Novel reductive-reductive mode electrochemical detection of Rohypnol following liquid chromatography and its determination in coffee.

Rohypnol (flunitrazepam) has been successfully determined in coffee by high performance liquid chromatography dual electrode detection (LC-DED) in the dual reductive mode. Initial studies were performed to optimise the chromatographic conditions and these were found to be 50% acetonitrile, 50% 50 mM pH 2.0 phosphate buffer at a flow rate of 0.75 mL min(-1), employing a Hypersil C18, 5 µm, 250 mm × 4.6 mm column. Cyclic voltammetric studies were made to ascertain the redox behaviour of Rohypnol at a glassy carbon electrode over the pH range 2-12. Hydrodynamic voltammetry was used to optimise the applied potential at the generator and detector cells; these were identified to be -2.4 V and +0.8 V for the redox mode and -2.4 V and -0.1 V for the dual reductive mode respectively. A linear range of 0.5-100 µg mL(-1), with a detection limit of 20 ng mL(-1) was obtained for the dual reductive mode. Further studies were then performed to identify the optimum conditions required for the LC-DED determination of Rohypnol in beverage samples. A convenient and rapid method for the determination of Rohypnol in beverage samples was developed using a simple sample pre-treatment procedure. A recovery of 95.5% was achieved for a sample of white coffee fortified at 9.6 µg mL(-1) Rohypnol.

Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam.

Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between -10 °C and -35 °C, whereas for flunitrazepam on-column interconversion was observed at temperatures between -40 °C and -66 °C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At -20 °C the enantiomerization barriers, ΔG(≠), for diazepam, prazepam and tetrazepam were determined to be in the range 17.6-18.7 kcal/mol. At -55 °C ΔG(≠) for flunitrazepam was determined to be in the 15.6-15.7 kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases.

Nanoparticle-assisted MALDI-TOF MS combined with seed-layer surface preparation for quantification of small molecules.

Despite the advantages of simplicity and high-throughput detection that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has over other methods, quantitative analysis of low-molecular-weight analyte is hampered by interference from matrix-derived background noise and signal fluctuation due to the inhomogeneous MALDI sample surface. Taking advantage of improved sample homogeneity through matrix-conjugated magnetic nanoparticles (matrix@MNP) and the seed-layer method, we report a new strategy for the rapid identification and quantification of drugs in urine samples, using morphine and 7-aminoflunitrazepam (7-aminoFM2) as model compounds. To our knowledge, this is the first attempt using the seed-layer method for small molecule analysis. By applying the proposed seed-layer method, which was specifically optimized for the 2,5-dihydroxybenzoic acid@MNP (DHB@MNP) matrix, homogeneous sample crystallization examined by microscopy analysis was obtained that generated reproducible MALDI signals (RSD<10.0%). For urine sample analysis, simple liquid-liquid extraction as a sample pretreatment step effectively reduced the ion suppression effect caused by the endogenous components in urine; good recoveries (82-90%) were obtained with a small ion suppression effect (<14% of signal decrease). This newly developed method demonstrated good quantitation linearity over a range of 50-2000 ng mL(-1) (R(2)>0.996) with reduced signal variation (RSD<10.0%). The detection limit is 30 ng mL(-1) with good precision (intra-day, 2.0-9.3%; inter-day, 5.0-10.0%) and accuracy (intra-day, 95.0-106.0%; inter-day, 103.0-115.5%). The nanoparticle-assisted MALDI-TOF MS combined with seed-layer surface preparation provides a rapid, efficient and accurate platform for the quantification of small molecules in urine samples.

Characterization and evaluation of two-dimensional microfluidic chip-HPLC coupled to tandem mass spectrometry for quantitative analysis of 7-aminoflunitrazepam in human urine.

Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 µL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1-10 ng mL(-1) and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL(-1) for 7-aminoFM2. After consecutive liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (n = 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL(-1). Intra-day and inter-day precision were 2.0-7.1% and 4.3-6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (-1.3 to -13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.

Quantification of benzodiazepines in whole blood and serum.

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.

Flunitrazepam metabolism by cytochrome P450S 2C19 and 3A4.

