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.

Nitrobenzodiazepines: Postmortem brain and blood reference concentrations.

Reference concentrations are needed to evaluate postmortem toxicology results and usually femoral blood is the specimen of choice. However, brain tissue has been suggested as a viable alternative specimen, since postmortem blood concentrations can be difficult to interpret due to postmortem redistribution, among other factors. Here we present reference concentrations of postmortem brain and femoral blood of the nitrobenzodiazepines clonazepam, flunitrazepam, and nitrazepam that are of particular interest since they commonly are converted to their corresponding 7-aminometabolites in the postmortem situation. The drugs and metabolites were quantified in both matrices using LC-MS-MS in 69 cases. In 63 cases the compounds were judged not to have been of significance for the death (C cases), whereas they were considered to have been a contributing factor in 6 cases (B cases). No cases were observed with a nitrobenzodiazepine being the sole cause of death (A cases). The brain-blood ratios for clonazepam and nitrazepam were 5.5 and 4.7, respectively, while the brain-blood ratios for the 7-aminometabolites ranged from 0.4 to 0.5. Flunitrazepam only occurred as the 7-aminometabolite. A positive correlation between brain and blood concentrations was found with Spearman's rank correlation coefficients (rs) ranging from 0.77 to 0.96. The measured femoral blood concentrations agree with literature values, but only few brain concentrations were available for comparison. The drug-metabolite ratios for clonazepam and nitrazepam were 10-12 times higher in brain than in blood. The pre-analytical variation in brain of 5.9% was fairly low, suggesting that brain tissue is a useful alternative to blood. The reported brain and femoral blood concentrations serve as reference values in postmortem investigations.

The detection of multiple illicit street drugs in liquid samples by direct analysis in real time (DART) coupled to Q-orbitrap tandem mass spectrometry.

Direct analysis in real time coupled to Q-orbitrap tandem mass spectrometry (DART-MS) without requiring preparatory procedures was used to directly detect trace amounts of illegal street drugs, namely p-chloroamphetamine, p-fluoromethamphetamine, γ-hydroxybutyrate, ketamine, methamphetamine, 3,4-methylenedioxypyrovalerone, p-methylethcathinone, methylone, and nimetazepam, in solution and also in real drug samples. Exact mass determination of the drug samples was completed in less than 1min. With the ability to rapidly identify drugs, this technique shows great potential as a useful analytical tool in the analysis of illicit street drugs, and has the significant advantages of simplicity and sensitivity without the sample preparation needed by other methods.

Detection of Nitrobenzodiazepines and Their 7-Amino Metabolites in Oral Fluid.

Clonazepam, nitrazepam and flunitrazepam are frequently used benzodiazepines, both as prescribed medication and as drugs of abuse. Little is, however, known about how these drugs are excreted in oral fluid. It has been claimed that the parent drugs are more likely to be detected in oral fluid than the 7-amino metabolites. The aim of this study was to investigate whether the parent drugs or the 7-amino metabolites of the nitrobenzodiazepines were most frequently detected in authentic oral fluid samples. Oral fluid samples were collected from patients undergoing opioid maintenance treatment. Cases where clonazepam, nitrazepam, flunitrazepam and/or their metabolites were detected were included. The samples were collected using the Intercept Oral Specimen Collection Device. A cutoff concentration of 1 nM (∼0.3 ng/mL) in oral fluid-buffer mixture was applied for all the substances. A total of 1,001 oral fluid samples were positive for clonazepam and/or 7-aminoclonazepam; both substances were detected in 707 samples, only the parent drug in 64 cases and only the metabolite in 230 cases. For nitrazepam, both substances were detected in 139 samples; only the parent drug in 16 cases and only the metabolite in 56 cases. Flunitrazepam only was not detected in any sample; both substances were detected in one of these cases, and only the metabolite in three cases. This study revealed that 7-amino metabolites were more likely to be detected in oral fluid than the parent drugs.

Silver nanoparticle enhanced chemiluminescence method for the determination of nitrazepam.

