All solid-state miniaturized potentiometric sensors for flunitrazepam determination in beverages.

Flunitrazepam is one of the frequently used hypnotic drugs to incapacitate victims for sexual assault. Appropriate diagnostic tools should be available to victims regarding the growing concern about "date-rape drugs" and their adverse impact on society. Miniaturized screen-printed potentiometric sensors offer crucial point-of-care devices that alleviate this serious problem. In this study, all solid-state screen-printed potentiometric flunitrazepam sensors have been designed. The paper device was printed with silver and carbon ink. Formation of an aqueous layer in the interface between carbon-conducting material and ion-sensing membrane nevertheless poses low reproducibility in the solid-contact electrodes. Accordingly, poly(3,4-ethylenedioxythiophene) (PEDT) nano-dispersion was applied as a conducting hydrophobic polymer on the electrode surface to curb water accumulation. Conditioning of ion-sensing membrane in the vicinity of reference membrane has been considered carefully using special protocol. Electrochemical characteristics of the proposed PEDT-based sensor were calculated and compared favorably to PEDT-free one. The miniaturized device was successfully used for the determination of flunitrazepam in carbonated soft drinks, energy drink, and malt beverage. Statistical comparison between the proposed sensor and official method revealed no significant difference. Nevertheless, the proposed sensor provides simple and user-friendly diagnostic tool with less equipment for on-site determination of flunitrazepam.

Liquid chromatography-electron ionization tandem mass spectrometry with the Direct-EI interface in the fast determination of diazepam and flunitrazepam in alcoholic beverages.

This is the first application based on electron ionization (EI) using a Direct-EI LC interface and MS/MS to detect unequivocally target compounds in a very small real sample. The determination and quantification of benzodiazepines in very small residues of beverages, collected at the scene of drug-facilitated crimes are mandatory in legal procedures. A specific and sensitive analytical instrumentation is needed, involving little or no sample preparation. Here, a direct flow injection analysis of alcoholic beverages spiked with commercially available drugs containing diazepam and flunitrazepam is presented. The method proposed is very fast and requires neither sample preparation nor chromatographic separation. Linearity (R(2) ) was between 0.9977 and 0.9992; LOD and LOQ spanned from 0.01 to 0.02 ng/µL and from 0.1 to 0.5 ng/µL, respectively; intra- and interday repeatabilities were between 1 and 8%. No matrix effects were observed from the comparison of the linear regression curves obtained in real fortified samples and in pure ethanol. Vodka, whisky, and white wine specimens were fortified with commercial drugs, Valium(®) and Rohypnol(®) , at two different concentrations (20 and 50 ng/µL) to simulate the typical amounts found in adulterated real samples and analyzed to demonstrate the method applicability to forensic analyses.

Identification and quantification of 35 psychotropic drugs and metabolites in hair by LC-MS/MS: application in forensic toxicology.

Despite a non-invasive sampling, hair samples are generally collected in limited amounts for an obvious esthetic reason. In order to reduce the required quantity of samples, a multianalytes method allowing simultaneous identification and quantification of 35 psychoactive drugs was developed. After incubation of 50 mg of hair in a phosphate buffer pH 5 for one night at room temperature, the substances of interest were extracted by a simple liquid-liquid extraction step, with a dichloromethane/ether mixture (70:30, v/v). After evaporation under a gentle stream of nitrogen and reconstitution in formate buffer (2 mM, pH 3)/acetonitrile (90:10, v/v), twenty microliter were injected into the LC-MS/MS system for a chromatographic run of 29 min using an Atlantis T3 column (150 × 2.1 mm, 3 µm) (Waters Corp, Milford, USA) and a gradient mixture of 2 mM, pH 3.0 ammonium formate, and 2 mM, pH 3.0 ammonium formate/acetonitrile. The data acquisition was performed in scheduled MRM mode. Intra- and inter-day precisions, estimated using the coefficient of variation and relative bias, were lower than 20 % for all concentration levels, except for two compounds. The limits of detection and quantification ranged from 0.5 to 10 pg/mg. After complete validation, this method has been successfully used in several forensic cases, three of which are reported.

Production of monoclonal antibody against clonazepam for immunoassay of benzodiazepine drugs in swine tissues.

