TY - JOUR
AU - Kintz, Pascal
AB - To the Editor: Benzodiazepines are a class of drugs widely prescribed for the treatment of anxiety, insomnia, epilepsy, muscle spasms or alcohol withdrawal. Reactive to GABA type A receptors, benzodiazepines have a rapid and weakly toxic symptomatic efficacy. This class of drugs is often diverted from the medical use both by opiates addicts or recreative abusers. Indeed, the extensive research around their chemical structures resulted in a wide variety of active compounds that never got a marketing authorization and which are now picked up by online e-merchants of new psychoactive substances and sold under the name “designer benzodiazepines”. The first example of illegal use of non-prescribed benzodiazepine was phenazepam, developed in the Soviet Union in the 1970s. Since the end of year 2010, designer benzodiazepines were proposed as recreational drugs. These drugs were etizolam, metizolam, pyrazolam, flubromazepam, flubromazolam, clonazolam, meclonazepam and nifoxipam. These compounds are sold as tablets, powder or blotters, at very affordable prices. The deleterious effects of designer benzodiazepines depend on the dose consumed. The most common side effects at low doses are drowsiness, fatigue and lethargy. At higher doses, motor coordination disorders, dizziness, mood swings and euphoria may occur. The slow elimination of some benzodiazepines causes their accumulation in lipid-based tissues, which can lead to a delayed overdose in case of repeated consumption. After a long period of consumption, the abuser can develop tolerance and dependence. Sudden drug discontinuation can cause a withdrawal symptom. Severe effects occur when designer benzodiazepines are consumed in combination with opioids. Both pharmacological classes induce a synergistic effect and a respiratory depression (1). Abuse is widespread among poly-consumers, because designer benzodiazepines balance the euphoria due to opioids. Some benzodiazepines have a marked amnesic effect and the consumer loses memory for a few hours after administration (2). The detection of designer benzodiazepines in biological fluids is challenging owing to the very few available data. Some of these compounds have been detected using immunoassays such as ELISA with high cross-reactivity, demonstrating that designer benzodiazepines can be detected during standard blood or urine drug screening. A few authors have developed methods to study the elimination rate and the metabolism of specific drugs, including etizolam (3, 4), metizolam (5–7), meclonazepam (4, 8), diclazepam (4, 9, 10), flubromazolam (11, 12), pyrazolam (11), flubromazepam (13), adinazolam, cloniprazepma, fonazepam, 3-hydroxyphenazepam and nitrazolam (7). Currently, there is no citation and no data for flunitrazolam testing in blood, urine and alternative biological specimens, such as oral fluid (PubMed consultation on 8 November 2017). Flunitrazolam was discovered in the 1960s but has never been marketed for unknown reasons. Its chemical structure is close to flunitrazepam, although flunitrazolam has a triazolo cycle. Scientists have very little information about doses, effects and tolerability. In order to be able to interpret data on flunitrazolam and to obtain information on its detectability in oral fluid, one of the authors (male, 56 years old, 85 kg) ingested one pink tablet bought on the internet and declared to contain 0.25 mg of active material. Oral fluid was collected over 8 h using the NeoSalTM (Neogen) device, used as recommended by the manufacturer by handing the pad in the mouth for 2 min. Immediately thereafter, the pad was placed into the buffer, present in a plastic tube. No blood sample was collected, as this requires specific authorizations due to the invasive nature of this specimen. All oral fluid samples were stored at +4°C until analysis. For identification and characterization of flunitrazolam, nuclear magnetic resonance (NMR) spectroscopy was applied (5). No significant impurity was detected in the tablet. In addition, the dosage of one tablet, using the ERETIC (Electronic Reference To access In vivo Concentrations) method, revealed a dosage of 0.23 mg of flunitrazolam, in agreement with the announced 0.25 mg. All oral fluid samples were submitted to UPLC–MS-MS analysis on an Acquity class I ultra-high performance liquid chromatography coupled to a Xevo TQD tandem mass spectrometer (UPLC–MS-MS) from Waters (Milford, MA, USA), using a screening method devoted to the identification and quantification of designer benzodiazepines. Flunitrazolam was extracted from 500 μL of mixed oral fluid and buffer in presence of 1 ng of diazepam-d5 used as internal standard, 500 μL saturated borate buffer pH 9.5 and 2.5 mL of a mixture of ether/dichloromethane/hexane/isoamyl alcohol (50:30:20:0.5). After 15 min of agitation at room temperature, centrifugation (3,000 rpm for 15 min) and evaporation to dryness at 45°C, the residue was reconstituted in 50 μL of 5 mM ammonium formate buffer adjusted at pH 3. Chromatography was achieved using a Waters Acquity HSS C18 column (150 × 2.1 mm × 1.8 μm) maintained at 50°C in a thermostatically controlled oven. A gradient elution was performed using formate buffer adjusted to pH 3 (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The flow rate was 0.4 mL/min. The initial gradient