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In this work, we propose an innovative high throughput (HT) apparatus using the bar adsorptive microextraction (BAμE) technique, which enables the simultaneous enrichment of up to 100 samples. This novel configuration was combined with microliquid desorption and high-performance liquid chromatography-diode array detection to monitor trace levels of eight benzodiazepines (diazepam, prazepam, bromazepam, oxazepam, lorazepam, alprazolam, temazepam and loflazepate) in biological samples. The proposed methodology was fully developed, optimized and validated, resulting in suitable intraday and interday precision (RSD ≤ 15%), with recovery yields ranging from 33.0% to 104.5%. The lower limits of quantification were between 20.0 and 100.0 µg L−1, using 1.0 mL of urine and 0.5 mL of plasma or serum samples. The application of the proposed methodology to real matrices resulted in average sample preparation time of around 2 min per sample, demonstrating that it is user-friendly, cost-effective and a rapid decision-making tool, whenever large number of samples are involved.

The analysis of the class of benzodiazepines (BDZs) is of a great importance on the levels of forensic, clinical and pharmaceutical uses. The study is focusing on the separation of three BDZs which are diazepam, clonazepam, and bromazepam using a green micellar mobile phase. It was found that the non-ionic surfactant Brij-35 can replace the organic solvent, as it decreases the retention time significantly and contributes to environmentally friendly or Eco-friendly separation procedures. The study was achieved on monolithic C18 column. Combination of sodium dodecyl sulphate (SDS): polyoxyethylene(23)lauryl ether (Brij-35) in a ratio of 50:50% v/v, was used as a mobile phase. The UV detection was set at 240 nm at a flow rate of 1.0 mL/min. The separation of diazepam, clonazepam and bromazepam was achieved in less than ten minutes even though there is no organic modifier in the mobile phase. A detailed study for the chromatographic parameters in this micellar system is carried out, in addition to a comparison with that achieved with other systems containing a single surfactant with low percent of organic modifier or other traditional mobile phase systems. Additionally, assessment of the greenness of the method is carried out using analytical eco-scale and the developed micellar HPLC method was proved to be an excellent green method for the analysis of the three drugs.

In Forensic Toxicology it is sometimes impossible to obtain a valid blood sample to perform toxicological analysis due to several factors like advanced state of decomposition, severe burns, bleed to death…. Pericardial Fluid has already been studied during the last years and has been proposed as a valid specimen for toxicological tests. Over the years, the consumption of benzodiazepines spread among the drug dependent population and became noticeable in drug facilitated assault cases and road accidents. Improvement of the analytical methodology required for detecting the presence of these drugs in biological samples is of great importance for forensic toxicology, in order to correctly diagnose an exposure or a poisoning. In this study, 9 benzodiazepines (diazepam, nordiazepam, midazolam, bromazepam, oxazepam, temazepam, lorazepam, clonazepam and alprazolam) have been determined in pericardial fluid. For this purpose a solid phase extraction (SPE) was carried out using Bond Elut Certify cartridges. After the derivatization of six of the nine benzodiazepines, gas chromatography coupled to a selective mass detector was used as the technique for the separation of the analytes. The method developed was fully validated for the 9 analytes and was applied to real samples of pericardial fluid received at the Forensic Toxicology Service of the University of Santiago de Compostela. Finally, they were compared with blood results looking for the existence of a possible correlation between both biological samples.

In this study, aqueous acidities of several benzodiazepine (BZD) drugs including Oxazepam, Clonazepam, Nitrazepam, Flunitrazepam, Diazepam, Alprazolam, Estazolam, Temazepam, Medazepam and Bromazepam were successfully computed using two common thermodynamic cycles. To that, calculated gas phase Gibbs free energies at PBE1PBE/6-311+G(d,p) level of theory and computed solvation Gibbs free energies at (CPCM/IEF-PCM: UAKS, UAHF and UA0)/HF/6-31+G(d) levels of theory were combined. The most accurate pKa values were obtained with thermodynamic cycle involving water and using CPCM-UA0 solvation model with MAD = 0.4. Moreover, for the first time, aqueous pKa value of Halazepam was predicted using the best method equal to 3.1.

