Ultrasound-assisted electromembrane extraction of clonazepam from plasma and determination using capillary electrophoresis.

In this work, ultrasound-assisted electromembrane extraction (UA-EME) coupled with capillary electrophoresis (CE) and diode array detection (DAD) was developed for the determination of clonazepam from plasma samples. A comparative study was carried out between conventional EME and UA-EME methods to investigate the influence of the ultrasound waves on the extraction efficiency. The central composite design was used for the optimization of the variables affecting these methods to achieve the best extraction efficiency. Under optimal extraction conditions, the UA-EME provided better extraction recovery in a shorter time (58% in 13 min) than the EME method (42% in 30 min). Ultrasound reduces the extraction time and increased recovery by reducing the thickness of the barrier layer. In addition, this method provided a higher pre-concentration factor (203) and a lower limit of detection (3 ng mL-1) with good repeatability (RSDs were less than 10.11%).

Diagnostic evaluation and management of seizures and status epilepticus in children with known epilepsy or new-onset seizures: A retrospective and comparative analysis.

OBJECTIVES: The purpose of this study was to describe and compare the initial management, including clinical/biological investigation and treatment, of new-onset seizures and status epilepticus (SE) in children versus seizures and SE in those with known epilepsy. METHODS: This was a retrospective, single-center, observational study conducted in an urban pediatric hospital in Paris. All patients, aged from 1 month to 18 years, admitted to the pediatric intensive care unit, the high-dependency care unit, and those who required hospitalization in the short-term unit of the emergency department between January 1 and December 31, 2014 for seizures and/or SE were included. RESULTS: We analyzed the data of 190 children: new-onset seizures (N=118; group A) versus those with known epilepsy (N=72; group B). At least one diagnostic test was performed on 156 patients (82.1%) (group A, N=104, 88.1%; group B, N=52, 72.2%; P=0.05). In group B, blood levels of antiepileptic drugs were measured in 14 of the 38 patients with SE, of whom six were under dosed. Treatments were: first line, diazepam (group A, 80%; group B, 46%; P<0.001); second line, diazepam (group A, 56%; group B, 34%; P=0.02) or clonazepam (group A, 24%; group B, 46%; P=0.001); third line, phenytoin (group A, 54%; group B, 22%; P<0.001) or clonazepam (group A, 18%; group B, 61%; P<0.001). CONCLUSION: Diagnostic evaluation and treatment should be individualized for children with known epilepsy.

Screen printed carbon electrode modified with a copper@porous silicon nanocomposite for voltammetric sensing of clonazepam.

The work describes an electrochemical sensor for the determination of the tranquilizer clonazepam (CZP) in serum and pharmaceutical preparations. A screen printed carbon electrode (SPCE) was modified with copper nanoparticles anchored on porous silicon (PSi). The surface of the SPCEs modified with the Cu/PSi nanostructure was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoemission spectroscopy, energy dispersive X-ray spectroscopy and field-emission scanning electron microscopy. Cyclic and differential pulse voltammetric methods were used for the electrochemical studies and electrochemical detection, respectively. Several parameters controlling the performance of the modified SPCE were optimized. The peak current values (at a potential of -0.52 V) were used to construct calibration plots. Under the optimum conditions, the calibration plot is linear in the 0.05-7.6 µM CZP concentration range, and the detection limit is 15 nM. The sensor is reproducible, repeatable, highly selective and sensitive. It was successfully applied to the determination of CPZ in spiked serum and in drugs. Graphical abstract Scheme of electrochemical reduction of clonazepam on the designed copper@porous silicon modified screen printed carbon electrode (CuNPs/PSi/SPCE). This electrode was employed for the determination of clonazepam in tablets and human blood plasma using differential pulse voltammetry.

Electrochemical determination of clonazepam drug based on glassy carbon electrode modified with Fe3O4/R-SH/Pd nanocomposite.

