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1.
Ultrason Sonochem ; 58: 104596, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31450358

ABSTRACT

Herein, we report a super-active electrocatalyst of copper(II) oxide nanoparticles (CuO NPs) decorated functionalized multiwalled carbon nanotubes (CuO NPs@f-MWCNTs) by the ultrasonic method. The as-synthesized CuO NPs@f-MWCNTs was characterized through the FESEM, XPS, XRD and electrochemical impedance spectroscopy (EIS). The combination of highly active CuO NPs and highly conductive f-MWCNTs film with rapid detection enables this nanohybrid to display excellent electrochemical performance towards anesthesia drug. Furthermore, the hybrid electrocatalyst modified SPCE was developed for the determination of flunitrazepam (FTM) for the first time. FTM is important anesthesia drug with high adverse effect in human body. Benefiting from the synergistic reaction of CuO NPs and f-MWCNTs, this nanohybrid exhibited high sensitivity and specificity towards FTM electro-reduction. The CuO NPs@f-MWCNTs film modified SPCE exhibits outstanding electrochemical activity including excellent reproducibility, wide linear range from 0.05 to 346.6 µM with nanomolar limit of detection for FTM detection. Further, the as-modified CuO NPs@f-MWCNTs/SPCE has been applied to determination of FTM in biological and drug samples with satisfactory recovery results, thereby showing a notable potential for extensive (bio) sensor applications.


Subject(s)
Anti-Bacterial Agents/analysis , Copper/chemistry , Electrochemistry/instrumentation , Flunitrazepam/analysis , Limit of Detection , Nanospheres/chemistry , Nanotubes, Carbon/chemistry , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/urine , Chemistry Techniques, Synthetic , Electrodes , Flunitrazepam/blood , Flunitrazepam/chemistry , Flunitrazepam/urine , Humans , Nanotechnology , Time Factors
2.
Article in English | MEDLINE | ID: mdl-25579112

ABSTRACT

This study establishes a novel calibration method for pre-equilibrium hollow-fiber liquid-phase microextraction (PE-HF-LPME), where the time constant of the extraction of the analyte from sample matrix to the extraction phase (organic solvent) is obtained from a simple concentration curve. Comparing to the traditional kinetic calibration method, where the time constant was obtained from the extraction time profile, the new calibration approach shows improved accuracy and precision. More importantly, deuterated standards are not required in the new method, thus significantly improving its cost-effectiveness and extending its applicability to a wide range of analytes lack of deuterated analogs serving as internal standards. In addition, mass spectrometry is not necessary for the quantification of analytes with the new calibration method, which may further extend the applicability of PE-HF-LPME to some laboratories without mass spectrometers. This study has been substantiated with both theoretical and experimental evidences. Further, the feasibility of the method for real biological samples was demonstrated by measuring the free concentration of flunitrazepam in urine and plasma samples and its drug-protein binding ratio in plasma. The results showed that the method had a short analysis time and was easily implemented with high accuracy and good reproducibility.


Subject(s)
Liquid Phase Microextraction/methods , Body Fluids/chemistry , Calibration , Flunitrazepam/blood , Flunitrazepam/urine , Reproducibility of Results
3.
Leg Med (Tokyo) ; 17(3): 150-6, 2015 May.
Article in English | MEDLINE | ID: mdl-25542365

ABSTRACT

A rapid and sensitive method was developed for the determination of benzodiazepines and benzodiazepine-like substances (BZDs) by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of BZDs. Determination of 8 BZDs (with two of their metabolites) belonging to top 12 medical drugs detected in poisonous cases in Japan, was performed using diazepam-d5 as the internal standard. The limit of detection of zolpidem was 0.07ng/ml with its quantification range of 0.2-20ng/ml in blood, in the best case, and the limit of detection of flunitrazepam was 2ng/ml with its quantification range of 6-200ng/ml in blood, in the worst case. The spectra of zopiclone in MALDI-MS and MS/MS were different from those in electrospray ionization MS and MS/MS. Present method provides a simple and high throughput method for the screening of these BZDs using only 20µl of blood. The developed method was successfully used for the determination of BZDs in biological fluids obtained from two victims.


