is the primary component. Our results indicate that the number of hydrogen bonds between 2HPβCD/water slightly decreases in the presence of BZDs, but the hydrogen bond’s quality remains constant.

Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023–0.043 pg/hair strand (0.077–0.36 pg/mg) for TZ, 0.11–0.63 pg/hair strand (0.44–5.2 pg/mg) for EZ, 0.14–2.6 pg/hair strand (0.56–22 pg/mg) for FNZ, 0.33–1.7 pg/hair strand (1.3–17 pg/mg) for NZ and 20–40 pg/hair strand (120–270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3–9.2 pg/hair strand (9.2–82 pg/mg) and 2.4–9.1 pg/hair strand (8.0–55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15–29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

A New Charge Transfer Reaction for Spectrophotometric Determination of Nitrazepam Using Reverse Flow Injection Analysis

A new sensitive spectrophotometric method conjugated with reverse flow-injection analysis has been developed for the assay of nitrazepam (NZP) in the bulk drug and dosage form. The proposed method is based on the formation of charge-transfer complex between reduced NZP (donor) with a new reagent (acceptor) namely, 4,4'-tetramethyl-diaminodiphenyl-methane in the presence of NaIO4 as an oxidant. A violet-colored product was quantified spectrophotometrically at 560 nm. All the chemical and physical conditions of the reaction have been studied carefully. Under the selected optimum conditions, Beer’s law was obeyed in the concentration range of 5–300 µg/mL. The limits of detection and quantification were 5 and 16 µg/mL, respectively, with a sampling frequency of 62 sample/h. The proposed method was further successfully applied for the assay of NZP in commercial dosage forms and compared statistically with the reference method using t- and F-tests. The method was characterized by its accuracy and simplicity.

Elucidation of Degradation Behavior of Nitrazepam and Other Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (II).

The degradation behavior of eight benzodiazepines (BZPs): alprazolam, etizolam, diazepam, triazolam, nitrazepam (NZP), flunitrazepam (FNZ), bromazepam, and lorazepam, in artificial gastric juice was monitored by a LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. For drugs that were degradable, such physicochemical parameters as reaction rate constant were measured to evaluate the effect of storage conditions on drug degradability, such as whether the degradation proceeds faster by increasing storage temperature, or whether the degradation reaction is reversible by adjusting pH. As a result, it was confirmed that although the eight BZPs degraded in artificial gastric juice, most of them could be restored when pH was increased, and the restoration rates differed depending on the pH and the type of BZP. As for NZP, an Arrhenius plot was drawn to obtain the physicochemical parameters, such as activation energy and activation entropy involved in the degradation reaction, and the reaction kinetics was discussed. In addition, two substances were confirmed as the degradation products of NZP in artificial gastric juice: one was a reversible degradation product (A) (intermediate) and the other was an irreversible degradation product (B) (final degradation product). The intermediate was identified as 2-amino-N-(2-benzoyl-4-nitrophenyl)-acetamide, and the final degradation product was 2-amino-5-nitrobenzophenone. Therefore, when detecting NZP in human stomach contents, such as during judicial dissection, it would be prudent to target NZP as well as the intermediate (A) and the final degradation product (B).

Spectrophotometric and Reverse Flow Injection Method Determination of Nitrazepam in Pharmaceuticals Using O-Coumaric Acid as a New Chromogenic Reagent

A spectrophotometric- reverse flow injection analysis (rFIA) method has been proposed for the determination of Nitrazepam (NIT) in pure and pharmaceutical preparations. The method is based upon the coupling reaction of NIT with a new reagent O-Coumaric acid (OCA) in the presence of sodium periodate in an aqueous solution. The blue color product was measured at 632 nm. The variation (chemical and physical parameters) related with reverse flow system were estimated. The linearity was over the range 15 - 450 µg/mL of NIT with detection limits and limit of quantification of 3.425 and 11.417 µg mL-1 NIT,respectively. The sample throughput of 28 samples per hour was achieved. Suggested method was successfully applied for the determination of NIT in its dosage forms.

Application of cerebral regional oxygen saturation monitoring in neonatal blood exchange transfusion