Herein, fabrication and electrochemical evaluation of a new sensing platform, which is sensitive to clonazepam (CLZP) drug, has been illustrated. By using Fourier transform infrared spectrometry (FT-IR), electron microscopy (EM) techniques and vibrating sample magnetometer (VSM), synthesized nanocomposite, consisting of Fe3O4, 3-aminopropyltriethoxysilane (APTES), cyanuric chloride (CC), 2-mercaptoethanol and Pd (Fe3O4/R-SH/Pd), was characterized. Then using the common drop-casting method, the new nanocomposite was cast on the surface of bear glassy carbon electrode. The influence of effective experimental factors was investigated to optimize the voltammetric response (current) of the modified electrode for electrochemical studies and determining the CLZP. Under optimal conditions, by the designed sensor a linear relationship was found between reduction peak current and CLZP concentrations in the range of 10 nM to 1 µM CLZP, with a detection limit of 3.02 nM. This system was also employed for measuring CLZP in human serum as well as pharmaceutical samples effectively. In addition, the fabricated sensor showed good sensitivity, long-term stability, and reproducibility. Through statistical student's t-test and F-test, the insignificant systematic error was found between measured and real values and also between voltammetric method at the surface of the proposed electrode and standard HPLC method.

Validation of the Neogen ELISA Benzodiazepine Kit using Clonazepam as the Target Molecule for Blood and Urine.

This study demonstrates the validation of a semi-quantitative method for the rapid screening of whole blood and urine specimens using clonazepam as the target molecule for the Neogen® Benzodiazepine kit. Decision points were validated at 10.0 ng/mL for whole blood and 25.0 ng/mL for urine. The validation design was based on the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation and included the evaluation of sensitivity, precision, specificity, carryover, hook effect, drift, ruggedness/robustness and a case sample evaluation. The experimental limit of detection for clonazepam was determined to be at least 5.0 ng/mL in whole blood and at least 10.0 ng/mL in urine. Excellent precision was demonstrated when the assay was evaluated using the mean of three replicates from five separate runs (n = 15) at the decision point and at concentration levels ±50% and +100% of the decision point. Although the method was optimized and exceptional precision was demonstrated at each level, the current SWGTOX validation requirements for a valid decision point were not fulfilled. However, both the blood and urine matrix did meet the proposed revision of the SWGTOX requirements for determining a valid decision point promulgated by the American Academy of Forensic Sciences Standards Board and the assay was reliably able to detect benzodiazepines without interference from matrix components or other compounds routinely detected in authentic case samples. Case sample results were comparable with those obtained when the samples were initially screened using oxazepam as the target molecule. The Neogen® Benzodiazepine kit using clonazepam as the target molecule exhibited cross-reactivity for 29 different benzodiazepines and demonstrated excellent precision and sensitivity in both whole blood and urine, making it an efficient and reliable method to screen for benzodiazepines, even though the validation did not fulfill current SWGTOX requirements for a valid decision point.

Klonopin assay using modified electrode with multiwalled carbon nanotubes and poly melamine nanocomposite.

Developing of cheap, sensitive and stable sensors plays a significant role in pharmaceutical and clinical applications. Considering the effective role of Klonopin (KNP) in the treatment of epilepsy, KNP quantification in its production process for dose adjustments and checking the purity and also after its usage by patents for bioavailability testing and effectiveness assay is vital. In present work, an efficient electrochemical sensor based on poly melamine and multiwalled carbon nanotubes nanocomposite (PMela/CNTs) was constructed which displayed effective electrochemical response toward KNP. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square-wave voltammetry (SWV) experiments were applied for performance evaluation of the PMela/CNTs modified electrode and electrochemical redox behavior of KNP. Distinguish synergetic effect was observed between CNTs and poly melamine in response to KNP electrochemical redox reaction. A linear detection range of 0.05 to 10 µM with the detection limits of 63 nM was achieved for KNP analysis. The practical application of the PMela/CNTs modified electrode revealed satisfactory results for quantification of KNP in biological fluids.

A relative study on sonochemically synthesized mesoporous WS2 nanorods & hydrothermally synthesized WS2 nanoballs towards electrochemical sensing of psychoactive drug (Clonazepam).