Subject(s)
Benzodiazepines/blood , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Azabicyclo Compounds/blood , Benzodiazepines/metabolism , Flunitrazepam/blood , Humans , Piperazines/blood , Pyridines/blood , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Zolpidem
4.
J Anal Toxicol ; 38(9): 686-95, 2014.
Article in English | MEDLINE | ID: mdl-25015743

ABSTRACT

The long-term stability of benzodiazepines, opioids, central stimulants and medicinal drugs in authentic postmortem blood samples was studied. All together, 73 samples were reanalyzed after storage at -20°C for 16-18 years. At reanalysis samples containing diazepam, nordiazepam and flunitrazepam demonstrated only small changes during long-term storage when mean and median drug concentrations were compared, while clonazepam concentrations tended to decrease. Samples containing amphetamine, morphine, codeine and 'acidic' medicinal drugs as paracetamol and meprobamate also showed small changes over 16-18 years in mean and median drug concentrations at a group level. For many drugs, however, single samples could demonstrate marked concentration changes, both increases and decreases during storage. For 'alkaline' medicinal drugs, concentration losses were observed in most cases.


Subject(s)
Analgesics, Opioid/blood , Benzodiazepines/blood , Blood Preservation/methods , Central Nervous System Stimulants/blood , Amphetamine/blood , Amphetamine/chemistry , Analgesics, Opioid/chemistry , Benzodiazepines/chemistry , Central Nervous System Stimulants/chemistry , Codeine/blood , Codeine/chemistry , Diazepam/blood , Diazepam/chemistry , Flunitrazepam/blood , Flunitrazepam/chemistry , Forensic Toxicology/methods , Freezing , Humans , Morphine/blood , Morphine/chemistry , Nordazepam/blood , Nordazepam/chemistry , Substance Abuse Detection/methods , Time Factors
5.
Anal Bioanal Chem ; 406(18): 4443-51, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24788888

ABSTRACT

The analysis of opioids, cocaine, and metabolites from blood serum is a routine task in forensic laboratories. Commonly, the employed methods include many manual or partly automated steps like protein precipitation, dilution, solid phase extraction, evaporation, and derivatization preceding a gas chromatography (GC)/mass spectrometry (MS) or liquid chromatography (LC)/MS analysis. In this study, a comprehensively automated method was developed from a validated, partly automated routine method. This was possible by replicating method parameters on the automated system. Only marginal optimization of parameters was necessary. The automation relying on an x-y-z robot after manual protein precipitation includes the solid phase extraction, evaporation of the eluate, derivatization (silylation with N-methyl-N-trimethylsilyltrifluoroacetamide, MSTFA), and injection into a GC/MS. A quantitative analysis of almost 170 authentic serum samples and more than 50 authentic samples of other matrices like urine, different tissues, and heart blood on cocaine, benzoylecgonine, methadone, morphine, codeine, 6-monoacetylmorphine, dihydrocodeine, and 7-aminoflunitrazepam was conducted with both methods proving that the analytical results are equivalent even near the limits of quantification (low ng/ml range). To our best knowledge, this application is the first one reported in the literature employing this sample preparation system.


Subject(s)
Analgesics, Opioid/analysis , Cocaine/analysis , Gas Chromatography-Mass Spectrometry/methods , Solid Phase Extraction/methods , Substance Abuse Detection/methods , Acetamides/chemistry , Analgesics, Opioid/blood , Analgesics, Opioid/urine , Automation , Cocaine/blood , Cocaine/urine , Codeine/analogs & derivatives , Codeine/analysis , Codeine/blood , Codeine/urine , Flunitrazepam/analogs & derivatives , Flunitrazepam/analysis , Flunitrazepam/blood , Flunitrazepam/urine , Fluoroacetates/chemistry , Humans , Limit of Detection , Methadone/analysis , Methadone/blood , Methadone/urine , Morphine/analysis , Morphine/blood , Morphine/urine , Morphine Derivatives/analysis , Morphine Derivatives/blood , Morphine Derivatives/urine , Reproducibility of Results , Robotics/instrumentation , Robotics/methods , Trimethylsilyl Compounds/chemistry
6.
Analyst ; 138(20): 6185-91, 2013 Oct 21.
Article in English | MEDLINE | ID: mdl-23971077

ABSTRACT

The electroanalytical sensing of Rohypnol® (flunitrazepam) is reported for the first time utilising screen-printed graphite electrodes without the requirement for any additional pre-treatment or modification. The methodology is shown to be useful for quantifying low levels (µg mL(-1)) of Rohypnol® in not only buffered solutions but also two internationally favoured drinks: Coca Cola™ and the alcopop WKD™ without any sample pre-treatment. The current analytical approaches for the sensing of Rohypnol® are also summarised within this paper. The niche of this electroanalytical protocol is the lack of the requirement of any pre-treatment of the sample/beverage or electrode modification (cleaning, pre-treatment etc.) for the determination of Rohypnol® in beverages and offers a potential rapid, cost-effective, yet suitably sensitive and accurate screening solution to the problem posed by coloured drinks to products such as the colour changing 'Smart Cup'.


