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Fonazepam (desmethylflunitrazepam) and nifoxipam (3-hydroxy-desmethylflunitrazepam) are benzodiazepine derivatives and active metabolites of flunitrazepam. They recently invaded the drug arena as substances of abuse and alerted the forensic community after being seized in powder and tablet forms in Europe between 2014 and 2016. A review of all the existing knowledge of fonazepam and nifoxipam is reported, concerning their chemistry, synthesis, pharmacology and toxicology, prevalence/use, biotransformation and their analysis in biological samples. To our knowledge, fonazepam and nifoxipam-related intoxications, lethal or not, have not been reported in the scientific literature. All the available information was gathered through a detailed search of PubMed and the World Wide Web.

Flunitrazepam (FNTZ) is prescribed to treat insomnia due to its sedative effects. These effects have been abused in cases of drug-facilitated sexual assault (DFSA), where the drug is slipped into a drink, typically containing the compounds ethanol and caffeine. Both of these compounds have been shown to interact with CYP3A4, a liver enzyme responsible for the metabolism of FNTZ. Steady-state kinetics of CYP3A4 conversion of FNTZ were measured to evaluate what effects caffeine and/or ethanol have on enzyme activity. The rate of formation for the two metabolites, 3-hydroxyflunitrazepam (3-OH FNTZ) and desmethylflunitrazepam (DM FNTZ), was shown to decrease in the presence of caffeine or ethanol. The conversion rate of 3-OH FNTZ had a reduction in kcat of about 77% when incubated with 14 mM caffeine. DM FNTZ had similar but less drastic effects, with a kcat reduction of about 19% with 14 mM caffeine. After a replot, the Kis of 3-OH were determined to be 9.1 mM and 224.1 mM for caffeine and ethanol, respectively. Alprazolam (ALP) and diazepam (DZ), two other drugs in the family that includes FNTZ were also analyzed. ALP to 4-hydroxy alprazolam (4-OH ALP) conversion was found to be reduced by 40%, and DZ to nordiazepam conversion was found to be reduced by 20% in the presence of 200 μM and 20mM ethanol. When taken together, the combination of the inhibitory potential of these compounds can begin to provide an explanation of the effects in cases of DFSA.

Flunitrazepam, a hypnotic benzodiazepine, is widely prescribed around the world for the treatment of insomnia and as a preanesthetic. In vitro studies have shown that the metabolism of flunitrazepam to desmethylflunitrazepam and 3-hydroxyflunitrazepam is mediated in part by the polymorphic enzyme CYP2C19. The objective was to examine the role of CYP2C19 activity in determining flunitrazepam kinetics in vivo. Sixteen healthy volunteers (14 genotypic extensive metabolizers and 2 poor metabolizers) were recruited who had a wide range of CYP2C19 activity (0.50-28.8), as determined by the omeprazole/ 5-hydroxyomeprazole ratio (OMR) at 3 hours following administration of omeprazole, 20 mg orally. Each subject received flunitrazepam, 1 mg orally. Blood samples were collected immediately before and up to 48 hours after drug administration and were assayed by HPLC for flunitrazepam and its metabolites, 7-aminoflunitrazepam, desmethylflunitrazepam, and 3-hydroxyflunitrazepam. Spearman correlations were determined for OMR and pharmacokinetic parameters. With increasing OMR (decreasing CYP2C19 activity), the ratio of flunitrazepam to both desmethylflunitrazepam and 3-hydroxyflunitrazepam AUCs increased ( r = 0.55, p = 0.03 and r = 0.65, p = 0.01, respectively). However, variation in CYP2C19 activity did not significantly affect the AUCs of flunitrazepam or its metabolites. The authors conclude that CYP2C19 contributes to the metabolism of flunitrazepam to desmethylflunitrazepam and 3-hydroxyflunitrazepam in vivo, but these data suggest that its role is minor and that differences in CYP2C19 activity do not likely substantially influence its clinical effects.

A gas chromatographic-mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, alpha-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and alpha-hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia beta-glucuronidase for 1 h at 56 degrees C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20x4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25x4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125x3 mm I.D. with 5 microm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile-20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.

GC-MS screening conditions were developed for 15 low-dosed benzodiazepines, covering alprazolam, flunitrazepam, flurazepam, ketazolam, lorazepam and triazolam, and the corresponding metabolites alpha-hydroxyalprazolam, 4-hydroxyalprazolam; 7-aminoflunitrazepam, desmethylflunitrazepam, 7-aminodesmethylflunitrazepam; hydroxyethylflurazepam, N-desalkylflurazepam; oxazepam and alpha-hydroxytriazolam, respectively. Benzodiazepines are analyzed on a polydimethylsiloxane column in both the scan and the multiple ion monitoring modes using on-column injection to attain maximal sensitivity. The reactive compounds are acetylated with pyridine and acetic anhydride for 20 min. The derivatives are stable for at least 4 days. The relative standard deviation observed with standard compounds at the low nanogram-level ranged from 1.13 to 4.87% within-day and from 1.12 to 4.94% between-day. Unequivocal identification potential, high chromatographic resolution and sensitivity are combined with minimal thermal degradation. The presented screening conditions provide the basis for a unique routine screening method for low-dosed benzodiazepines with a broad polarity range.