In this paper, mesoporous tungsten sulfide electrocatalyst (MP-WS2) were developed through a facile sonochemical technique (SC) and utilized as an electrocatalyst for the sensitive electrochemical detection of Psychoactive drug. The as-prepared SC-MP-WS2 NRs and HT-WS2 NPs (hydrothermally synthesized) were characterized using XRD, Raman, XPS, FESEM, HRTEM, BET, EDX, and electrochemical analysis, which exposed the formation of WS2 in the form of mesoporous nanorods in shape. Further, the use of the as-developed SC-MP-WS2 NRs and HT-WS2 NPs as an electrocatalyst for the detection of clonazepam (CNP). Interestingly, the SC-MP-WS2 NRs modified screen-printed carbon electrode (SC-MP-WS2 NRs/SPCE) exhibited an excellent electrocatalytic performance, and enhanced reduction peak current when compared to HT-WS2 NPs with unmodified electrode. Moreover, as-prepared SC-MP-WS2 NRs/SPCE displayed wide linear response range (10-551 µM), lower detection limit (2.37 nM) and high sensitivity (24.32 µAµM-1cm-2). Furthermore, SC-MP-WS2 NRs/SPCE showed an excellent selectivity even in the existence of potentially co-interfering compounds. The proposed sensor was successfully applied for the determination of CNP in biological and drug samples with acceptable recovery.

Spectrofluorometric determination of clonazepam in dosage forms: Application to content uniformity testing and human plasma.

The present paper describes a developed and validated simple, highly sensitive and cost-effective spectrofluorometric method for determination of clonazepam (CNP). The proposed method depends on forming a highly fluorescent product through the reduction of CNP with Zn/HCl. The produced fluorophore exhibits a strong fluorescence at λem 350 nm after excitation at λex 250 nm. The use of carboxymethylcellulose (CMC) greatly enhanced the fluorescence intensity of the produced fluorophore to the extent of about 100%. Calibration curve showed good linear regression (r2  > 0.9998) within test ranges of 20-400 ng ml-1 with a lower detection limit of 0.67 ng ml-1 and lower quantification limit of 2.22 ng ml-1 upon using CMC. The method was successfully applied to the analysis of CNP in its pharmaceutical formulations and the results were in agreement with those obtained using a reference method. Furthermore, the content uniformity testing of the tablets was also performed. The application of the proposed method was extended to determine CNP in spiked human plasma sample as a preliminary investigation and the results were satisfactory.

A Case Report of Clonazepam Dependence: Utilization of Therapeutic Drug Monitoring During Withdrawal Period.

Clonazepam is long-acting benzodiazepine agonist used in short-acting benzodiazepine withdrawal; however, recent observations suggest the existence of its abuse. We demonstrate a 40-year-old man with a 20-year history of psychiatric care with recently benzodiazepine dependence (daily intake of ∼60 mg of clonazepam and 10 mg of alprazolam). High serum levels of both drugs were analyzed 3 weeks before admission to hospitalization (clonazepam 543.9 ng/mL, alprazolam 110 ng/mL) and at the time of admission (clonazepam 286.2 ng/mL, alprazolam 140 ng/mL) without any signs of benzodiazepine intoxication. Gradual withdrawal of clonazepam with monitoring of its serum levels and increase of gabapentin dose were used to minimize physical signs and symptoms of clonazepam withdrawal. Alprazolam was discontinued promptly. Clinical consequences of the treatment were controllable tension, intermittent headache, and rarely insomia. It is the first case report showing utilization of therapeutic drug monitoring during withdrawal period in the patient with extreme toleration to severe benzodiazepine dependence.

Development of a voltammetric assay, using screen-printed electrodes, for clonazepam and its application to beverage and serum samples.