Subject(s)
Beverages/analysis , Electrochemical Techniques/methods , Flunitrazepam/analysis , Forensic Toxicology/methods , Graphite/chemistry , Electrodes , Flunitrazepam/blood , Humans
7.
J Chromatogr A ; 1266: 10-6, 2012 Nov 30.
Article in English | MEDLINE | ID: mdl-23116799

ABSTRACT

Hollow fiber liquid-phase microextraction (HF-LPME) has been demonstrated to potentially become a mainstream sample preparation technique for complex samples. Nevertheless, the need for a relatively long extraction time is considered to be the major disadvantage of this method. Lengthy extractions may cause the loss of the extraction phase and may change the contents of biological samples via the action of enzymes. Therefore, control calibrations for particular biological systems must be made. In this study, a theoretical model of the mass transfer dynamics of two-phase HF-LPME was proposed, and the kinetic calibration (KC) of this method for plasma and urine samples was validated. The theoretical results were validated by examining the kinetics of the extraction and back-extraction processes of HF-LPME. The KC-HF-LPME method was successfully used to correct for matrix effects in plasma and urine samples during flunitrazepam analysis. The free amount of flunitrazepam was extracted from plasma for 10 min and analyzed by gas chromatography/mass spectrometry. The amount of pre-added standard and the standard remaining in the extraction phase after extraction were used for the quantification of flunitrazepam in plasma and urine samples. The new method not only significantly shortens the extraction time but also provides a new opportunity to determine the free concentration of analyte in biological systems.


Subject(s)
Liquid Phase Microextraction/methods , Models, Theoretical , Calibration , Female , Flunitrazepam/blood , Flunitrazepam/urine , Gas Chromatography-Mass Spectrometry/methods , Humans , Kinetics , Limit of Detection , Linear Models , Reproducibility of Results
8.
Talanta ; 99: 649-59, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22967607

ABSTRACT

A simple, high-throughput, sensitive LC-ESI-MS/MS method is presented for the simultaneous determination of methadone (MET), flunitrazepam (FNZ) and their major metabolites, EDDP (2-ethilidene-1,5-dimethyl-3,3-diphenylpyrrolidone) and 7-aminoflunitrazepam (7-AFNZ), respectively, in human, rat and rabbit plasma. The isolation of the selected compounds involved a liquid-liquid extraction with ethyl acetate at a basic pH. Good chromatographic separation was achieved on a HSS T3 column (1.8 µm particle size), with a 3 min gradient elution using a mixture of acetonitrile with 0.1% formic acid (solvent A) and 5mM ammonium acetate (solvent B) as the mobile phase. The tandem mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with ionization of the analytes in positive mode. The assay was fully validated according to current acceptance criteria for bioanalytical methods validation. It was proved to be linear in the range of 0.5-250 ng/mL, with adequate accuracy and precision over this range. Based on accuracy and CV% values the LOQ and ULOQ values were set at 0.509 ng/mL and 2036 ng/mL for MET, 0.520 ng/mL and 2080 ng/mL for EDDP, 0.524 ng/mL and 2096 ng/mL for FNZ and 0.528 ng/mL and 2114 ng/mL for 7-AFNZ, respectively. The method was tested for potential matrix effects, without observing significant ion suppression. The investigated compounds stability was examined in plasma at room temperature and after three freeze-thaw cycles and in the final extract when maintained at 4 °C in the autosampler. Potential stability issues were observed only for FNZ at room temperature. The method was successfully applied to quantify the selected compounds in human, rat and rabbit plasma samples, after exposure to FNZ or simultaneous exposure to FNZ and MET.


