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.

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.

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination.

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.

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.

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS).

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, N-glucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.

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.

Quantification of 7-aminoflunitrazepam in human urine by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry.

Using a simple liquid-liquid extraction (LLE) procedure for sample pretreatment, 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The linearity was found in the range of 0.1-50ngmL-1 with a method detection limit (signal-to-noise ratio of 3) estimated at 0.05ngmL-1. Using the proposed method, good precision and recovery were also found in spiked urine samples at the levels of 0.5, 5.0, and 50ngmL-1 (intra-day/inter-day precision: 0.6-1.8% / 0.1-0.8%; post-spiked/pre-spiked recovery: 95.4-102.9% / 96.3-102.5%). In addition, acceptable relative differences (-24.2 - 0.8%) were observed by analyzing clinical urine samples using this monolith-based capillary LC-MS/MS method compared with the results obtained by the routine GC-MC method. Using the monolithic column, no noticeable deterioration of separation efficiency or carry-over was observed for more than 200 injections of urine samples. The applicability of the developed monolith-based capillary LC-MS/MS method was demonstrated by quantifying 7-aminoFM2 in various clinical urine samples. Based on these experimental results, the proposed LLE-monolith-based capillary LC-MS/MS method shows the potential for routine determination of drug metabolites in human urine for clinical and forensic applications.

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.

Development of immunoaffinity solid phase microextraction probes for analysis of sub ng/mL concentrations of 7-aminoflunitrazepam in urine.

We report on the development of solid phase microextraction probes for drug analysis, prepared with antibodies specific for benzodiazepines covalently immobilized to the surface. In the technique, immobilized antibody probes are exposed to a sample containing the drug for 30 min. Extracted drugs are subsequently desorbed from the probes in 500 microL of methanolic desorption solution, which is dried, reconstituted in a small volume of injection solution and analysed by LC-MS/MS. The antibodies were characterized both before and after immobilization, to facilitate the rational selection of antibodies for such analyses. Polyclonal and monoclonal antibodies were compared as was the impact of affinity purification of the polyclonal antibody to isolate the drug-specific fraction. The probes were evaluated for utility in analyzing 7-aminoflunitrazepam at sub ng/mL concentrations in urine, which is expected to be found several days after a single oral dose of 2 mg of flunitrazepam. Such analyses are required in monitoring for abuse of this drug, both in terms of 'club drug' use and in cases of drug-facilitated sexual assault. In these cases drug concentrations in blood and urine are much lower than in chronic abuse cases and are difficult to analyse by conventional methods. The method developed has a limit of detection of 0.02 ng/mL, with accuracy ranging from 1% to 27% and precision (% R.S.D.) ranging from 2% to 10% between the lower and upper limits of quantitation for the analysis of 7-aminoflunitrazepam in urine. The dynamic range of the method is from 0.02 ng/mL, which is limited by the instrument sensitivity, to 0.5 ng/mL, which is approaching the capacity of the probes. This would allow for quantitative analysis of samples at concentrations below that measurable by many other methods for general benzodiazepines analysis from urine, and a highly selective screen for samples at higher concentrations. The method has similar limits of detection to the most sensitive literature methods specifically designed for such analysis but with the advantage of significantly simplified sample preparation. This simplification makes the technique more amenable for use by both professionals and non-professionals.

Extraction and analysis of flunitrazepam/7-aminoflunitrazepam in blood and urine by LC-PDA and GC-MS using butyl SPE columns.

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.

Performance characteristics of 7-aminoflunitrazepam specific enzyme-linked immunosorbent assays.