In this paper, novel univariate and multivariate regression model updating methods were developed and validated for the simultaneous determination of ternary mixture of bromazepam, diazepam and clonazepam. The univariate methods used are the mean centering of ratio spectrophotometric method and iso-absorptive point coupled with ratio-subtraction and ratio difference spectrophotometric method. Linearity, ranges, precision and accuracy of these methods were determined. The multivariate methods adopted are the principal component regression and partial least-squares. These models were estimated using nineteen mixtures as calibration set and six mixtures as validation set. The minimum root square error of predication were 0.1215 and 0.0568 for BMZ, 0.0598 and 0.0712 for DIZ and 0.0867 and 0.0753 for CNP by PLS and PCR respectively. PCR and PLS methods were successfully applied for the analysis of BMZ, DIZ and CNP in their dosage forms. Content uniformity testing of the studied pharmaceutical tablets by PLS and PCR was also determined. MCR, IPRSRD and PLS model updating enabled the determination of BMZ, DIZ and CNP in spiked human urine. Moreover, PLS model updating was applied to drug-dissolution rate testing of BMZ and DIZ in their commercial tablets. The obtained results were statistically compared with those obtained by reported methods giving a conclusion that there is no significant difference regarding accuracy and precision.

Dispersive liquid-liquid microextraction with and without ultrasound assistance (DLLME, UA-DLLME) and microextraction with packed sorbent (MEPS) methods for the extraction and determination of eight different benzodiazepines (BDZ) (chlordiazepoxide, flurazepam, bromazepam, oxazepam, lorazepam, clobazam, clonazepam, and flunitrazepam) in three commercial non-alcoholic and light alcoholic beverages were optimized and compared. Benzodiazepines are frequently used for their extensive diffusion and strong numbing effect in drug-facilitated crimes (DFC). The tiny small amount of sample required for DLLME and MEPS extraction makes them very suitable for specimens collected at the crime scene of DFCs. Microextraction techniques are of increasing interest thanks to their accordance to green analytical chemistry (GAC) guidelines providing good recovery values. Ultrasound assistance (UA-DLLME) was used to investigate whether this type of energy can improve the recoveries of the analytes. Analyses of the extracts were performed with reverse-phase capillary high-performance liquid chromatography with UV detection (HPLC − UV), thanks to low environmental impact, robustness, diffusion, and affordability. Recovery percentages at three different concentrations in the three beverages were between 14.30% and 103.28% with intraday and interday RSD lower than ± 2.78%. The same samples were extracted using a MEPS protocol, and the results were compared with those obtained with DLLME. MEPS gave recoveries between 20.90% and 101.88% for all matrices showing a better performance than DLLME at higher concentrations, though lower recoveries were observed with diluted samples.

The study aims the development and validation of a LC–MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long-term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1–2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam-D5) and sonicated up to two hours. The solution was then direct injected in the LC–MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7-aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water.

Bromazepam, citric acid and their binary mixtures were analysed by Differential Scanning Calorimetry. For the bromazepam molar fraction 0.7, the mixture presents a eutectic which suggests an increase in solubility at this composition. The mixture’s greater solubility has been confirmed by a series of dissolution tests. Fourier Transform Infrared spectroscopy and X-ray diffraction tests have been performed in order to investigate the interactions between citric acid and bromazepam.

4-methylpentedrone (4-MPD) is a new psychoactive substance (NPS) belonging to the cathinone class. We report an original case of death mainly involving 4-MPD, along with cocaine, sildenafil, bromazepam and nevirapine. The investigation data and the autopsy findings indicated fatal intoxication in a chemsex context (drug use during sex). 4-MPD concentrations were determined in peripheral blood (1285 ng/mL), cardiac blood (1128 ng/mL), urine (>10,000 ng/mL), bile (1187 ng/mL) and vitreous humor (734 and 875 ng/mL in left and right samples, respectively) by gas chromatography (GC) coupled to tandem mass spectrometry. 4-MPD metabolites were explored by GC coupled to high resolution mass spectrometry. Due to the paucity of data concerning 4-MPD, its use and effects were gathered from online user testimonies. This case illustrates the toxicity of this infrequent pentedrone derivate and confirms the significant overdose risk associated with chemsex.