Subject(s)
Blood Chemical Analysis/methods , Flunitrazepam/analogs & derivatives , Flunitrazepam/blood , Methadone/blood , Pyrrolidines/blood , Animals , Chromatography, High Pressure Liquid , Female , Flunitrazepam/pharmacokinetics , Flunitrazepam/toxicity , Humans , Male , Methadone/pharmacokinetics , Methadone/toxicity , Pyrrolidines/pharmacokinetics , Pyrrolidines/toxicity , Rabbits , Rats , Reproducibility of Results , Tandem Mass Spectrometry , Time Factors , Toxicity Tests
9.
J Forensic Leg Med ; 19(2): 77-82, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22281215

ABSTRACT

The use of psychoactive substances to improve social relations and increase body energy, in Rave Culture, has raised many legal and health public concerns, both for illicit trade and consumption. Therefore, forensic toxicology plays an important role in this area, mainly linked to the detection and quantitation of these substances, both in vivo and in post-mortem samples. In fact, at the moment, forensic sciences have been under public authorities' scrutiny and critical look, due to the increasing attention of the media and public opinion, always applying for the use of scientific knowledge to help solving forensic cases. However, forensic toxicology results are only reliable to solve legal cases if all the analytical methodologies used are appropriately validated. In this work, a methodology for the extraction and analysis of 7-aminoflunitrazepam, buprenorphine, flunitrazepam, ketamine, methadone, phencyclidine (PCP) and d-propoxyphene was developed for whole blood samples, with solid phase extraction (SPE), using OASIS(®) MCX SPE columns, and gas chromatography coupled to mass spectrometry. The procedure presented here proved to be reliable, specific, selective and sensitive, with good LODs and LOQs and good precision.The adoption of a SPE procedure with an automatic SPE extraction device, allowed an increased level of automation in sample treatment, being contemporarily less time-consuming, increasing productiveness, and allowing good recovery and appropriate selectivity being, also, simple and reproducible. The simultaneous detection and quantitation of all compounds by the same extraction and detection methodology is crucial and has a great potential for forensic toxicology and clinical analysis.


Subject(s)
Illicit Drugs/blood , Narcotics/blood , Buprenorphine/blood , Dextropropoxyphene/blood , Flunitrazepam/analogs & derivatives , Flunitrazepam/blood , Forensic Toxicology , Gas Chromatography-Mass Spectrometry , Humans , Ketamine/blood , Limit of Detection , Methadone/blood , Phencyclidine/blood , Solid Phase Extraction
10.
Am J Forensic Med Pathol ; 33(4): 293-6, 2012 Dec.
Article in English | MEDLINE | ID: mdl-21372658

ABSTRACT

A 52-year-old woman was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.


Subject(s)
Anti-Anxiety Agents/poisoning , Flunitrazepam/analogs & derivatives , Flunitrazepam/poisoning , Triazolam/analogs & derivatives , Triazolam/poisoning , Anti-Anxiety Agents/blood , Anti-Anxiety Agents/urine , Chromatography, High Pressure Liquid , Drug Overdose , Female , Flunitrazepam/blood , Flunitrazepam/urine , Forensic Toxicology , Humans , Mass Spectrometry , Middle Aged , Triazolam/analysis , Triazolam/blood , Triazolam/urine
11.
Forensic Sci Int ; 202(1-3): e9-11, 2010 Oct 10.
Article in English | MEDLINE | ID: mdl-20494535

ABSTRACT

Topiramate belongs to a new group of anticonvulsive drugs primarily applied in treatment of epilepsy and in preventive therapy of migraines. Topiramate is structurally unrelated to other antiepileptic drugs and acts by multiple neurostabilizing mechanisms. However, the pharmacology of topiramate appears to be complex and some of its pharmacodynamic actions still remain to be elucidated. This case report documents a fatal intoxication involving topiramate. A 41-year old woman with a known history of psychiatric disorder was found unresponsive by her husband. Resuscitation efforts did not succeed and the woman was pronounced dead at the intensive care unit four hours later. At the scene, drug packages of topiramate, citalopram and flunitrazepam were found. Autopsy including histological examination revealed morphological signs of an acute intoxication and shock. A comprehensive toxicological analysis with GC-MS was performed on the deceased's autopsy samples (femoral blood, bile, kidney, gastric content). The results revealed the presence of topiramate at a concentration of 49mg/L in the femoral blood sample, thus clearly exceeding the therapeutic range. Additionally, citalopram (0.85mg/L) and flunitrazepam in traces (<2µg/L) were detected in peripheral blood. Based on the autopsy findings and toxicological results, the cause of death was primarily attributed to an intoxication with topiramate in combination with citalopram.