We report on a simple and sensitive chemiluminescence (CL) method to determine nitrazepam. This method is based on the fact that rhodamine 6G (Rh6G) enhanced the weak CL emission of the reaction of hexacyanoferrate with nitrazepam, and that it was further enhanced by silver nanoparticles (AgNPs). The effects of the concentrations of K3Fe(CN)6, Rh6G, AgNPs and NaOH on the CL reaction were investigated. Under the optimum conditions, the CL intensity was proportional to the concentration of nitrazepam in the range from 1.0 nM to 10.0 µM. The detection limit (3σ) was at 0.1 nM. The relative standard deviation was 2.1% (at a 0.1 µM concentration and for n = 11). The method was successfully applied to the determination of nitrazepam in Coca-Cola beverage, urine and plasma, and the recovery was 98 - 103%. We also considered the possible CL reaction mechanism.

[Simultaneous determination of 10 unapproved sedative drugs in feeds by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry].

A new analytical method using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed for screening and confirmation of 10 unapproved sedative drugs in feeds. The samples were extracted using the solution of methanol-0.1 mol/L HCl (9:1, v/v), and the extracts were centrifuged and then directly purified through MCX cartridges. The identification and detection were achieved in positive electrospray ionization (ESI) mode using Q-TOF-MS. The potential of UPLC-Q-TOF MS for confirmatory analysis was shown by determining the accurate mass of all the compounds and fragment ions upon collision-induced-dissociation (CID) at different energies. The extra mass measurement errors for all the sedative drugs were found to be within 5 ppm. The calibration graphs were linear in the concentration range of 5-100 microg/L with the correlation coefficients more than 0.99 for the 10 drugs. The limits of quantification (LOQ, S/N = 10) were 8 microg/kg for nitrazepam, zolpidem and thioridazine; 10 microg/kg for thriazolam, estazolam, diazepam, promethazine, chlorpromazine and midazolam; 20 microg/kg for clozapine. The recoveries for all the compounds in feeds were 60.6%-108.5% with the relative standard deviations less than 10% at the spiked levels of LOQ, 2LOQ and 4LOQ.

Measurement of benzodiazepines in urine by liquid chromatography-tandem mass spectrometry: confirmation of samples screened by immunoassay.

BACKGROUND: Liquid chromatography linked to tandem mass spectrometry (LC/MS/MS) provides the ability to identify a range of benzodiazepines in accordance with European Union criteria and is an attractive method for the confirmation of benzodiazepines following immunoassay screening. METHODS: An LC/MS/MS method to detect and quantitate the six most common benzodiazepines/metabolites (diazepam, nitrazepam, nordiazepam, oxazepam, temazepam and 7-aminonitrazepam) was developed together with a qualitative screening method for a further 11 benzodiazepines/metabolites. These methods were used for confirmation of 250 urine samples submitted for routine drug screening by immunoassay for benzodiazepines (100 samples positive for a benzodiazepine, assay cut-off >200 microsg/L). RESULTS: The lower limits of detection and quantitation were less than 2.5 and 5 microg/L for the six most common benzodiazepines. Recoveries ranged between 97% and 102% and calibration curves were linear to at least 4000 microg/L (r = 0.99). Intra and inter-assay imprecision were <10% (n = 10) and <20% (n = 15), respectively. Confirmation of benzodiazepines using LC/MS/MS was achieved for 89% of the immunoassay-positive urine samples. Of the immunoassay-negative urine samples, 31% of these demonstrated a benzodiazepine using LC/MS/MS. CONCLUSION: The validated LC/MS/MS method developed is effective for the confirmation of immunoassay screening results for benzodiazepines. The lower limit of detection and assay specificity offers a longer window of detection and more detailed clinical information compared with immunoassay screening.

Optimisation of high-performance liquid chromatography with diode array detection using an automatic peak tracking procedure based on augmented iterative target transformation factor analysis.

An automated method for the optimisation of high-performance liquid chromatography is developed. First of all, the sample of interest is analysed with various eluent compositions. All obtained data are combined into one augmented data matrix. Subsequently, augmented iterative target transformation factor analysis performs the integrated tasks of curve resolution and peak tracking. Since this type of curve resolution processes all data at once, it can deal with strong peak overlap and reveal the correspondence of compounds between runs, i.e. peak tracking. The retention time and peak width at half height for each component of the sample are determined for every eluent composition. Next, models are built for the retention time and the peak width at half height. These models are used to predict the resolution and the analysis time for each point in factor space. Finally, a multi-criterion decision-making method, Pareto optimality, is used to find the optimum. The method completes all calculations within a few minutes and without user intervention. By means of this procedure, a mixture of three benzodiazepines is successfully separated using a ternary mobile phase. There are two requirements for the automated optimisation method to work correctly. Firstly, all components of the sample must have sufficiently different spectra. Secondly, each compound should have the same spectrum under all experimental conditions.