The aims were to examine the kinetics of the oxidative metabolism of flunitrazepam in vitro when flunitrazepam was dissolved in dimethylformamide and acetonitrile, and to determine which cytochrome P450 isoform(s) are involved. The kinetics of the formations of 3'-hydroxyflunitrazepam and desmethyl-flunitrazepam were non-linear and best estimated using the Hill equation. Inhibition of their formation was studied using specific chemical inhibitors, expressed enzyme systems and specific antibodies. Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 165-338 and 179 391 microM, 22-81 and 3-10 nmol x mg protein(-1) x h(-1), 6-17 and 0.9-1.9 microl x mg protein(-1) x h(-1), and 2.3-3.6 and 1.6-2.6 respectively when dimethylformamide was the organic solvent. When acetonitrile was the solvent, Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 173-231 and 74-597 microM, 35-198 and 2.7-48 nmol.mg protein(-1) x h(-1), 1347 and 0.7-6.3 microl.mg protein(-1) x h(-1), and 1.5-3.6 and 1.1-2.7 respectively. CYP2C19, CYP3A4 and CYP1A2 mediated the formation of both 3'-hydroxyflunitrazepam and desmethylflunitrazepam. Investigators need carefully to consider the choice of organic solvent to avoid false CYP identification.

A high-performance liquid chromatographic method for the simultaneous determination of flunitrazepam and four metabolites, desmethylflunitrazepam (DMF), 7-aminodesmethylflunitrazepam (7-NH2DMF), 7-aminoflunitrazepam (7-NH2F) and 3-hydroxyflunitrazepam (3-OHF), in serum is described. The method involves a simple extraction from alkalinized plasma (pH 9.5) into diethyl ether-chloroform (80:20, v/v). Prazepam was used as an internal standard for the quantification of the five compounds. Separation was achieved with a 10 microns RSil CN column (300 x 3.9 mn I.D.). The detection wavelength was set at 242 nm. The limits of detection ranged from 2.5 to 5 micrograms/l with a limit of quantification of 10 micrograms/l for all analytes.

International audience; 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.

Screening for benzodiazepines is routinely performed by immunochemical analysis, such as enzyme immunoassay. However, the wide range of therapeutic and toxic concentrations of the different benzodiazepines and their extensive metabolism and conjugation hamper the design of an immunoassay protocol that can detect all benzodiazepines and their corresponding urinary metabolites. Multiple commercially available immunoassay methods exist, but false-negative (FN) results are still encountered, even with some frequently prescribed benzodiazepines. Several earlier investigations focused on determining how a particular benzodiazepine reacts in the different types of immunoassays, but only a few investigators have tried to improve the detection of some benzodiazepines, either by incorporating an enzymatic hydrolysis step before screening (1)(2) or by adding β-glucuronidase to the immunoassay reagents (3). These studies were performed with supplemented samples or with samples collected after controlled intake of therapeutic doses of one single benzodiazepine by healthy volunteers. We evaluated the relevance of hydrolyzing urine samples before screening with the Emit® d.a.u.TM Benzodiazepine Assay by analyzing a large number (n = 530) of authentic patient urine samples, reflecting actual practice. All urine samples were screened before (Emit) and after (Emit-H) enzymatic hydrolysis and subsequently analyzed by a sensitive gas chromatographic–mass spectrometric (GC-MS) method specifically developed for the analysis of benzodiazepines as a reference method (4)(5). The Emit d.a.u. Benzodiazepine Assay reagent sets were from Syva Co. (Dade Behring) and were used on a Cobas Mira Instrument (Roche) according to the instructions of the manufacturer. The applied hydrolysis, solid-phase extraction, and derivatization procedures were validated for alprazolam, α-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, N -desalkylflurazepam, ketazolam, lorazepam, lormetazepam, oxazepam, triazolam, and α-hydroxytriazolam. Because oxazepam and nordiazepam …

Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called "date-rape" drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gammahydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to * This paper was presented at the joint 1998 meeting of the International Association of Forensic Toxicologists (TIAFT) and Society of Forensic Toxicologists in Albuquerque, New Mexico. f Author to whom correspondence should be addressed. 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish interand intraday variabilities. Two suicide cases along with one alleged date-rape case and one of an emergency room patient whose blood screened positive for benzodiazepines were analyzed. All the hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. Two cases, including the daterape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite.