Subject(s)
Anticonvulsants/poisoning , Fructose/analogs & derivatives , Adult , Anti-Anxiety Agents/blood , Anticonvulsants/analysis , Bile/chemistry , Citalopram/blood , Female , Flunitrazepam/blood , Forensic Toxicology , Fructose/analysis , Fructose/poisoning , Gas Chromatography-Mass Spectrometry , Gastrointestinal Contents/chemistry , Humans , Kidney/chemistry , Selective Serotonin Reuptake Inhibitors/blood , Topiramate
12.
Methods Mol Biol ; 603: 107-19, 2010.
Article in English | MEDLINE | ID: mdl-20077063

ABSTRACT

The benzodiazepines are a large, commonly prescribed family of psychoactive drugs. We describe a method permitting the simultaneous detection and quantification of 12 benzodiazepines in serum using ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS). Analytes included alprazolam, temazepam, oxazepam, nordiazepam, clonazepam, lorazepam, diazepam, chlordiazepoxide, midazolam, flunitrazepam, 7-aminoclonazepam, and 7-aminoflunitrazepam. Sample pretreatment is simple consisting of protein precipitation using cold acetonitrile (ACN) mixed with the deuterated internal standards. Samples were capped and vortexed for 5 min to ensure maximum precipitation. Following a 5-min centrifugation period, 400 microL of the supernatant was transferred to a clean tube and evaporated down under nitrogen. Samples were reconstituted in 200 microL of a deionized water:ACN (80:20) mixture and transferred to appropriate vials for analysis. Chromatographic run time was 7.5 min, and the 12 analytes were quantified using multiple reaction monitoring (MRM) and 6-point calibration curves constructed for each analyte at concentrations covering a clinically significant range.


Subject(s)
Benzodiazepines/blood , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Alprazolam/blood , Chlordiazepoxide/blood , Clonazepam/analogs & derivatives , Clonazepam/blood , Flunitrazepam/analogs & derivatives , Flunitrazepam/blood , Humans , Lorazepam/blood , Midazolam/blood , Nordazepam/blood , Oxazepam/blood , Reproducibility of Results
13.
Przegl Lek ; 66(6): 301-10, 2009.
Article in Polish | MEDLINE | ID: mdl-19788134

ABSTRACT

The Main Department of Police in Poland notes about 2000 rapes a year. Some of the crimes are performed with "Date Rape Drugs". The term means substances helping comitting a rape such as GHB (gamma hydroxybutyric acid), ketamine, flunitrazepam and other benzodiazepines derivatives, MDMA ("ecstasy"), marihuana, amphetamine. The substances are often joined with alcohol. The victims are usually young women, and not all the cases are recorded by the police or physicians, because the victims often do not remember details of the event. The toxicological analysis of blood or urine would be helpful to explain the circumstances of the case and to prove using "Date Rape Drug". The samples for toxicological determinations should be collected as soon as possible (24 to 72 hours after admission). Preventing violence with "Date Rape Drugs" include wide education by media, police, teachers and parents. The purpose of the research was to check the level of knowledge about "Date Rape Drugs". The consciousness of risk behavior when the kind of substances is used and the ways of preventing the risk of being a sexual victim were checked. Material for the research were the results of questionnaire prepared by The Department of Medicine Sociology Collegium Medicum Jagiellonian University in Krakow, carried out on 740 students. Most of respondents (77%) were women. The age of respondents was between 19-36 years (mean 21.41; SD - 1.29). The results of the research showed, that respondents didn't have completed knowledge about "Date Rape Drugs". They did not know the ways of recognizing and preventing the risk of being given this kind of substances. The main source of information about "Date Rape Drugs" were internet and colleagues. There is a need to start education about "Date Rape Drugs" by serious institutions such as the police and schools in Poland. This is the best way to prevent young people against a risk of being given "Date Rape Drugs" and being a victim of sexual crimes.


Subject(s)
Health Knowledge, Attitudes, Practice , Rape/prevention & control , Sex Offenses/prevention & control , Substance Abuse Detection/methods , Substance-Related Disorders/diagnosis , Substance-Related Disorders/epidemiology , Adolescent , Adult , Amphetamine-Related Disorders/blood , Amphetamine-Related Disorders/diagnosis , Amphetamine-Related Disorders/urine , Crime Victims , Female , Flunitrazepam/blood , Flunitrazepam/urine , Humans , Hydroxybutyrates/blood , Hydroxybutyrates/urine , Illicit Drugs/blood , Illicit Drugs/urine , Incidence , Ketamine/blood , Ketamine/urine , Male , Marijuana Abuse/diagnosis , Marijuana Abuse/epidemiology , Marijuana Abuse/prevention & control , Poland/epidemiology , Rape/statistics & numerical data , Risk-Taking , Sex Offenses/statistics & numerical data , Students , Young Adult
14.
J Chromatogr A ; 1216(12): 2241-7, 2009 Mar 20.
Article in English | MEDLINE | ID: mdl-19185867