This paper describes the development of an electrochemical assay based on screen-printed carbon sensors for the determination of clonazepam in serum and in wine. The cyclic voltammetric behaviour of the drug was investigated and the effects of pH and scan rate on the peak current and peak potential determined. Two reduction peaks were recorded on the initial negative going scan, which were considered to result from the 2e(-), 2 H(+) reduction of the 4,5-azomethine and from the 4e(-), 4 H(+) reduction of the 7-NO2 to a hydroxylamine. On the return positive going scan an oxidation peak was seen, which was considered to result from the 2e(-), 2 H(+) oxidation (O1) of the hydroxylamine to the corresponding nitroso species. At pH 11 the solution of clonazepam was found to turn from clear to yellow in colour and the voltammetric signal of the O1 oxidation process was found to be adsorptive in nature, this was exploited in the development of an adsorptive stripping voltammetric assay. Experimental conditions were then optimised for the differential pulse adsorptive voltammetric measurement of clonazepam in wine and serum samples. It was shown that these analyses could be performed on only 100µL of sample which was deposited on the sensor surface. Mean recoveries of 79.53% (%CV=9.88%) and 88.22% (%CV=14.1%) were calculated for wine fortified with 3.16µg/mL and serum fortified with 12.6µg/mL.

GABAergic modulation in central sensitization in humans: a randomized placebo-controlled pharmacokinetic-pharmacodynamic study comparing clobazam with clonazepam in healthy volunteers.

Positive allosteric modulators of GABAA receptors (GAMs) acting at specific subtypes of GABAA receptors effectively restore compromised spinal pain control in rodents. Studies addressing a similar antihyperalgesic effect in humans are sparse and are hampered by sedative effects of nonselective GAMs available for use in humans. We present results from a randomized controlled double-blind crossover study in 25 healthy volunteers, which addressed potential antihyperalgesic actions of clobazam (CBZ) and clonazepam (CLN) at mildly sedating equianticonvulsive doses. Clobazam was chosen because of its relatively low sedative properties and CLN because of its use in neuropathic pain. Tolterodine (TLT) was used as an active placebo. The primary outcome parameter was a change in the area of cutaneous UVB irradiation-induced secondary hyperalgesia (ASH), which was monitored for 8 hours after drug application. Sedative effects were assessed in parallel to antihyperalgesia. Compared with TLT, recovery from hyperalgesia was significantly faster in the CBZ and CLN groups (P = 0.009). At the time point of maximum effect, the rate of recovery from hyperalgesia was accelerated by CBZ and CLN, relative to placebo by 15.7% (95% confidence interval [CI] 0.8-30.5), P = 0.040, and 28.6% (95% CI 4.5-52.6), P = 0.022, respectively. Active compounds induced stronger sedation than placebo, but these differences disappeared 8 hours after drug application. We demonstrate here that GAMs effectively reduce central sensitization in healthy volunteers. These results provide proof-of-principle evidence supporting efficacy of GAMs as antihyperalgesic agents in humans and should stimulate further research on compounds with improved subtype specificity.

Detection Times of Diazepam, Clonazepam, and Alprazolam in Oral Fluid Collected From Patients Admitted to Detoxification, After High and Repeated Drug Intake.

BACKGROUND: Clonazepam, diazepam, and alprazolam are benzodiazepines with sedative, anticonvulsant, and anxiolytic effects, but their prevalence in drug abuse and drug overdoses has long been recognized. When detection times for psychoactive drugs in oral fluid are reported, they are most often based on therapeutic doses administered in clinical studies. Repeated ingestions of high doses, as seen after drug abuse, are however likely to cause positive samples for extended time periods. Findings of drugs of abuse in oral fluid collected from imprisoned persons might lead to negative sanctions, and the knowledge of detection times of these drugs is thus important to ensure correct interpretation. The aim of this study was to investigate the time window of detection for diazepam, clonazepam, and alprazolam in oral fluid from drug addicts admitted to detoxification. METHODS: Twenty-five patients with a history of heavy drug abuse admitted to a detoxification ward were included. Oral fluid was collected daily in the morning and the evening and urine samples every morning for 10 days, using the Intercept device. Whole blood samples were collected if the patient accepted. The cutoff levels in oral fluid were 1.3 ng/mL for diazepam, N-desmethyldiazepam, and 7-aminoclonazepam and 1 ng/mL for clonazepam and alprazolam. In urine, the cutoff levels for quantifications were 30 ng/mL for alprazolam, alpha-OH-alprazolam, and 7-aminoclonazepam, 135 ng/mL for N-desmethyldizepam, and 150 ng/mL for 3-OH-diazepam and for all the compounds, the cutoff for the screening analyses were 200 ng/mL. RESULTS: The maximum detection times for diazepam and N-desmethyldiazepam in oral fluid were 7 and 9 days, respectively. For clonazepam and 7-aminoclonazepam, the maximum detection times in oral fluid were 5 and 6 days, respectively. The maximum detection time for alprazolam in oral fluid was 2.5 days. New ingestions were not suspected in any of the cases, because the corresponding concentrations in urine were decreasing. Results from blood samples revealed that high doses of benzodiazepines had been ingested before admission, and explains the longer detection times in oral fluids than reported previously after intake of therapeutic doses of these drugs. CONCLUSIONS: This study has shown that oral fluid might be a viable alternative medium to urine when the abuse of benzodiazepines is suspected.