Quantitative determination of diazepam, nitrazepam and flunitrazepam in tablets using thin-layer chromatography-densitometry technique.

A simple and reliable assay for diazepam, nitrazepam and flunitrazepam in tablets by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC)-densitometry is described. A quantity of a ground tablet mass, equal to the average weight of one tablet was sonicated in MeOH, mixed with appropriate internal standard, filtered and either injected directly into the liquid chromatograph, or after evaporation and reconstitution of an aliquot of the extract, was spotted on a silica gel thin-layer plate. A variable UV detector, operated at 254 nm was employed in both procedures. A C18, reversed phase 7 microm column was used for HPLC analysis; the mobile phase was a 1:1 (v/v) mixture of MeOH (40 degrees C) and 0.01 M phosphate buffer (pH 7, 80 degrees C). The TLC plate was developed in an unsaturated chromatographic chamber containing 100 ml chloroform-acetone (9:1); at room temperature, the mobile phase was allowed to travel 15 cm. The percentage of the active ingredient content of each tablet obtained by both procedures, was in the range of the stated amount except for one brand of diazepam tablets which contained approximately 23% less active ingredient than the minimum prescribed amount. The TLC densitometry, although yields slightly higher values than the HPLC method, is preferred due to its simplicity, ease and low cost.

Tissue distribution of nitrazepam and 7-aminonitrazepam in a case of nitrazepam intoxication.

We report a case of nitrazepam poisoning in which the distribution of nitrazepam and 7-aminonitrazepam was determined in body fluids and tissues. A 52-year-old woman was found dead in a shallow ditch (approximately 5 cm in depth), in the winter. Ambient temperature was 2-8 degrees C. The postmortem interval was estimated to be approximately 1 day and no putrefaction was observed. The cause of death was thought to be drowning due to nitrazepam overdose and cold exposure. Blood concentrations of nitrazepam and 7-aminonitrazepam were very site dependent (0.400-0.973 microg/ml and 0.418-1.82 microg/ml). In addition, the concentration of the same analytes in the bile were 4.08 and 1.67 microg/ml, respectively, and in the urine: 0.580 and 1.09 microg/ml, respectively. A high accumulation of both substances was observed in various types of brain tissue (2.17-6.22 microg/g and 2.49-5.11 microg/g). Only small amounts of nitrazepam and 7-aminonitrazepam were detected in the liver (0.059 and 0.113 microg/g, respectively). Large differences in the observed concentrations of nitrazepam and 7-aminonitrazepam among arterial and venous blood samples were thought to be mainly due to dilution of arterial blood by water entering the circulation through lungs at the time of death. Bacterial metabolism of nitrazepam may also have contributed to the observed differences.

[Poisonings with psychotropic drug mixtures: analysis using the thin-layer chromatography method]. / Apsinuodijimu psichotropiniu vaistu misiniu tyrimas plonasluoksnes chromatografijos metodu.

It is observed increase in number of remedy intoxications in Lithuania, especially important is intoxication with psychotropic drugs and their mixtures. The thin-layer chromatography (TLC) method was proposed for separation and identification of drugs in mixture aminazine: nitrazepam: barbamylum. The mixture of these drugs excreted from body fluids (blood and urine) was investigated by TLC. Most acceptable these mobile phases: 1. Diethyl ether: dioxane--40:60. Rf values for drugs: aminazine 0.23-0.25; barbamylum 0.85-0.9; nitrazepam 0.75-0.8. 2. Diethyl ether: NH3 (25%): benzenum--80:10:10. Rf values for drugs: aminazine 0.55-0.58; barbamylum 0.32-0.35; nitrazepam 0.17-0.2.

[Ultraviolet spectrophotometric determination of psychotropic drug mixtures]. / Psichotropiniu vaistu misinio tyrimas ultravioletines spektrofotometrijos metodu.