With 7-aminoflunitrazepam (7-amino-FM2)-specific ELISAs now readily available from several commercial sources (e.g., Cozart Bioscience, Immunalysis, this study was conducted to evaluate the performance characteristics of these products when applied to the two-step testing protocol as commonly practiced in today's workplace drug-testing programs. Cross-reacting characteristics of these two assays toward a list of 25 benzodiazepines were evaluated. These assays were then applied to the analysis of urine specimens collected from patients treated with flunitrazepam (FM2) and/or other benzodiazepines. Resulting data were evaluated against gas chromatography-mass spectrometry (GC-MS) test data to ascertain corresponding cutoffs suitable for the two-step immunoassay/GC-MS testing strategy. Both Cozart and Immunalysis ELISAs are highly specific to 7-amino-FM2, with the latter reagent generating slightly higher responses. Diazepam and FM2 (parent compound) are the only compounds with significant cross-reacting characteristics. With the ELISA reagents' optimal dynamic ranges set between 0 and 25 ng/mL, urine specimens should be diluted by a factor of 5 prior to ELISA testing. If 30 ng/mL 7-amino-FM2 is adapted as the GC-MS cutoff, the corresponding ELISA cutoffs range is approximately 100-200 (or 20-40 when diluted by a factor of 5) ng/mL. Reagent lot and specimen characteristics (with or without the presence of cross-reacting compounds) affect the correlation of data derived from ELISA and GC-MS tests.

Postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in body fluids and solid tissues in an autopsy case: Usefulness of bile for their detection.

We experienced an autopsy case of a woman in her 70s, in which the direct cause of her death was judged as asphyxia due to the occlusion of food in the trachea. The postmortem interval was estimated at about 2days. The specimens dealt with were femoral vein blood, right heart blood, left heart blood, bile, brain, lung, heart muscle, liver, spleen, kidney, pancreas, skeletal muscle, and adipose tissue. By tentative drug screening, we found a high concentration of 7-aminoflunitrazepam in the femoral vein blood, which lead us to examine the postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in her body fluids and solid tissues. The extraction of flunitrazepam, 7-aminoflunitrazepam and internal standard nimetazepam was performed by a modified QuEChERS method, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, we used the standard additional method to overcome the matrix effects. The concentration of 7-aminoflunitrazepam were generally much higher than those of the parent drug flunitrazepam for most specimens except for the adipose tissue, showing that flunitrazepam is readily metabolized to its 7-amino metabolite after absorption into the body both antemortem and postmortem. The outstandingly highest concentration of 7-animoflunitrazepam was found in the bile, followed by the kidney, pancreas, left heart blood, spleen and liver. Although a majority of flunitrazepam was converted to 7-aminoflunitrazepam, the flunitrazepam concentration was highest in the pancreas, followed by the spleen, bile, left heart blood, and brain. In contrast to the results on synthetic cannabinoids, the levels of flunitrazepam and 7-animoflunitrazepam in the adipose tissue were relatively low. The present study showed that the bile may be a useful specimen for detection of unchanged benzodiazepines/their metabolites to be collected at autopsy.

Pharmacokinetics of flunitrazepam following single dose oral administration in liver disease patients compared with healthy volunteers.

The pharmacokinetic behaviour of flunitrazepam and its main active metabolite, N-desmethyl flunitrazepam, was investigated in 12 patients with liver disease (cirrhosis or hepatitis) compared to 6 healthy volunteers. A gas-liquid chromatographic method allowing for simultaneous determination of flunitrazepam and N-desmethyl flunitrazepam in plasma samples was developed. The accuracy and the precision near the quantification limit of ca. 1 ng/ml were better than 5%. Plasma levels of flunitrazepam were not significantly altered by hepatic failure, whereas plasma levels of N-desmethyl flunitrazepam were lower in patients than in healthy subjects. Pharmacokinetic parameters did not differ significantly between healthy subjects and liver disease patients: the oral clearance was 3.5 +/- 0.8, 3.5 +/- 1.9 and 4.0 +/- 1.2 ml/min/kg, respectively in healthy subjects, patients with hepatitis and patients with cirrhosis. The apparent elimination half-life was 22 +/- 5 h in healthy subjects, 25 +/- 10 h in patients with hepatitis and 20 +/- 6 h in patients with cirrhosis. However, the expected increase of the drug free fraction during liver disease could decrease the therapeutic and toxic ranges of flunitrazepam in these patients.

Simultaneous determination of flunitrazepam and its metabolites in plasma and urine by HPLC/DAD after solid phase extraction.