was 87% phase A and the final gradient, at 15 min, was 5% phase A. An injection volume of 10 μL was used in all cases. A Xevo TQD triple quadrupole mass spectrometer was used for the detection of the molecule. Ionization was achieved using electrospray in the positive ionization mode (ES+). The following conditions were found to be optimal for the analysis of flunitrazolam and the internal standard: capillary voltage at 1.5 kV; source block temperature at 149°C; desolvation gas nitrogen heated at 600°C and delivered at a flow rate of 1,000 L/h. In order to establish appropriate multiple reaction monitoring condition, the cone voltage was adjusted to maximize the intensity of the protonated molecular ion and collision induced dissociated of both species was performed. Cone voltage and collision energy were adjusted to optimize the signal for the two most abundant product ions of flunitrazolam: m/z 337.9 > 263.9 (60 V and 34 eV) and 337.9 > 102.8 (60 V and 40 eV), and the most abundant product ion of internal standard m/z 291.9 > 197.8 (56 V and 32 eV). MassLynx 4.1 software was used for quantification. Because of the nature of the study (detectability of flunitrazolam in oral fluid), a simple analytical validation was achieved. Linearity was observed for flunitrazolam concentrations ranging from 10 to 1,000 pg/mL, with a correlation coefficient of 0.999 (y = 0.0003x − 0.0006). QC samples (100 and 200 pg/mL), analyzed in duplicate in five independent experimental assays, were used for determination a coefficient of variation for precision and accuracy. These CVs were lower than 20%. The limit of detection and the lower limit of quantification were 5 and 10 pg/mL, respectively. Under the used chromatographic conditions, there was no interference with the analyses by chemicals or any extractable endogenous materials present in oral fluid. The matrix effect (<20%) was investigated with spiked flunitrazolam at 200 pg/mL in 15 blank mixed oral fluid and buffer samples. It is generally accepted that chemical testing of oral fluid is an objective mean of diagnosis of drug use and can demonstrate a recent exposure. Flunitrazolam was detectable in oral fluid between 1 and 8 h, with estimated concentrations ranging between 7 and 178 pg/mL. The excretion pattern of flunitrazolam is presented in Figure 1. Tmax was obtained at 3 h. A typical oral fluid chromatogram is presented in Figure 2. As it was described for other benzodiazepines (14), the excretion of flunitrazolam in oral fluid is very weak. None of the oral fluid concentration was >200 pg/mL. This should prevent its detection during routine analyses. Furthermore, contamination of the oral cavity during the collection of the specimens cannot be ruled out, and effects of collection methods on drug concentrations in oral fluid are not well described in the scientific literature. It was not possible to evaluate the amount of collected oral fluid, even by weighing the test tube before and after the centrifugation, as the volume of the buffer varies from device to device. Figure 1. View largeDownload slide Concentration time profile of flunitrazolam excretion in saliva after administration of a single 0.25 mg tablet to a male volunteer. Figure 1. View largeDownload slide Concentration time profile of flunitrazolam excretion in saliva after administration of a single 0.25 mg tablet to a male volunteer. Figure 2. View largeDownload slide Chromatogram obtained after extraction of saliva at H+90 min. Flunitrazolam concentration was 94 pg/mL. From top to bottom: two transitions for flunitrazolam (m/z: 337.9 > 102.8 and 337.9 > 263.9) and one transition for diazepam-d5 (m/z: 291.9 > 197.8). Figure 2. View largeDownload slide Chromatogram obtained after extraction of saliva at H+90 min. Flunitrazolam concentration was 94 pg/mL. From top to bottom: two transitions for flunitrazolam (m/z: 337.9 > 102.8 and 337.9 > 263.9) and one transition for diazepam-d5 (m/z: 291.9 > 197.8). The designer benzodiazepines are a growing class of NPS. With the introduction of new regulations concerning the first designer benzodiazepines (phenazepam and etizolam), users are turning to less known molecules, for which very little data have been published, including their effects, their toxicities and the analytical methods necessary to detect them (15). It appears that the use of alternative matrices, such as oral fluid, is steadily gaining recognition in forensic, occupational and traffic medicine. Although limited to a single administration to one volunteer, this article presents for the first time the detection of flunitrazolam in a human biological specimen. It can be used as a basis to develop a procedure in blood or urine. There is still no case of flunitrazolam poisoning described in the literature and the risk of missing it during conventional screening is large, irrespective of the analytical technique used. 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TI - Characterization of Flunitrazolam, a New Designer Benzodiazepine, in Oral Fluid After a Controlled Single Administration
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bky012
DA - 2018-02-17
UR - https://www.deepdyve.com/lp/oxford-university-press/characterization-of-flunitrazolam-a-new-designer-benzodiazepine-in-a5eiG9HE1u
SP - 1
EP - e60
VL - Advance Article
IS - 6
DP - DeepDyve
ER -