ABSTRACT

A new polyvinylidene difluoride (PVDF) hollow fiber (200 microm wall thickness, 1.2mm internal diameter, 0.2 microm pore size) was compared with two other polypropylene (PP) hollow fibers (200, 300 microm wall thickness, 1.2mm internal diameter, 0.2 microm pore size) in the automated hollow fiber liquid-phase microextraction (HF-LPME) of flunitrazepam (FLNZ) in biological samples. With higher porosity and better solvent compatibility, the PVDF hollow fiber showed advantages with faster extraction efficiency and operational accuracy. Parameters of the CTC autosampler program for HF-LPME in plasma and urine samples were carefully investigated to ensure accuracy and reproducibility. Several parameters influencing the efficiency of HF-LPME of FLNZ in plasma and urine samples were optimized, including type of porous hollow fiber, organic solvent, agitation rate, extraction time, salt concentration, organic modifier, and pH. Under optimal conditions, extraction recoveries of FLNZ in plasma and urine samples were 6.5% and 83.5%, respectively, corresponding to the enrichment factor of 13 in plasma matrix and 167 in urine matrix. Excellent sample clean-up was observed and good linearities (r(2)=0.9979 for plasma sample and 0.9995 for urine sample) were obtained in the range of 0.1-1000 ng/mL (plasma sample) and 0.01-1000 ng/mL (urine sample). The limits of detection (S/N=3) were 0.025 ng/mL in plasma matrix and 0.001 ng/mL in urine matrix by gas chromatography/mass spectrometry/mass spectrometry.


Subject(s)
Automation , Chemical Fractionation/methods , Flunitrazepam/blood , Flunitrazepam/urine , Polyvinyls/chemistry , Gas Chromatography-Mass Spectrometry , Hydrogen-Ion Concentration , Microscopy, Electron, Scanning , Reproducibility of Results , Sensitivity and Specificity , Sodium Chloride/chemistry , Solvents/chemistry , Tandem Mass Spectrometry , Time Factors
15.
J Chromatogr B Analyt Technol Biomed Life Sci ; 877(23): 2275-83, 2009 Aug 01.
Article in English | MEDLINE | ID: mdl-19110476

ABSTRACT

A LC-APPI-MS method was developed and validated for the detection of alprazolam, flunitrazepam and their major metabolites in haemolysed blood. Samples were diluted with water (2:1, v:v) and extracted with a hydrophobic-lipophilic balanced copolymer. The method was fully validated according to ICH guidelines and SFSTP protocols. Deuterated internal standards of both parent drugs were used and good quantitative performance was achieved in terms of trueness and precision (repeatability and intermediate precision) since accuracy profiles were achieved within the acceptance limits (+/-30% for biological samples). The LC-APPI-MS method was linear over the concentration range of 1-1000 and 3-1000 ng/mL, for alprazolam and flunitrazepam, respectively. Lower limits of quantification as low as 1 ng/mL in haemolysed blood were reached and the method was successfully applied to the quantification of alprazolam, flunitrazepam and their major metabolites in real toxicological samples.


Subject(s)
Alprazolam/blood , Anti-Anxiety Agents/blood , Chromatography, Liquid/methods , Flunitrazepam/blood , Spectrometry, Mass, Electrospray Ionization/methods , Humans , Sensitivity and Specificity
16.
Toxicol Sci ; 106(1): 64-73, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18703559