A high performance liquid chromatography (HPLC) assay was developed for the determination of flunitrazepam (FNZ) and its metabolites in urine and plasma. The analytes and the internal standard (triazolam, TRZ) were extracted by Sep-Pak C18 SPE-cartridge and separated utilizing a 5 microm ChromSpher C8 glass column with a gradient mobile phase containing methanol and 0.125% (v/v) of isopropylamine in water. Diode array detection (DAD) was carried out at a monitoring wavelength of 240 nm and a reference wavelength of 550 nm. Standard curves were linear from their quantitation limits until 200 ng ml(-1) urine or 250 ng ml(-1) plasma for 7-amino-desmethyl-flunitrazepam (ADF), 7-amino-flunitrazepam (AF), 7-acetamino-flunitrazepam (ACF) and until 400 ng ml(-1) urine or 500 ng ml(-1) plasma for FNZ, 1-desmethyl-flunitrazepam (DF), and 3-hydroxyl-flunitrazepam (HF). The intraday and interday coefficients of variation ranged from 2.04 to 9.07% and from 2.64 to 14.10%, respectively in urine and from 5.13 to 8.60% and from 7.27 to 10.46%, respectively in plasma. The developed method is used in forensic toxicology and is also applicable to pharmacokinetic studies in man.

Testing human hair for flunitrazepam and 7-amino-flunitrazepam by GC/MS-NCI.

To validate information on flunitrazepam use, we investigated human hair for flunitrazepam and its major metabolite 7-amino-flunitrazepam by gas chromatography coupled to mass spectrometry in negative chemical ionization mode of detection. Samples were twice decontaminated with methylene chloride, pulverized in a ball mill and 50 mg of powdered hair were incubated in Soerensen buffer (pH 7.6) in the presence of diazepam-d5 used as internal standard. After liquid-liquid extraction of the incubation medium with diethylether-chloroform (80:20, by vol.), the organic phase was evaporated and the dry extract was derivatizated with heptafluorobutyric anhydride. Benzodiazepines were separated on a 30 m capillary column and detected using single ion monitoring. Among 40 hair samples tested (obtained from drug addicts deceased by heroin overdose), 14 were positive for both flunitrazepam and 7-amino-flunitrazepam and 12 for 7-amino-flunitrazepam only. Concentrations ranged from 31 to 129 pg/mg (mean: 60 pg/mg) and from 3 to 161 pg/mg (mean: 46 pg/mg) for flunitrazepam (14 cases) and 7-amino-flunitrazepam (26 cases), respectively. This first report described the detection of flunitrazepam and 7-amino-flunitrazepam in hair of chronic abusers. Due to the low concentrations observed, negative chemical ionization appears to be the alternative to test flunitrazepam and other benzodiazepines in hair.

Screening for drugs of abuse in hair with ion spray LC-MS-MS.

Analyzing hair for many substances can be tedious and expensive, and a rapid screening method should prove helpful. Generally, screening has been performed using immunological tests, mainly in workplace drug testing, where the number of samples has been high. The aim of this study was to develop an LC-MS-MS method for the simultaneous analysis of several drugs of abuse in human hair as an alternative to immunological screening tests. In 75 randomly selected autopsy cases, hair was analyzed in addition to the usual specimens of blood and urine. The method included nicotine, cotinine, morphine, codeine, 6-acetylmorphine, ethylmorphine, amphetamine, methamphetamine, MDA, MDMA, benzoylecgonine, cocaine, 7-aminoflunitrazepam and diazepam. The LC-MS-MS analysis was performed on a SCIEX API 2000 MS-MS instrument equipped with an electrospray interface. To 20-50 mg of hair, 0.5 ml of mobile phase A (acetonitril:methanol:20 mM formate buffer, pH 3.0 (10:10:80)) and 25 microl of internal standard were added and the sample was incubated in a water bath at 37 degrees C during 18 h. Using a threshold of 20 ng/sample, equivalent to 1 ng/mg if 20mg hair is used, 26 positive results were found in 16 cases. Three of the 26 positive detections could not be confirmed by GC-MS. Two of the cases were not previously known as drug users. Of the 59 negative cases, only one case had a positive blood sample showing 0.01 and 0.07 microg/g femoral blood of 6-acetylmorphine and morphine, respectively. This might indicate drug abstinence resulting in decreased tolerance or even a "first time" use of heroin resulting in death. We conclude that the use of hair analysis in postmortem cases can reveal both unknown drug use, as well as confirm a period of drug abstinence prior to an acute fatal overdose. The proposed LC-MS-MS method showed high sensitivity, was very easy to perform and seemed appropriate for screening purposes.