Four simple, sensitive and selective stability indicating spectrophotometric methods are presented for quantitative determination of the benzodiazepine drug; bromazepam (BMZ) and one of its reported potential impurities and degradation product; 2-(2-amino-5-bromobenzoyl) pyridine (ABP) in methanol. Method A, is isoabsorptive point coupled with D0 method, where good linearity was obtained by measuring the absorbance of BMZ at 264 nm (Aiso) in the concentration range of 2–25 μg mL−1, and the absorbance of ABP at its λmax 396 nm in concentration range of 0.5–24 μg mL−1. Method B, is ratio subtraction; the absorbance was measured at 233 nm for BMZ using 20 μg mL−1 of ABP, while ABP was determined directly at its λmax 396 nm using methanol as a solvent. Method C, was based on measuring the total peak amplitude of the first derivative of the ratio spectra (DD1) of BMZ from 301 to 326 nm using 10 μg mL−1 of ABP as a divisor and determination of ABP at peak amplitude of 293 nm using 5 μg mL−1 of BMZ as a divisor. In method D, ratio difference method, good linearity was achieved for determination of BMZ and ABP by measuring the differences between the amplitudes of ratio spectra at 312 nm and 274 nm and differences between the amplitudes of ratio spectra at 274 nm and 312 nm, respectively. The stability of BMZ was investigated under different ICH recommended forced degradation conditions. The suggested methods were then successfully applied for determination of BMZ in its pharmaceutical formulations.

Although information exists regarding the rate of benzodiazepines (BZDs) use in different countries, little information is available concerning the BZDs consumption in Morocco. To describe prescription rate in Morocco, a retrospective descriptive analysis of BZDs and their agonists use with the instituteIQIVIA database was performed during the period 2004-2017. The obtained data provide a dynamic approach to total BZDs consumption using an annual collection of sales data in Morocco and were expressed in terms of daily defined doses/ 1000 inhabitants / day. Data analysis showed that the major BZDs sold in Morocco were Alprazolam, Bromazepam, Nordazepam, Lorazepam, Parazepam, Diazepam and two benzodiazepine agonists, Zolpidem and Zopiclone. The Bromazepam was the molecule the most consumed during 2004-2016. In 2017, Alprazolam was the most consumed followed by Bromazepam, Nordazepam, Zolpidem, Lorazepam, Parazepam, Diazepam, Dipotassium clorazepate, Dipotassium Clorazypate and Zopiclone with 0.94, 0.91, 0.6, 0.55, 0.45, 0.32, 0.18, 0.18, 0.07 and 0.05 daily defined doses/ 1000 inhabitants / day respectively. The total amount consumed each year for all BZDs and their agonists in Morocco was 2.69, 2.77, 3, 3.17, 3.32, 3.54, 3.61, 3.81, 4.06, 4.30, 4.06, 3.94, 3.78 and 3.66 daily defined doses/ 1000 inhabitants / day, respectively during 2004-2017. To our knowledge, this is the first study that describes the consumption of BZDs and their agonists (Zolpidem, Zopiclone) in Morocco. This data may help the analytical toxicology laboratory and health organizations operating in the field of analytical biochemistry to develop specific BZDs quantification and detection methods needed for the Moroccan population.

Objectives Description of a fatal intoxication due to Kratom, including results of mitragynine quantitative analysis. Case history A 36-year-old man with a history of heroin, cocaine and drug (oxazepam and bromazepam) use was found dead in his bed. A plastic bag containing 50 g of an unidentified green powder was found near the corpse. The results of the autopsy revealed asphyxia with pulmonary edema, pancreatitis and petechial gastritis. Methods Toxicological screening was performed for ethanol, drugs and drugs of abuse by LC-MS/MS and GC-MS, in biological samples (peripheral blood, urine, gastric fluid, bile) and powder. Furthermore, compounds identified by screening were quantified in blood by LC-MS/MS. Results Mitragynine was identified in blood, urine and powder. A lethal concentration of mitragynine and oxazepam was found, associated with cocaine and central nervous system depressants (methadone, morphine, bromazepam). The postmortem peripheral blood concentrations are presented in Table 1 . Conclusion Kratom (Mitragyna speciosa), a plant traditionally used in Southeast Asia for its medicinal value in treating diarrhoea, fever or pain, is now popular in Western countries as a recreational drug or as a self-treatment for opioid dependence. Mitragynine, the main alkaloid found in Kratom, is an opiate mu receptor agonist and kappa receptor antagonist. It is described to produce stimulant effects at low doses, and sedative and/or euphoric effects at high doses. An increasing number of deaths is reported after Kratom use, especially in association with other central nervous system depressant drugs, with mitragynine concentrations ranging from 16 to 4800 ng/mL [1] . In this case, mitragynine and oxazepam were found in lethal concentration and were associated with other depressants of the central nervous system (methadone, morphine, bromazepam). Corroborated by the asphyxia syndrome observed at autopsy, all these data were in favor of the causal role of Kratom in the death.