ABSTRACT

High-dosage buprenorphine (BUP) consumed concomitantly with benzodiazepines (BZDs) including flunitrazepam (FZ) may cause life-threatening respiratory depression despite a BUP ceiling effect and BZDs' limited effects on ventilation. However, the mechanism of BUP/FZ interaction remains unknown. We hypothesized that BUP may alter the disposition of FZ active metabolites in vivo, contributing to respiratory toxicity. Plasma FZ, desmethylflunitrazepam (DMFZ), and 7-aminoflunitrazepam (7-AFZ) concentrations were measured using gas chromatography-mass spectrometry. Intravenous BUP 30 mg/kg pretreatment did not alter plasma FZ and 7-AFZ kinetics in Sprague-Dawley rats infused with 40 mg/kg FZ over 30 min, whereas resulting in a three-fold increase in the area under the curve (AUC) of DMFZ concentrations compared with control (p < 0.01). In contrast, BUP did not significantly modify plasma DMFZ concentrations after intravenous infusion of 7 mg/kg DMFZ, whereas resulting in a similar peak concentration to that generated from 40 mg/kg FZ administration. Regarding the effects on ventilation, BUP (30 mg/kg) as well as its combination with FZ (0.3 mg/kg) significantly increased PaCO(2), whereas only BUP/FZ combination decreased PaO(2) (p < 0.001). Interestingly, FZ (40 mg/kg) but not DMFZ (40 mg/kg) significantly increased PaCO(2) (p < 0.05), whereas DMFZ but not FZ decreased PaO(2) (p < 0.05). Thus, decrease in PaO(2) appears related to BUP-mediated effects on DMFZ disposition, although increases in PaCO(2) relate to direct BUP/FZ additive or synergistic dynamic interactions. We conclude that combined high-dosage BUP and FZ is responsible for increased respiratory toxicity in which BUP-mediated alteration in DMFZ disposition may play a significant role.


Subject(s)
Analgesics, Opioid/toxicity , Buprenorphine/toxicity , Flunitrazepam/analogs & derivatives , Flunitrazepam/toxicity , GABA Modulators/toxicity , Pulmonary Ventilation/drug effects , Respiratory Insufficiency/chemically induced , Analgesics, Opioid/administration & dosage , Animals , Biotransformation , Buprenorphine/administration & dosage , Carbon Dioxide/blood , Drug Interactions , Flunitrazepam/administration & dosage , Flunitrazepam/blood , Flunitrazepam/pharmacokinetics , GABA Modulators/administration & dosage , GABA Modulators/blood , GABA Modulators/pharmacokinetics , Hydrogen-Ion Concentration , Infusions, Intravenous , Male , Oxygen/blood , Rats , Rats, Sprague-Dawley , Respiratory Insufficiency/blood , Respiratory Insufficiency/physiopathology
17.
Forensic Sci Int ; 159(2-3): 83-91, 2006 Jun 02.
Article in English | MEDLINE | ID: mdl-16087304

ABSTRACT

Benzodiazepines are sedatives used for anxiolysis, hypnosis, muscle relaxation and the treatment of epilepsy. Paradoxical reactions including agitation, talkativeness, confusion, disinhibition, aggression, violent behavior and loss of impulse control may, however, occur in some subjects. It has been claimed that high doses of flunitrazepam may cause aggression on a more regular basis in all individuals. The present study makes use of a Norwegian forensic toxicological database containing analytical results from drivers suspected of driving under the influence and suspects of violent crime to analyze the relationship between behavior and blood flunitrazepam concentration. Four-hundred and fifteen cases of drivers suspected of driving under the influence and seven cases of suspects of violent crime were studied. These selected cases had flunitrazepam as the only drug in blood samples and had been evaluated by a clinical test for impairment (CTI) performed by a police physician at the time of blood sampling. The impaired drivers had higher blood flunitrazepam concentrations than the not impaired drivers. Multivariate analysis revealed that both blood flunitrazepam concentration and age of the suspected drivers had independent impact on impairment, indicating tolerance with age. Most of the effects measured were sedative effects of flunitrazepam and these effects were related to flunitrazepam level. Possible paradoxical reactions were observed in a subgroup of 23 individuals (6%), but these reactions did not relate to blood flunitrazepam concentration. The suspects of violent crime showed similar degree impairment and had not more paradoxical reactions than the suspected drugged drivers. The findings were in agreement with other research that claims paradoxical reactions should be viewed as a reaction in certain individuals, and does not support the notion that flunitrazepam in high concentration produces aggression in all individuals taking the drug.


Subject(s)
Anti-Anxiety Agents/adverse effects , Automobile Driving/statistics & numerical data , Conduct Disorder/epidemiology , Flunitrazepam/adverse effects , Substance-Related Disorders/epidemiology , Violence/statistics & numerical data , Adult , Aggression , Anti-Anxiety Agents/blood , Conduct Disorder/blood , Conduct Disorder/etiology , Conduct Disorder/psychology , Crime/statistics & numerical data , Databases, Factual , Female , Flunitrazepam/blood , Forensic Psychiatry , Humans , Male , Norway/epidemiology , Substance-Related Disorders/blood , Substance-Related Disorders/etiology , Substance-Related Disorders/psychology
18.
Forensic Sci Int ; 157(2-3): 156-62, 2006 Mar 10.
Article in English | MEDLINE | ID: mdl-15955650

ABSTRACT

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.