The objective of the present study was to produce a generic monoclonal antibody for immunoassay of residues of benzodiazepine drugs in swine tissues. Clonazepam was used to synthesize a hapten that was coupled to bovine serum albumin as an immunogen for the production of monoclonal antibody. Results showed that the obtained monoclonal antibody was able to recognize five benzodiazepine drugs simultaneously (clonazepam, flunitrazepam nitrazepam, diazepam, and oxazepam). The cross-reactivities were in the range of 24-100% and the limits of detection were in the range of 0.2-1.5 ng mL(-1) depending on the drug. Then a competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of five benzodiazepines in swine tissues (muscle, liver and kidney). The recoveries of five analytes from the fortified blank samples were in the range of 74.5-96.5% with coefficients of variation lower than 16.7%. Therefore, this immunoassay could be used as a rapid and simple method for the screening of residues of five benzodiazepine drugs in animal-derived foods.

Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices.

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.

Forensic electrochemistry: the electroanalytical sensing of Rohypnol® (flunitrazepam) using screen-printed graphite electrodes without recourse for electrode or sample pre-treatment.

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'.

A new screening method for flunitrazepam in vodka and tequila by fluorescence spectroscopy.

A new screening method for flunitrazepam in colourless alcoholic beverages based on a spectroscopic technique is proposed. Absorption and steady-state fluorescence of flunitrazepam and its protonated form with various acids were investigated. The redshift of the wavelength of maximum absorption was distinctively observed in protonated flunitrazepam. An emissive fluorescence at 472 nm was detected in colourless spirits (vodka and tequila) at room temperature. 2-M perchloric acid was the most appropriated proton source. By using electron ionization mass spectrometry and time-dependent density functional theory calculations, the possible structure of protonated flunitrazepam was identified to be 2-nitro-N-methylacridone, an acridone derivative as opposed to 2-methylamino-5-nitro-2'-fluorobenzophenone, a benzophenone derivative.

Rapid determination of flunitrazepam in alcoholic beverages by desorption electrospray ionization-mass spectrometry.

Desorption electrospray ionization-mass spectrometry (DESI-MS), a novel ambient ionization technique, was used in this study for determining flunitrazepam in various alcoholic beverages. Using this technique, no pretreatment of the samples was necessary and identification of the drug was accomplished in individual samples in minutes. In addition, the acquired mass spectra provide the information of the identity of the drink based on the detected characteristic ions from the matrices. This study also demonstrates the capability of DESI-MS to perform quantitative analysis of simulated evidence samples with a limit of quantification of 3µg/mL. Furthermore it has been shown that this method can be used for high-throughput analysis whereby six samples were analyzed in a row within 6 minutes and no observable sample carry-over was noted. DESI-MS shows potential as a rapid, sensitive, and selective technique for forensic analysis of spiked beverages which are typical evidence of drug facilitated sexual assault and robbery cases.

A case of drowning whilst under the influence of brotizolam, flunitrazepam and ethanol.

A case of drowning involving brotizolam, flunitrazepam and ethanol ingestion was presented. Quantitative toxicological analysis showed that the concentrations of brotizolam, 7-aminoflunitrazepam (a metabolite of flunitrazepam) and ethanol in the femoral blood were 0.025 microg/ml, 0.094 microg/ml and 0.29 mg/ml, respectively, and these drugs were also detected in the stomach contents. We concluded that the cause of death was drowning whilst under the influence of combined use of brotizolam, flunitrazepam and ethanol.

[Analysis of the binding capacity of the benzodiazepine site of gabaa receptor in mice C57BL/6 and BALB/C pretreated with anxiolytics].

The level of specific 3H-flunitrazepam binding in synaptosomal membranes of C57BL/6 and BALB/c mice brain underwent to the stress of different types has been studied. Mild stress (Elevated Plus Maze) was shown to induce the decrease of benzodiazepine binding in BALB/c mice only, while the strong one (Exposure to a predator) was revealed to cause this decrease in both strains. Behavioral effects of different non-benzodiazepine drugs possessing anxiolytic properties (Afobazol, Ladasten and Noopept) was accompanied with the normalization of the level of benzodiazepine reception, reduced by the stress of both modalities.

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.

Chemical submission: results of 4-year French inquiry.