Objective To observe the changes in cerebral regional oxygen saturation (CrSO2) during neonatal blood exchange transfusion(BET) and its effect on the level of bilirubin in neonates with severe hyperbilirubinemia. Methods From January 2017 to March 2018, 52 newborns with severe hyperbilirubinemia were hospitalized in the Department of Neonatology, Children′s Hospital of Fudan University.Every newborn was treated with BET.Near infrared spectroscopy was used to monitor CrSO2 in the process of BET.The monitoring lasted from 2 hours before the beginning of BET to 2 hours after the completion of BET.The CrSO2 were recorded every 2 minutes and total surem bilirubin (TSB) and transcutaneous bilirubin(TCB) was measured.During this period, it is accompanied by the monitoring of neonatal body temperature, heart rate, respiration and bolld oxygen saturation(SpO2). The differences in CrSO2 changes at different time points during BET were compared.At the same time, the correlations between CrSO2 and blood oxygen saturation, TSB and TCB levels were analyzed.The results of repeated measurement analysis of variance compared between the two groups were corrected by Bonfferoni. Results Among the 52 children, there were 33 males (63.46%) and 19 females (36.54%). The gestational age, average birth weight and average head circumference of newborns were (38.6±2.1) weeks, (3 338±444) g and (33.6±3.2) cm, respectively.The Apgar score of newborn was (8.1±1.6) scores at 1 minute after birth.The level of TSB detected for the first time after admission was (457.9±97.8) μmol/L.The CrSO2 after BET (74.6%-76.0%) was significantly higher than that before BET (69.4%-69.0%), and the difference was statistically significant (P<0.05). Correlation analysis showed that during BET, CrSO2 showed a gradual upward trend, SpO2 also showed a synchronous increase, while the level of bilirubin showed a downward trend, and the downward trend of TSB level was more obvious than that of TCB. Conclusions CrSO2 can reflect the improvement of cerebral oxygenation during neonatal blood exchange transfusion and avoid cerebral hypoxia in the course of treatment. Key words: Cerebral regional oxygen saturation; Monitoring; High-risk infant; Cerebral protection; Severe hyperbilirubinemia

Reducing and Oxidizing Columns in the Flow Injection Determination of Nitrazepam in Pharmaceutical Formulations

Two flow injection analysis (FIA) systems for determining nitrazepam (NZP) in pharmaceutical formulations using two kinds of solid-phase reactors were developed. The first system involved on-line oxidation of the reagent using a mini column containing PbO2 immobilized on cellulose acetate, while the second one involved on-line reduction of drug using a mini-Jones reductor. Both methods are based on spectrophotometric method involving oxidative coupling reaction to phenothiazine with reduced NZP in the presence of a suitable oxidizing agent to form a green color product measured at 615 or 589 nm for the two methods, respectively. The calibration graphs were linear over the range 0.5 to 45 and 10 to 250 μg/mL with a relative standard deviation less than 3.6% (n = 32) and 2.4% (n = 35) for the two methods. Solid-phase reactors can be applied for the determination of NZP in a FIA with high sensitivity and significant advantages over conventional procedures.

DETERMINATION OF NITRAZEPAM IN PHARMACEUTICAL PREPARATIONS USING SOLID-PHASE REACTOR - REVERSE FLOW INJECTION ANALYSIS

Objective: A simple and fast reverse flow injection system including a solid-phase reactor containing PbO2 with spectrophotometric detection was suggested for the determination of nitrazepam (NIT) in pharmaceutical tablets.Methods: The method was based on oxidation of the reagent (phloroglucinol) with PbO2 immobilized in a polymeric matrix which was then coupled with reduced NIT in aqueous medium. The pink-colored product was measured at 530 nm.Results: The calibration graph was linear over the range of 50–400 μg/mL with a relative standard deviation of &lt;2% (n=29) and a sample throughput of 48 samples per hour. The variables of the solid-phase reactor such as composition, particle size, and length of the reactor were studied. The chemical and physical parameters, which affect the reverse flow method, were also studied.Conclusion: The oxidation reactor engaged with a flow system was successfully applied for the determination of NIT with good sensitivity and precision.

Identification of enzymes responsible for nitrazepam metabolism and toxicity in human

Nitrazepam (NZP) is a hypnotic agent that rarely causes liver injuries in humans and teratogenicity in rodents. In humans, NZP is primarily metabolized to 7-aminonitrazepam (ANZP) by reduction and subsequently to 7-acetylamino nitrazepam (AANZP) by acetylation. ANZP can be regenerated from AANZP by hydrolysis in rodents, but it is still unclear whether this reaction occurs in humans. In rodents, AANZP may be associated with teratogenicity, while in humans, it is known that drug-induced liver injuries may be caused by NZP reactive metabolite(s). In this study, we attempted to identify the enzymes responsible for NZP metabolism to obtain a basic understanding of this process and the associated metabolite toxicities. We found that the NZP reductase activity in human liver cytosol (HLC) was higher than that in human liver microsomes (HLM). We purified the responsible enzyme(s) from HLC and found that the NZP reductase was aldehyde oxidase 1 (AOX1). The role of AOX1 was confirmed by an observed increase in the NZP reductase activity upon addition of N1-methylnicotinamide, an electron donor of AOX1, as well as inhibition of this activity in HLC in the presence of AOX1 inhibitors. ANZP was acetylated to form AANZP by N-acetyltransferase (NAT) 2. An experiment using recombinant esterases in an inhibition study using HLM revealed that AANZP is hydrolyzed by arylacetamide deacetylase (AADAC) in the human liver. N-Hydroxylamino NZP, which is suspected to be a reactive metabolite, was detected as a conjugate with N-acetyl-l-cysteine through NZP reduction and ANZP hydroxylation reactions. In the latter reaction, the conjugate was readily formed by recombinant CYP3A4 among the various P450 isoforms tested. In sum, we found that AOX1, NAT2, AADAC, and CYP3A4 are the determinants for the pharmacokinetics of NZP and that they confer interindividual variability in sensitivity to NZP side effects.