Subject(s)
Anti-Anxiety Agents/blood , Anti-Anxiety Agents/urine , Flunitrazepam/analogs & derivatives , Flunitrazepam/blood , Flunitrazepam/urine , Chromatography, High Pressure Liquid/methods , Forensic Medicine/methods , Gas Chromatography-Mass Spectrometry , Humans , Nitrazepam/blood , Nitrazepam/urine
19.
Int J Legal Med ; 120(6): 323-30, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16220317

ABSTRACT

A high-performance liquid chromatography method for the determination of benzodiazepines and their metabolites in whole blood and serum using mass spectrometry (MS) and photodiode array (PDA) detection is presented. The combination of both detection types can complement each other and provides extensive case relevant data. The limits of quantification (LOQ) with the MS detection lie between 2 and 3 microg/l for the following benzodiazepines or metabolites: 7-amino-flunitrazepam, alprazolam, desalkyl-flurazepam, desmethyl-flunitrazepam, diazepam, flunitrazepam, flurazepam, alpha-hydroxy-midazolam, lorazepam, midazolam, nitrazepam, nordazepam and oxazepam, respectively 5 microg/l for lormetazepam and 6 microg/l for bromazepam. The LOQ of clobazam determined with the PDA detector is 10 microg/l. A convenient approach for determining the measurement uncertainty of the presented method--applicable also for other methods in an accreditation process--is presented. At low concentrations (<10 microg/l), measurement uncertainty was estimated to be about 50%, and at concentrations >180 microg/l, it was estimated to be about 15%. One hundred and twenty-eight case data acquired over 1 year are summarised.


Subject(s)
Benzodiazepines/blood , Serum/chemistry , Anti-Anxiety Agents/blood , Anti-Anxiety Agents/isolation & purification , Benzodiazepines/isolation & purification , Chromatography, High Pressure Liquid , Flunitrazepam/analogs & derivatives , Flunitrazepam/blood , Flunitrazepam/isolation & purification , Flurazepam/analogs & derivatives , Flurazepam/blood , Flurazepam/isolation & purification , Forensic Toxicology , Humans , Mass Spectrometry , Midazolam/analogs & derivatives , Midazolam/blood , Midazolam/isolation & purification , Molecular Structure
20.
Hum Psychopharmacol ; 20(8): 583-9, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16317801

ABSTRACT

1611 patients were included in this investigation. 16.7% of the patients were involved in traffic accidents, 38.2% were injured by a sudden fall, 3.5% were involved in an act of violence, 22.8% were injured by a sports related accident and 18.9% were hurt within a work-related accident. 19.5% of the patients tested positive for alcohol, 5.2% tested positive for benzodiazepines and 1.4% tested positive for both substances. Blood samples were positive for alcohol in 27% males and 7.7% females and for benzodiazepines in 6.3% males and in 3.5% females. The mean blood alcohol concentration (BAC) as well as the mean benzodiazepine plasma level were higher in patients injured in violent accidents compared to the other injury groups. This study provides epidemiologic information about the relationship between specific kinds of accidents and alcohol and/or benzodiazepine use in a large probability sample of emergency room patients. We found a high number of patients using alcohol, and a lower but still relavant number of benzodiazepine users in this large and unselected traumatology ER sample. This study adds evidence to the existing literature about the co-occurance of alcohol and/or benzodiazepine consumption and accident-related injuries.


Subject(s)
Accidents/statistics & numerical data , Diazepam/administration & dosage , Ethanol/administration & dosage , Flunitrazepam/administration & dosage , Accidents/classification , Adult , Age Factors , Aged , Alcohol Drinking/blood , Anti-Anxiety Agents/administration & dosage , Anti-Anxiety Agents/blood , Central Nervous System Depressants/administration & dosage , Central Nervous System Depressants/blood , Diazepam/blood , Emergency Service, Hospital/statistics & numerical data , Ethanol/blood , Female , Flunitrazepam/blood , Humans , Injury Severity Score , Male , Middle Aged , Sex Factors , Time Factors , Trauma Centers/statistics & numerical data
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