GC-MS determination of flunitrazepam and its major metabolite in whole blood and plasma.

A gas chromatography-mass spectrometry method was developed for the analysis of flunitrazepam (FN) and its major metabolite, 7-amino-flunitrazepam (7-amino-FN), in both plasma and whole blood. The method was based on acid hydrolysis of the samples after dilution with HPLC water followed by extraction and derivatization (heptafluorobutyrate) of the resulting benzophenones. Analysis of plasma and whole blood samples from subjects administered 2-mg doses of FN showed that FN was only detected in whole blood (LOD 5 ng/mL) and not in plasma. However, 7-amino-FN was detected in both plasma and whole blood, although the levels were much higher in plasma. 7-Amino-FN was detected for the entire period of specimen collection (12 h), but FN was only detected in whole blood for 4 h after ingestion with peak levels after 1 h.

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.

Improved screen and confirmation test of 7-aminoflunitrazepam in urine specimens for monitoring flunitrazepam (Rohypnol) exposure.

Confirmed and alleged misuses of flunitrazepam (FM2, Rohypnol) have brought about serious interest in the development of an analytical methodology that can be effectively used for preliminary screen and confirmatory test of FM2 (or its metabolites) in urine specimens under high-volume settings. Reported methods do not serve this need well for the following reasons: (1) common benzodiazepine (BZ) immunoassays (IAs) have broad cross-reactivities toward widely prescribed BZs (and their metabolites) and are therefore likely to generate an unacceptable number of false positives and (2) because FM2 is typically used at low doses (1-4 mg), IAs with low cross-reactivities toward FM2 (and its metabolites) are likely to generate false-negative results. In this current study, a familiar and effective two-step IA/gas chromatography-mass spectrometry (GC-MS) approach is successfully developed and applied to clinical specimens. Cross-reacting characteristics of the following BZ IAs toward various BZs (and their metabolites) are evaluated focusing on their effectiveness in serving as the preliminary test reagent in a two-step testing protocol: TDx, Beckman, CEDIA, Roche Cobas Integra, Emit II Plus, and Cozart ELISA. Although other IAs show broad cross-reactivities toward various BZs and their metabolites, diazepam is the only non-FM2 derived compound that exhibits noticeable cross-reactivity toward Cozart ELISA reagent. Cross-reactivity data and data derived from studies conducted on a limited number of clinical specimens demonstrate that, when used to monitor FM2 exposure in a large population group (including those exposed to other BZs), Cozart ELISA has the potential of being as effective as (or better than) those currently used in various workplace drug-testing programs for monitoring respectively targeted drugs. Data derived from this study further suggest that 50 ng/mL apparent 7-aminoflunitrazepam (Cozart ELISA) and 30 ng/mL free 7-aminoflunitrazepam (GC-MS) are potentially effective preliminary test and confirmation test cut-offs. To maximize efficiency, it is further suggested that urine specimens are first diluted by a factor of 5 for the preliminary test in which a 10-ng/mL 7-aminoflunitrazepam standard is used as the assay's cut-off standard.

Determination of flunitrazepam and its main metabolites in serum and urine by HPLC after mixed-mode solid-phase extraction.

A rapid and sensitive method is presented for the simultaneous determination of flunitrazepam, norflunitrazepam, 7-aminoflunitrazepam, and 7-acetamidoflunitrazepam in serum, plasma, and urine. The compounds were extracted by a mixed-mode solid-phase procedure following analysis by high-performance liquid chromatography and ultraviolet detection and using methylclonazepam as the internal standard. The method revealed high recoveries and showed good precision and linearity for all compounds. The limit of detection was at least 1 ng/ml serum (plasma) for all compounds.