Development of an ELISA for nitrazepam based on a monoclonal antibody

We report the production of a specific monoclonal antibody against Nitrazepam (NZP). First, the hapten 7-aminonitrazepam (7-ANZP) was synthesized, then the hapten was coupled with bovine serum albumin and ovalbumin by the diazotization method, and used as immunogen and coating antigen. Factors affecting binding, ionic strength, pH, and methanol concentration in phosphate-buffered saline, were optimized. The monoclonal antibody had a satisfactory IC50 of 0.2 ng/mL for NZP. The cross-reactivities with other analogs were all lower than 0.1% except for 23% with 7-NZP, which suggested that the enzyme-linked immunosorbent assay method possessed high specificity. The recoveries were in the range of 84–95%, indicating that the method could be applied for the detection of NZP in urine.

Dispersive micro-solid-phase extraction of benzodiazepines from biological fluids based on polyaniline/magnetic nanoparticles composite

In this study, diverse types of Fe3O4 nanocomposites modified by polyaniline, polypyrrole, and aniline–pyrrole copolymer were synthesized through chemical oxidative polymerization process for dispersive-μ-solid phase extraction (D-μ-SPE) in the presence of various dopants. The results showed that the nanocomposite modified by polyaniline with p-toluene sulfonic acid as a dopant demonstrated higher extraction efficiency for lorazepam (LRZ) and nitrazepam (NRZ). Also the synthesized magnetic sorbents were characterized. The nanocomposite sorbent in combination with high performance liquid chromatography–UV detection was applied for the extraction, preconcentration and determination of lorazepam and nitrazepam in urine and plasma samples. Different parameters influencing the extraction efficiency including: sample pH, amount of sorbent, sorption time, elution solvent and its volume, salt content, and elution time were optimized. The obtained optimal conditions were: sample pH, 6; amount of sorbent, 5mg; sorption time, 5.0min; elution solvent and its volume, 0.5mM cethyltrimethyl ammonium bromide in acetonitrile, 150μL; elution time, 2.0min and without addition of NaCl. The calibration curves were linear in the concentration range of 1–2000μgL−1. The limits of detection (LODs) were achieved in the range of 0.5–1.8μgL−1 for NRZ and 0.2–2.0μgL−1 for LRZ, respectively. The percent of extraction recoveries and relative standard deviations (n=5) were in the range of 84.0–99.0, 6.1–7.8 for NRZ and 90.0–99.0, 4.1–7.0 for LRZ, respectively. Ultimately, the applicability of the method was successfully confirmed by the extraction and determination of NRZ and LRZ in human urine and plasma samples.

Randomised clinical efficacy trial of topiramate and nitrazepam in treatment of infantile spasms.

Infantile spasms (IS) are among the most catastrophic epileptic syndromes of infancy. The purpose of this study was to compare efficacy and safety of topiramate (TPM) and nitrazepam (NZP) as first-line drugs in the treatment of IS. In a parallel single-blinded randomized clinical trial, 50 patients with IS referred to Pediatric Neurology Clinic of Shahid Sadoughi University of Medical Sciences, Yazd, Iran, were evaluated from September 2008 to March 2010. Patients were randomly assigned to two groups to be treated with TPM or with NZP for 6 months. The primary endpoint was efficacy in cessation of all spasms or reduction of more than 50% in weekly seizure frequency, which was evaluated before and 6 months after the drug use. Secondary outcome was clinical sideeffects of the drugs. Twenty boys (40%) and 30 girls (60%) with the mean age of 9.4±3.8 months were evaluated. Cessation of all spasms occurred in 12 (48%) infants in TPM group and 4(16%) in NZP group. Eight (32%) children in TPM group and 7 (28%) in NZP group had more than 50% reduction in spasms frequency. So, TPM was more effective. Side effects were seen in 32% of TPM and in 36% of NZP groups. Topiramate is an effective and safe drug, which might be considered as the firstline drug for the treatment of ISs.