A polydrug intoxication involving methoxetamine in a drugs and driving case.

Methoxetamine ((RS)2-(3-methoxyphenyl)-2-(ethylamino)cyclohexanone)) is becoming a drug of interest among practitioners of forensic toxicology. In this case report, we describe the case background, standard field sobriety tests, sampling, and analysis of this drug in a whole blood sample as well as screening methods and analysis from a driver operating under the influence of intoxicating substances. Methoxetamine was isolated from the blood sample using mixed mode solid phase extraction. After elution and evaporation, the residue was dissolved in mobile phase (consisting of acetonitrile and aqueous formic acid) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS). The case sample was found to contain clonazepam, 7-aminoclonazepam, carboxy-THC, Ddphenhydramine, and MDMA. The case sample was found to contain 10 ng/mL of the drug (methoxetamine) in whole blood. The results of this drug analysis and previous analyses are discussed in terms of this driver operating under the influence of drugs.

Selective extraction of clonazepam from human plasma and urine samples by molecularly imprinted polymeric beads.

A molecularly imprinted polymer (MIP) based on free-radical polymerization was prepared with 1-(N,N-biscarboxymethyl)amino-3-allylglycerol and N,N-dimethylacrylamide as functional monomers, N,N-methylene diacrylamide as the cross-linker, copper ion-clonazepam as the template and 2,2-azobis(2-methylbutyronitrile) as the initiator. The imprinted polymer was characterized by Fourier transform infrared spectroscopy, elemental analysis, thermo-gravimetric analysis, and SEM. The MIP of agglomerated microparticles with multipores was used for SPE. The imprinted polymer sorbent was selective for clonazepam. The optimum pH and sorption capacity were 5 and 0.18 mg/g at 20C, respectively. The profile of the drug uptake by the sorbent reflects good accessibility of the active sites in the imprinted polymer sorbent. The MIP-SPE was the most feasible technique for the extraction of clonazepam with a high recovery from human plasma and urine samples.

Positive lidocaine toxicology screen after J-Tip for venipuncture.

Venipuncture is common in children, and topical anesthetics are often used to alleviate the pain of the procedure. The J-Tip (National Medical Products, Inc, Irvine, Calif) device has become popular as a rapid and effective means of delivering lidocaine noninvasively. We report a case of a positive lidocaine blood toxicology screen after the use of the J-Tip device in a child pre-venipuncture. A repeat toxicology screen obtained 1 hour later by venipuncture without J-Tip use was negative. This report serves to remind clinicians that topical anesthetics may interfere with toxicology assays, leading to unreliable toxicology results.

Development and validation of an ultra-performance liquid chromatography/electrospray ionization-tandem mass spectrometry bioanalytical method for quantifying clonazepam in human plasma.