The mixture of psychotropic drugs aminazine, barbamylum and nitrazepam using ultraviolet (UV) spectrophotometric method was researched. As the solvent, most acceptable for identification and differentiation of these preparation was found 0.1N solution of sulphuric acid. By measuring the optical density of the solutions in different concentration the calibration diagrams for all preparations were made. The standard deviation, by determining the quantity of aminazine is 2.8; 4.0; 4.3; barbamylum--1.0; 2.5; 1.15; nitrazepam--2.64; 1.0; 1.35.

Electrospray ionization gas-phase electrophoresis under ambient conditions and it's potential or high-speed separations.

A moderately high resolution nanoelectrospray ionization gas-phase electrophoresis instrument was constructed and evaluated for simple high-speed separations of several groups of compounds. The insertion of a plate containing a 1.6 cm diameter exit orifice, 2.5 cm from the location of electrospray, allowed ions to be created and desolvated under ambient conditions with minimal solvent contamination to the drift tube. Ion separation selectivity is discussed and shown to be slightly altered by changing the drift gas flow rate. Issues of using gas-phase electrophoresis as a high-speed separation technique are discussed. Gas-phase electrophoresis-spectra of selected benzodiazepines, triazine herbicides, and simple combinatorial chemistry libraries are demonstrated.

Flow-injection fluorimetric determination of 1,4-benzodiazepines in pharmaceutical formulations after acid hydrolysis.

A simple, rapid and fully automated flow injection method with fluorimetric detection after hydrolysis with H2SO4 in ethanolic or methanolic medium at room temperature has been developed for the determination of 1,4-benzodiazepines (oxazepam, diazepam and nitrazepam) in pharmaceutical formulations. The calibration curves are linear in the ranges (mg ml(-1)) of oxazepam (0.025-0.150), diazepam (0.010-0.125) and nitrazepam (0.010-0.150), with detection limits of 0.01, 0.005 and 0.005 mg ml(-1), respectively, and RSD (1% (n = 10). The measurement throughput is 60 h(-1) using a 200-microl sample volume obtained by the direct dissolution of formulations in alcohol.

Stability of nitrobenzodiazepines in postmortem blood.

Studies were undertaken to determine the stability of nitrobenzodiazepines and their 7-amino metabolites in water and blood. At 22 degrees C nitrazepam and clonazepam were stable in sterile fresh blood containing preservative over 28 days, whereas 25% of flunitrazepam was degraded. At 37 degrees C all three drugs were substantially lost over 9 h (29-51%). There was only a small loss observed for the 7-amino metabolites and no substantial amounts of parent drug and 7-amino metabolite were degraded in water under these conditions. In the absence of preservative substantial amounts (25-50%) of parent drugs were lost in fresh blood over 10 days at 22 degrees C. In bacterially-contaminated postmortem blood all three drugs were completely degraded over 8 h at 22 degrees C with almost all drug completely converted to the respective 7-amino metabolite. These metabolites were also partially degraded (10-20%) over 45 h at 22 degrees C. All 3 nitrobenzodiazepines were stable in blood stored for up to 24 months at -20 degrees C, or 4 degrees C over 10 months. Their respective 7-amino metabolites were, however, relatively unstable at -20 degrees C with a significant loss (29%) after 2 months. At 4 degrees C a 21% loss occurred after 1 month. Freeze/thawing was found not to affect the concentration of nitrobenzodiazepine and 7-amino metabolites. These results show that the nitrobenzodiazepines and their metabolites are unstable chemically and metabolically in blood. We advise that blood collected for the purpose of nitrobenzodiazepine determinations should be preserved with sodium fluoride, stored at -20 degrees C and assayed as soon as practicable, preferably within a week of collection.

Postmortem distribution and redistribution of nitrobenzodiazepines in man.