We have identified CYP2C19 and CYP3A4 as the principal cytochrome P450s involved in the metabolism of flunitrazepam to its major metabolites desmethylflunitrazepam and 3-hydroxyflunitrazepam. Human CYP2C19 and CYP3A4 mediated the formation of desmethylflunitrazepam with Km values of 11.1 and 108 microM, respectively, and 3-hydroxyflunitrazepam with Km values of 642 and 34.0 microM, respectively. In human liver microsomes (n = 4) formation of both metabolites followed biphasic kinetics. Desmethylflunitrazepam formation was inhibited 31% by S-mephenytoin and 78% by ketoconazole, suggesting involvement of both CYP2C19 and CYP3A4. Formation of 3-hydroxyflunitrazepam was also significantly inhibited by ketoconazole (94%) and S-mephenytoin (18%). In support of these chemical inhibition data, antibodies directed against CYP2C19 and CYP3A4 selectively inhibited formation of desmethylflunitrazepam by 26 and 45%, respectively, while anti-CYP3A4 antibodies reduced 3-hydroxyflunitrazepam formation by 80%. Our data also suggest that CYP1A2, -2B6, -2C8, -2C9, -2D6, and -2E1 are not involved in either of these metabolic pathways. We estimate that the relative contributions of CYP2C19 and CYP3A4 to the formation of desmethylflunitrazepam in vivo are 63 and 37%, respectively, at therapeutic flunitrazepam concentrations (0.03 microM). We conclude that the polymorphic enzyme CYP2C19 importantly mediates flunitrazepam demethylation, which may alter the efficacy and safety of the drug, while CYP3A4 catalyzes the formation of 3-hydroxyflunitrazepam.

Rapid method for the solid-phase extraction and GC-MS analysis of flunitrazepam and its major metabolites in urine.

Recently, flunitrazepam (Rohypnol) has become an increasingly popular drug of abuse among young adults, who take it for its euphoric effects. In other cases, the drug has been used by rapists for its sedative and hypnotic effects that can induce a catatonialike trance and memory loss in potential victims; as a result, it has been nicknamed the "date-rape drug". For these reasons, the Drug Enforcement Administration recently considered adding the drug (a.k.a. "Roofies") to the same category as heroin and LSD. A selective and sensitive technique has been developed for extracting, detecting, and identifying flunitrazepam and its two major metabolites (7-aminoflunitrazepam and N-desmethylflunitrazepam) in human urine. Using a solid-phase extraction cartridge containing a "mixed-mode" bonded silica gel (Bond Elut Certify), flunitrazepam and its metabolites were selectively isolated from other urine components and quantitated and identified by gas chromatography-tandem mass spectrometry with a benchtop ion mass spectrometer. The extraction method is rapid, reproducible, and precise, and it has a broad linear working range. The overall extraction efficiency was found to be more than 90% for the parent drug as well as the two major metabolites.

On-line immunoaffinity extraction and HPLC analysis of flunitrazepam and its main metabolites in serum.

A sensitive, simple, and rapid method for the determination of flunitrazepam and its major metabolites (7-aminoflunitrazepam, 7-acetamidoflunitrazepam, and norflunitrazepam) in serum and plasma is presented. The on-line procedure uses an immobilized, highly reusable antibody against benzodiazepines for selective extraction from serum followed by analysis by high-performance liquid chromatography with ultraviolet detection. This reliable method provides a limit of detection of 1 ng/mL serum, and results are obtained in less than 40 min.

The detection and quantitation of 7-aminoflunitrazepam, the major urinary metabolite of flunitrazepam, by gas chromatography/mass spectrometry.

A sensitive gas chromatography/mass spectrometry method for the detection and quantitation of 7-aminoflunitrazepam, the major urinary metabolite of flunitrazepam, is described. The method is based upon solvent extraction followed by derivatisation with methyl-bis-trifluoroacetamide (MBTFA), to give a trifluoroacetyl derivative. A profile of 7-aminoflunitrazepam urinary concentrations following ingestion of 0.5 to 4 mg oral doses is reported. The method has been found to be reproducible and can be used for the confirmation of flunitrazepam administration.

Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair.

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, gamma-hydroxybutyrate, 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 7-aminoflunitrazepam (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 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish inter- and intraday variabilities. Two suicide cases along with one alleged date-rape case and one case 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 date-rape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite.

Gas chromatographic determination of bromo and fluoro derivatives of benzodiazepine in human body fluids.

A rapid, sensitive and accurate method for the determination of bromazepam and flunitrazepam in plasma and urine using gas chromatography has been developed. Bromazepam was extracted with diethyl ether and flunitrazepam with hexane at pH 7. A nitrogen detector was used to determine bromazepam and an electron-capture detector was used for flunitrazepam.