A sensitive, selective, and rapid ultra-performance LC (UPLC)/MSIMS method was validated for the confirmation and quantification of clonazepam in human plasma. The analyte was extracted from human plasma with diethyl ether, reaching an average recovery of 64.02 and 66.48% for clonazepam and the internal standard, respectively. The separation was performed on a Waters ACQUITY UPLC BEH C18 column (50 x 2.1 mm id, 1.7 microm particle size) with gradient elution at a flow rate of 0.25 mL/min using a 0.5% formic acid solution (mobile phase A) and acetonitrile-methanol-formic acid (75+25 + 0.5, v/v/v; mobile phase B). Detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode via electrospray ionization. Linear calibration curves were obtained in the concentration range of 0.3-50.0 ng/mL, with an LOQ of 0.3 ng/mL. The intraday and interday precision (CV) values were below 10%, and accuracy (relative error) ranged from -2.6 to 6.6% at all QC levels. The suggested method was successfully applied for the determination of clonazepam in human plasma in a bioequivalence study.

Melamine modified gold nanoprobe for "on-spot" colorimetric recognition of clonazepam from biological specimens.

Inexpensive, rapid, and reliable methods of detection are crucial to the control of toxicological investigations. Here we report a highly sensitive, selective and cost effective method for the detection of trace amounts of clonazepam based on gold nanoparticles (AuNPs) in the presence of melamine. Hydrogen bonding interactions between clonazepam and melamine resulted in the aggregation of AuNPs and a consequent color change of AuNPs from wine red to blue. The results showed that the absorption ratio (A636/A552) was linear for clonazepam concentrations in the range of 10 × 10(-8) to 1.0 × 10(-9) M (R(2) = 0.999). The detection limit was 8.9 × 10(-10) M (S/N = 3), which was much lower than that of most existing methods. Coexisting substances including alprazolam, diazepam, nitrazepam and lorazepam did not affect the determination of clonazepam. The sensor developed by this new approach could be used as a spot test and a good alternative means for on-site and real time screening of clonazepam. This proposed scheme was also supported by the use of real samples such as skeletal remains and blood to illustrate the applicability of the developed nanosensor by a series of experiments.

Computer-assisted design and synthesis of a highly selective smart adsorbent for extraction of clonazepam from human serum.

A computational approach was applied to screen functional monomers and polymerization solvents for rational design of molecular imprinted polymers (MIPs) as smart adsorbents for solid-phase extraction of clonazepam (CLO) form human serum. The comparison of the computed binding energies of the complexes formed between the template and functional monomers was conducted. The primary computational results were corrected by taking into calculation both the basis set superposition error (BSSE) and the effect of the polymerization solvent using the counterpoise (CP) correction and the polarizable continuum model, respectively. Based on the theoretical calculations, trifluoromethyl acrylic acid (TFMAA) and acrylonitrile (ACN) were found as the best and the worst functional monomers, correspondingly. To test the accuracy of the computational results, three MIPs were synthesized by different functional monomers and their Langmuir-Freundlich (LF) isotherms were studied. The experimental results obtained confirmed the computational results and indicated that the MIP synthesized using TFMAA had the highest affinity for CLO in human serum despite the presence of a vast spectrum of ions.

Clonazepam use for prevention of acute and delayed vomiting induced by Cisplatin-based chemotherapy for lung cancer.

We investigated the efficacy of clonazepam as an antiemetic in cisplatin-based chemotherapy for lung cancer. Seven patients experienced cisplatin-induced vomiting despite antiemetic therapy including 5-hydroxytryptamine(3) (5-HT(3)) antagonist and dexamethasone. Therefore, the antiemetic therapy including clonazepam, 5-HT(3) antagonist and dexamethasone was subsequently explored in the next course for the same seven patients. We administered clonazepam (0.5 or 1.0 mg/kg) once a day orally for 5 d from day one prior to chemotherapy. The grade of delayed vomiting, evaluated according to Common Terminology Criteria for Adverse Events Version 3.0, in the courses of therapy with clonazepam was significantly lower than without clonazepam (p=0.013). The patients whose serum clonazepam concentrations were below the lower limit of detection (3.0 ng/ml) experienced vomiting in three of three courses, whereas the patients whose serum clonazepam concentrations were higher than 4.3 ng/ml experienced no vomiting in six of seven courses. We observed that the symptom of cisplatin-induced delayed vomiting is controlled with serum clonazepam levels in the order of 10.0 ng/ml.