Forensic toxicological drug analyses of human specimens are usually performed immediately after autopsy or on frozen preserved tissues. Occasionally, cases require analysis of drugs from tissues fixed in formalin solution. To improve the estimation of the level of drug in tissues following formalin fixation, we studied drug concentrations in human tissues, liver and kidney, that were collected from a drug-positive autopsy case. Parts of tissues were preserved in formalin solution for 1, 3, 6 and 13 months. Tissues obtained before and after preservation, along with tissue-exposed fixatives, were assayed using gas chromatography–mass spectrometry; all of the samples were assayed for the presence of drugs and changes in the drug concentrations both before and after preservation in formalin. Concentrations of assayed drugs decreased upon fixation in formalin; levels of these drugs did not necessarily show further decreases during subsequent storage in fixative, up to 13 months. Distinct trends in drug levels were found in liver and kidney. In liver, the levels of chlorpromazine, levomepromazine, and promethazine decreased to 23–39% at 1 month after preservation; all 3 of these drugs were detected at all tested time points of preservation. Bromazepam was not detected at 13 months after preservation. Milnacipran was the most unstable after preservation in formalin solution among all of the assayed drugs. In kidney, all assayed drugs exhibited reduced stability during preservation compared to levels in liver. Methamphetamine and methylenedioxymethamphetamine were not detected in any time points of tissues. The proportions of the drugs that remained within the tissues differed between liver and kidney. Also, S-oxide compounds of chlorpromazine and levomepromazine, which were not observed before preservation, were detected in fixed liver tissues and their fixatives at 3, 6 and 13 months of preservation. These results suggest that analyses in formalin-fixed tissues need to include analysis of various organ-tissues and their fixatives at multiple time points for the duration of preservation. These analyses should include detection of chemical degradation/denaturation products, such as S-oxides of chlorpromazine and levomepromazine.

Intoxication with calcium channel inhibitory drugs is rare but mortality rates reach 10 %. We report the case of a 5-year-old girl who had ingested five 240-mg tablets of extended-release verapamil (VLP) and a tablet of bromazepam. Thirty hours after the ingestion she had a vasoplegic shock, heart conduction disorders, and metabolic complications. She was treated in pediatric intensive care with continuous epinephrine and insulin and recovered completely 60h after the ingestion. This case underlines the danger of calcium channel blocker overdose, increased by the extended-release mechanism: the drug effect is to slow down gastric motility - which explains the latency of the symptoms - but this also increases the drug absorption, inducing a vicious circle. These mechanisms in calcium blocker intoxication need to be kept in mind in order to decrease the mortality of such accidents.

The use of synthetic cannabinoids is an emerging problem in contemporary society, and the detection techniques currently used do not meet the requirements of a drug test. We report herein an inexpensive, in situ method for the detection of the synthetic cannabinoid AB-001 in herbal samples. The method exploits a colorimetric kit consisting in a 96 plate well containing a bench stable modified Ehrlich reagent adsorbed on silica. The colour derived by the interaction of AB-001 with the detection matrix is read on a RGB (Red, Green, and Blue) base by using the camera of a smartphone, thus making the method accessible to everyone. A calibration curve of the analyte in the range 1–200 μg was realized, with a MQL (method quantitation limit) of 1 μg and positive recovery tests from samples of tobacco and damiana spiked with different amounts of AB-001. The method presents selectivity for AB-001 with respect to other indole-based cannabinoids (e.g. JWH-018, JWH-302, whose response is ca 5% of the one produced by the same concentration of AB-001) and other common drugs (e.g. acetaminophen, ascorbic acid, bromazepam, fluoxetine), these last not interfering up to a 100:1 drug:AB-001 ratio. This new colorimetric approach would be considered a category C screening test, and therefore recommended as part of a comprehensive analytical scheme for the identification of AB-001.