A high-performance liquid chromatographic assay for the quantification of the oxidative metabolites of flunitrazepam, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in human liver microsomal incubations was developed. Both metabolites were quantifiable in a single assay following a solvent extraction and reversed-phase high-performance liquid chromatography with UV detection. Standard curve concentrations for both metabolites ranged from 0.2 to 10 microM. Assay performance was determined using quality control samples and the intra- and inter-day accuracy and precision as determined by the coefficient of variations which were less than 15% (0.5-6 microM) for both metabolites. This method provides good precision and accuracy for use in kinetic studies of the oxidative metabolism of flunitrazepam in human liver microsomes.

Nanogram quantities of flunitrazepam and its major metabolite, desmethylflunitrazepam, can be reliably quantitated in human plasma using electron-capture gas-liquid chromatography. After addition of methylnitrazepam as the internal standard, plasma samples are extracted with an organic solvent (benzene:isoamyl alcohol). The organic extract is separated, evaporated to dryness, reconstituted, and injected onto the chromatograph using an automatic sampling system which allows up to 100 analyses/24 h. The sensitivity limits are 0.5 ng/ml for flunitrazepam and 1-2 ng/ml for desmethylflunitrazepam. Use of the method is illustrated in a study of the pharmacokinetic properties of flunitrazepam following a single 1.0-mg intravenous dose.

Photodecomposition of flunitrazepam (FNZP) and its photolabeling to human serum albumin (HSA) under oxygen-rich and -poor conditions were studied. The kinetics of both photodecomposition and photolabeling in oxygen-flushed solutions correspond completely with those found under normal aerobic conditions, as usually applied in photoaffinity labeling (PAL) of benzodiazepine receptor sites. This correspondence also holds for the photoproducts found. Nitroreduction products photochemically formed in the absence of oxygen, appeared not likely to contribute in PAL, in contrast to what has been suggested earlier. FNZP photodecomposes up to about 55% into N -desmethylflunitrazepam (D-FNZP) under aerobic conditions. In this case photolabeling to HSA proceeds far more efficiently, and results in twice as much covalent binding, as in an oxygen-poor environment. D-FNZP also photobinds to HSA to an extent comparable to that of FNZP. The results may in part explain the low percentage (20–25%) of photolabeled sites in PAL with [methyl- 3 H]FNZP by: (i) loss of label by photodecomposition of FNZP into-D-FNZP; (ii) photobinding of (unlabeled) D-FNZP leading to a decrease of receptor sites available for labeled FNZP; and (iii) destruction of receptor sites by singlet oxygen produced by photoexcited nitrobenzodiazepine.

A simple, sensitive, and rapid method for analysis of clonazepam in blood by gas-liquid chromatography with electron-capture detection is described. The method employs neutral benzene extraction from plasma and concentration into a small volume for autoinjection into the electron capture detector-equipped chromatograph. This method consistently provides blank plasma samples free of contaminants in the areas corresponding to the retention times for clonazepam and desmethylflunitrazepam (the internal standard). Sensitivity is 1 ng/mL, and standard curves are linear from 1 to 200 ng/mL. The applicability of the method to single-dose pharmacokinetics is illustrated.

A rapid method for the determination of flunitrazepam and desmethylflunitrazepam in human serum in the range 10–300 ng/ml is described. Both drugs are isolated from biological material by means of a single extraction, part of the organic phase is evaporated to dryness and the residue is dissolved in a small volume of benzene. Without further purification, the substance is determined gas chromatographically with an electron-capture detector configuration of 63 Ni-type. The method permits the quantitative determination of at least 25–300 ng/ml with an overall recovery of flunitrazepam of 99.7 ± 4.9% and of desmethylflunitrazepam of 98.6 ± 7.8% from serum. All calculations were carried out by a data system that was programmed for this purpose. The limit of detection for flunitrazepam is of the order of 1 ng/ml in serum. The method is sufficiently sensitive and specific for therapy control purposes. The time needed for an analysis is less than 1 h.

A high-performance liquid chromatographic method for the simultaneous determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma is described. After extraction with an organic solvent, the compounds and their internal standard (7-amino-methylclonazepam or Ro 5-3384) are derivatized with fluorescamine and chromatographed on a reversed-phase muBondapak C18 column using pH 8 buffer solution-acetonitrile (3:1) as mobile phase. The detection is performed by a fluorometer at excitation and emission wavelengths of 390 and 470 nm, respectively. The sensitivity limit is about 1 ng/ml of plasma for both 7-amino-flunitrazepam and 7-amino-desmethylflunitrazepam. The method has been applied to the determination of plasma levels of these substances during pharmacokinetic studies of flunitrazepam, desmethylflunitrazepam and 7-amino-flunitrazepam.