Psychoactive substances may be administered without the knowledge of a victim in order to induce incapacitation and thus facilitate criminal actions. The characteristics of the victims and the drugs used in such suspected chemical submissions (CS) were analyzed in 309 cases collected from October 2003 to December 2007 through a national survey. Out of 309 cases, 158 met all criteria of CS. The victims were mostly female (n = 89, 56%). The type of aggression was mostly sexual assault (in 79 cases 50%). Benzodiazepines and related drugs were detected in 129 victims (82%) and were mostly clonazepam, zolpidem, and bromazepam whereas flunitrazepam and gamma hydroxybutyrate, well known for their use in CS, were identified in 11 (7%) and five (3%) of the 158 victims. CS is not an anecdotal phenomenon in France. Information for health professionals and workers in forensic structures as well as education of the general population associated with preventive measures such as drug dosage form changes should contribute to improved care management of victims and decreased risk.

Withdrawn: Hair Analysis for Drug-Facilitated Crime: The Critical Role of Hair Growth Rate.

Hair analysis is increasingly used in detecting drug-facilitated crime (DFC) claiming success in identifying even single dose exposures. The calculation of accurate deposition time of the drug in hair is typically based on the assumption of mean hair growth of 1 cm/month. We describe a case of potential exposure to flunitrazepam. Assuming the literature average hair growth rate of 1 cm/month, the alleged victim had measurable amounts of the 7 amino flunitrazepam a month after the alleged drug exposure. However, in this case, due to hair dying, the true growth rate could be quantified at 1.5 cm/month. This difference has led to different interpretation from the one based on the average assumed hair growth of 1 cm/month. In conclusion, hair growth rate can be a critical variable in verifying the alleged time of drug exposure.

Lab-on-a-screen-printed electrochemical cell for drop-volume voltammetric screening of flunitrazepam in untreated, undiluted alcoholic and soft drinks.

Flunitrazepam, also known as "Rohypnol" or "Rophy" among other trade and street names, is an extremely potent benzodiazepine that is prescribed to treat severe insomnia. Due to these attributes, flunitrazepam, when is surreptitiously administered to an alcoholic or soft drink, is associated with "drug-facilitated sexual assault". We report here for the first time, a low cost lab-on-a-screen-printed electrochemical cell (SPC) based on iron-sparked graphite working electrode modified with glucose oxidase (GOx) and glucose hydrogel droplets (GluHD) for the detection of flunitrazepam. Iron-spark modification increases the response of the sensor by ca. 3-fold compared with that of the plain electrode, while an in situ deoxygenation process, based on GOx-glucose enzyme reaction, depletes dissolved oxygen. As a result, the method enables interference free voltammetric measurements of the electro reduction of the nitro group of flunitrazepam at ca. -0.71 to -0.78 V vs. Ag printed pseudo reference electrode depending on the sample's matrix, and the detection of the drug at the sub-millimolar level. GOx/GluHD-FeSPC was directly applied to the drop-volume (∼60 µL) detection of flunitrazepam to a wide range of untreated and undiluted spiked samples (Pepsi cola®, Vodka, Whisky, Tequila, Gin, and Rum) of different acidity (pH 2.3-8.4), and alcohol content up to 40% v/v. Data demonstrate the excellent performance of the sensor for point-of-need screening of flunitrazepam and suggest that GOx/GluHD-FeSPC holds promise as an effective analytical tool to prevent phenomena of covert drug administration.

Facile synthesis of copper(II) oxide nanospheres covered on functionalized multiwalled carbon nanotubes modified electrode as rapid electrochemical sensing platform for super-sensitive detection of antibiotic.

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.

Forensic toxicological implication of an autopsy case of mixed drug overdose involving clomipramine, chlorpromazine and flunitrazepam.

A case of fatal poisoning involving clomipramine, chlorpromazine and flunitrazepam is presented. Quantitative toxicological analysis showed that the concentrations of clomipramine, chlorpromazine and 7-aminoflunitrazepam (a metabolite of flunitrazepam) in the femoral blood were 3.24 microg/ml, 0.36 Kg/ml and 0.61 microg/ml, respectively, and large amounts of drugs were also detected from the stomach contents. We concluded that the cause of death was due to the combined use of clomipramine, chlorpromazine and flunitrazepam.

Field-test of a date-rape drug detection device.