The distribution of the nitrobenzodiazepines, flunitrazepam, clonazepam and nitrazepam, and their respective 7-amino metabolites were examined in blood, serum, vitreous humor, liver, bile and urine of decedents taking these drugs. Peripheral blood, serum and liver concentrations were not significantly different to each other. However, vitreous concentrations were one-third of blood, while bile concentrations were 5-12 fold higher. Blood, serum and vitreous contained predominantly the 7-amino metabolite, liver contained only the metabolite, while bile contained significant concentrations of both the parent drug and the 7-amino metabolite. Urine contained only small concentrations of parent drug, however, as expected a number of metabolites were detected. Redistribution studies compared the drug concentrations of femoral blood, taken at body admission to the mortuary, with femoral blood taken at autopsy approximately 39 h later in 48 cases. The concentrations of 7-amino metabolites were not significantly different, however the concentrations of parent nitrobenzodiazepines were significantly higher in the admission specimens. In 6 cases in which subclavian blood was taken, the concentrations were not significantly different to the concentrations in admission blood. Similar findings were observed when femoral and subclavian blood concentrations were compared in 6 cases. There was also no apparent difference in total blood concentrations of nitrobenzodiazepines when blood concentrations taken in hospital shortly prior to death were compared to postmortem blood. Postmortem diffusion into peripheral blood is therefore not a confounding factor in the interpretation of nitrobenzodiazepine concentrations.

Determination of droperidol in plasma by liquid chromatography.

A reversed-phase high performance liquid chromatographic method is described for the determination of droperidol concentrations in plasma. Following extraction, separation of droperidol and the internal standard flurazepam was achieved with a Spherisorb Nitrile, 5 microns, S5CN 250 mm x 4.6 mm column at 200 nm. The mobile phase was phosphate buffer (0.05 M, pH 2.4), acetonitrile and ethanol (65:20:15, v/v/v). The assay was rapid, sensitive and linear over the range 2-4000 ng ml-1. Precision of the assay expressed as the intra- and inter-day relative standard deviations (%RSD) did not exceed 10%. Flunitrazepam, midazolam and nitrazepam were also resolved with this technique and did not interfere with droperidol or flurazepam. Resolution of all five compounds was complete in less than 6 min. The assay was used to study the pharmacokinetics of high dose droperidol infusions during and after cardiac surgery.

[Determination of benzodiazepines by thermospray liquid chromatograph-mass spectrometer. Part 1. Nitrazepam, estazolam, bromazepam, flunitrazepam].

Thermospray liquid chromatography mass spectrometric method is described for the determination of the benzodiazepines (Nitrazepam, Estazolam, Bromazepam, Flunitrazepam). Reversed-phase liquid chromatography was performed using a 15 cm Shim-pack CLC-ODS (Shimadzu) column, with acetonitrile-water (40:60) + 0.1 M ammonium acetate as mobile phase, at a flow rate of 1.0 ml/min. The temperature of the vaporizer, block and TH of the source block were 166, 270 and 275 degrees C, respectively. Positive ion thermospray mass spectra by thermospray ionization (TSP ionization) mode or thermospray on filament ionization (filament-on ionization) mode were obtained. Formation of the MH+ ion was observed as a base peak under TSP ionization and filament-on ionization conditions and fragment ions were very few. On both ionization mode, peaks representing nitrazepam as MH+ at m/z 282 at a retention time (R.T.) of 6.4 min, from estazolam as MH+ at m/z 295 at an R.T. of 6.4 min, from bromazepam as MH+ at m/z 316 at an R.T. of 4.5 min and from flunitrazepam as MH+ at m/z 314 at an R.T. of 8.8 min. The detection limit for all the benzodiazepines under investigation was less than 0.5 ng (S/N = 9.4 +/- 4.6) using selected ion monitoring.

Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol-high-pH buffer eluent. Changes in selectivity with the age of the stationary phase.

The long-term reproducibility of the separation of basic drugs by high-performance liquid chromatography using a silica column with a methanol-high-pH eluent was examined. The relative capacity factors of specific tertiary amines changed systematically compared with the other drugs on storage of the columns for 1 year. The changes were enhanced on storing the column in mobile phase and were accelerated by prolonged washing with water. A comparison of freshly packed columns suggested that similar changes also occurred with the dry silica on storage. These effects may be a contributor to previously observed batch-to-batch differences in the silica.

[Spectrofluorometric analysis of select 1,4-benzodiazepine derivatives]. / Spektrofluorymetryczne oznaczanie, wybranych pochodnych 1,4-benzodiazepiny.

The optimal conditions for spectrofluorimetric determination of oxazepam and nitrazepam in substance and pharmaceutical preparations were determined. Simultaneous comparison with spectrophotometric method was performed.