Synthesis and Characterization of Poly (3-hexylthiophene) and Used as Modified Electrode for Determination of Antidepressants in Pharmaceutical Formulations and Urine Samples

Conducting poly (3-hexylthiophene) (P3HT) was synthesized electrochemically in aqueous medium and characterized by electrochemical impedance spectroscopy, Tafel, SEM and TEM. The antidepressants drugs diazepam (DIZ), nitrazepam (NITZ) and imipramine (IMI) were analyzed on nanosize P3HT modified glassy carbon electrode. The voltammetric behavior of the three antidepressants was studied in aqueous acid, neutral, and alkaline conditions. One well-defined reduction peak each for DIZ and NITZ and one oxidation peak for IMI were observed in the cyclic voltammograms. The influence of pH, scan rate on peak current was studied extensively. A systematic study of the experimental parameters that affect the differential pulse stripping voltammetric response was carried out and optimized conditions which yield maximum peak current were arrived. The calibration was made under optimum conditions. The range of study for DIZ, NITZ and IMI was 100–1100, 150–1100 and 200–1300 μg/L and lower limit of determination was 75, 100, and 150 μg/L respectively. The suitability of the method for the determination of the three antidepressants in pharmaceutical preparations and urine samples was also ascertained.

Spectrophotometric Determination of Nitrazepam in Pharmaceutical Tablets Using Flow Injection Analysis

Nitrazepam (NZP) was determined spectrophotometrically in the pure form and in the pharmaceutical tablets using flow injection analysis (FIA). The method was based on oxidative coupling organic reaction of reduced NZP with promethazine hydrochloride in the presence of sodium periodate to give a green solution having an absorbance maximum at 613 nm. The various chemical and physical variables were optimized. The calibration graph of NZP is linear from 1 to 50 μg mL-1 with detection limit (S/N = 3) was 0.9284 μg mL-1. The method was successfully applied to the analysis of NZP in pharmaceutical tablets. The results obtained by applying the proposed FIA method were in good agreement with those obtained by British Pharmacopoeia method at the 95% confidence.

Nitrazepam withdrawal/flumazenil

Nitrazepam withdrawal/flumazenil

Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines

Nitrobenzodiazepine (NBDZ) is a sedative-hypnotic drug used in the treatment of anxiety and sleep problems. Overdose of NBDZ may cause severe neurological effects, especially for people in drug abuse or addiction. In the present study, we investigated NBDZ nitroreduction in rat enteric contents and characterized the role of enterobacterial nitroreductase in the reductive pathway. Nitroreduction of flunitrazepam (FZ) was studied in the microsomal membrane fractions of rat liver, jejunum and jejunal microflora using HPLC analysis. In the jejunal microflora, FZ was demonstrated to be significantly reduced to its amino derivative under anaerobic condition. Escherichia coli type I nitroreductase NfsB (EC 1.5.1.34) was found in rat jejunal microflora via PCR technique and Western blotting. The participation of NfsB in FZ nitroreduction was demonstrated from inhibition studies. Kinetic study of the purified recombinant NfsB indicated that nitroreduction of FZ, nitrazepam (NZ) and clonazepam (CZ) are mediated by NfsB, where CZ shows lower k cat/ K M ratio than that of the other two. Finally, two other nitroreductases E. cloacae NR (EC 1.6.99.7) and S. typhimurium Cnr were also found to be responsible for FZ nitroreduction. These results provide that the reduction of NBDZ in normal flora is catalyzed by type I nitroreductase NfsB.

Long-term stability of various drugs and metabolites in urine, and preventive measures against their decomposition with special attention to filtration sterilization

The long-term stability of drugs and metabolites of forensic interest in urine, and preventive measures against their decomposition have been investigated, with special attention to filtration sterilization. An aseptic urine collection kit, which was recently developed based on filtration sterilization, was utilized for the aseptic collection and storage of urine samples. For evaluating preservation measures, methamphetamine (MA), amphetamine (AP), nitrazepam (NZ), estazolam (EZ), 7-aminoflunitrazepam (7AF), cocaine (COC), and 6-acetylmorphine (6AM) were spiked into urine at 500 ng/mL each, and were monitored for 6 months at 25, 4, and −20 °C, after the addition of NaN 3 and/or filtration sterilization using the aseptic collection kit. In severely contaminated urine with bacteria, there were significant losses of 7AF and NZ, and slight decomposition of MA and AP at 25 °C. However, such degradation was successfully suppressed by the use of the kit, though the use of the kit and NaN 3 were preferred for 7AF. The kit was also effective in preventing the hydrolyses of COC and 6AM, while it was suggested that the common preservative NaN 3 can accelerate the hydrolysis of such ester-type drugs and metabolites.

Nitrazepam Synthesized by Amination of 2-(2-Chloroacetamido)-5-nitrobenzophenone with Hexamethylenetetramine

Nitrazepam Synthesized by Amination of 2-(2-Chloroacetamido)-5-nitrobenzophenone with Hexamethylenetetramine