Drink Safe Technology Version 1.2 is an inexpensive color-change reagent test marketed internationally for use by consumers in settings such as a night club to detect potentially incapacitating concentrations of gamma-hydroxybutyric acid (GHB) and ketamine in beverages. The objective of this study was to compare product performance in the laboratory and performance in the hands of consumers in the field. Product performance in the laboratory adhered to the protocol defined by the manufacturer. Product performance in the hands of consumers in field settings allowed browsing participants to pipette an aliquot of their own drinks into randomly coded vials containing authentic drugs, or pure water, so as to yield the same concentrations of GHB or ketamine specified in the manufacturer-defined protocol, or blanks. Consumers were to proceed according to the directions printed on the product, and to record their results on a card with a code corresponding with the vial to which they had added an aliquot of their beverage. Diagnostic performance was calculated using two-way analysis. In the laboratory, Drink Safe Technology Version 1.2 reliably detected GHB and ketamine at concentrations specified by the manufacturer's protocol. The reactive color change denoting a positive test for GHB was rapid, but a positive test for ketamine required substantially more time to resolve. Nonetheless, test accuracy following the manufacturer's protocol in the laboratory was 100%. In the field, based on 101 paired-test results recorded by consumers, the test efficiency was 65.1%, sensitivity 50%, and specificity 91.6%. The product performed much better in the laboratory than it did in the hand of consumers in the field. There seems to be considerable potential for consumers to misinterpret a test result. The potential for consumers to record a false-negative test result for a spiked drink is cause for concern.

An estimate of the proportion of drug-facilitation of sexual assault in four U.S. localities.

In recent years, drugs including flunitrazepam, gamma-hydroxybutyrate, ketamine, and ethanol, have become popularly associated with drug-facilitated sexual assault. Other drugs are also candidates as factors in "drug facilitated sexual assault" (DFSA). The true extent of DFSA is not known, and is difficult to estimate. We recruited sexual assault complainants at four clinics in different parts of the U.S. to anonymously provide urine and hair specimens, and to answer questions about suspected drugging, drug use, and the sexual assault incident. Urine and hair specimens were tested for 45 drugs, including ethanol, and those pharmacologically capable of inducing sedation, amnesia, or impairment of judgment. Analytical test results were used to estimate the proportion of subjects, and the proportion of all complainants to the clinic in the same time period, who were victims of DFSA. Overall, cases of 43% of 144 subjects, and 7% of 859 complainants, were characterized as DFSA. Subjects underreported their use of drugs. The role of toxicological results and history in characterizing DFSA cases is discussed.

The detection of flunitrazepam in beverages using portable Raman spectroscopy.

Portable Raman spectroscopy has been used for the detection of the date-rape drug flunitrazepam in spiked beverages that may be involved in cases of drug-facilitated sexual assault. Solutions of flunitrazepam with different concentrations were prepared in water and for each beverage type. Although some bands attributable to the beverage matrix are present, they did not interfere with the identification of the drug. Definitive evidence for contamination of the spiked drink concerned can be acquired within 10 s. The data can be acquired in situ and sample extraction and/or preparation steps are unnecessary. The ability of portable Raman spectrometers to interrogate spiked alcoholic beverages with flunitrazepam has been demonstrated. Copyright © 2016 John Wiley & Sons, Ltd.

Sweeping technique combined with micellar electrokinetic chromatography for the simultaneous determination of flunitrazepam and its major metabolites.

A sweeping technique, in conjunction with micellar electrokinetic chromatography, for the simultaneous determination of flunitrazepam and its major metabolites, 7-aminoflunitrazepam and N-desmethylflunitrazepam, is described. The optimized conditions for the sweeping and separation were a pH 9.5 buffer, 25mM borate, 50mM cetyltrimethylammonium bromide, 30% MeOH (v/v), and a 151-mm injection length. The calibration functions were all linear with the coefficient of determination (r(2)) exceeding 0.996 for the three target compounds. Using the sweeping procedure, the limits of detection were determined to be 13.4, 5.6, and 12.0ng/mL for flunitrazepam, 7-aminoflunitrazepam, and N-desmethylflunitrazepam, respectively, and the sensitivity enhancement for each compound was within the range of 110-200 fold. The RSDs for the retention time and the peak area were less than 4.10%. The optimized sweeping method was also used to examine a spiked urine sample. We conclude that sweeping with micellar electrokinetic chromatography has considerable potential use in clinical and forensic analyses of flunitrazepam and its metabolites.