Aims To investigate the use of biochemical markers in combination for the postmortem diagnosis of hypothermia. To estimate physiological values for lactate in postmortem samples by an internal study. Case report Case #1: a 31-year-old female was found dead and naked. She had been undergoing treatment for thyroid disease. Case #2: a 55-year-old female was found dead in the wild. She had been undergoing psychiatric treatment for depression. Case #3: a 35-year-old male was found naked in a river. Alcoholic. Case #4: a 60-year-old female was found on her back in a field after an accident on the public highway. She had multiple sclerosis. Case #5: a 21-year-old male was found dead next to the highway in a sleeping bag. Case #6: a 77-year-old male was found dead hang on a mouldboard plough five days after his disappearance. Case #7: a 49-year-old female was found dead in the bush after hospitalization for depression. Methods Analysis of the biochemical markers (glucose, lactate, HbA1c and TSH) was conducted on vitreous humor, peripheral blood by electrochemiluminescence Roche Cobas® 6000. Results All values were Table 1 . An internal study was performed on 160 postmortem samples from different contexts of death (homicide, suicide, injuries) to propose physiological values for lactate in vitreous humor, which are unknown. Contrary to the other parameters whose physiological values were Conclusion To identify the cause of death, different parameters must be considered. The combination of results for biochemical markers and other molecules such as acetone and isopropanol are the proof of ketoacidosis [1] . In addition, the effect of high levels of bromazepam was discussed in case #1. Furthermore, the differences between cases of diabetic ketoacidosis by hyperglycemia or insulin overdose were reported [2] . The negative lactate values in these cases were one of the most important factors in identifying the cause of death. In fact, the collapse of lactate values in vitreous humor might be a pathognomonic sign of hypothermia and could establish a postmortem diagnosis.

Objective The most common mental diseases in pregnant women are depression and anxiety disorders (estimated prevalence 6.5–12.9% and 15.6%, respectively [1] , [2] ). The drugs used in their treatment (antidepressants and benzodiazepines) can produce serious obstetric complications, as well as deleterious effects in fetal development and later in life [1] . The aim of this study was to compare maternal hair and meconium for detecting in utero drug exposure to benzodiazepines and antidepressants. Methods Maternal hair and meconium samples were collected after delivery from 145 mother–newborn dyads. Samples were analyzed for the determination of benzodiazepines (alprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, and zolpidem), antidepressants (amitriptyline, clomipramine, fluoxetine, paroxetine, venlafaxine, citalopram), and 6 metabolites by LC-MS/MS. Hair samples were segmented in 3 segments corresponding with the 3 trimesters (n = 99), but if the amount of hair was not enough a 8-cm segment was employed (n = 46). Maternal hair results (LOQ = 5–20 pg/mg) and meconium Results (LOQ = 5–20 ng/g) were compared. Results Forty-nine hair samples (33.8%) tested positive, 23 (15.9%) for benzodiazepines (19 segmented and 4 non-segmented) and 33 (22.8%) for antidepressants (23 segmented and 10 non-segmented). Specifically, the most frequently detected compounds in hair were nordiazepam (n = 13, > LOD-224.6 pg/mg), citalopram (n = 11, 30.2–7166.1 pg/mg), diazepam (n = 10, 6.6–879.5 pg/mg), amitriptyline (n = 8, 11.3–556.5 pg/mg), and venlafaxine (n = 8, 14.4–659.1 pg/mg). Twenty-eight meconium samples (19.3%) tested positive, 24 (16.6%) for benzodiazepines and 8 (5.5%) for antidepressants. The most frequently detect compounds in meconium were nordiazepam (n = 16, > LOD-125.1 ng/g), diazepam (n = 12, > LOD-68.6 ng/g), citalopram (n = 5, > LOD-2729.5 ng/g) and its metabolite desmethylcitalopram (n = 4, > LOD-1286.1 ng/g), and alprazolam (n = 3, 5.7–42.2 ng/g). The overall agreement between maternal hair and meconium results was 66.9%, but this value increased to 70.2% when only the hair results corresponding with the third trimester were compared with meconium. This good agreement was due to the high percentage of negative results (n = 84), but if only positive results were analyzed the percentage decreased to 19.7% and 42.6%, respectively. A better correlation was found between positive results in both specimens for benzodiazepines (39.1%) than antidepressants (18.2%). Moreover, when hair samples were positive in the three trimesters (n = 15), meconium was positive in 7 cases, and in the remaining cases the concentration in hair was decreasing along pregnancy, except in one case. Surprisingly, when benzodiazepines were administrated during delivery (n = 15), in 6 cases meconium tested positive at low concentrations (1.1–8.6 ng/g). Conclusion Maternal hair was more sensitive than meconium to detect consumption during pregnancy, while meconium was able to detect consumption during the third trimester and also during delivery.

In this study, an analytical methodology was developed for the determination of psycho-active drugs in the treated effluent of the University Hospital at the Federal University of Santa Maria, RS – Brazil. Samples were collected from point A (Emergency) and point B (General effluent). The adopted methodology included a pre-concentration procedure involving the use of solid phase extraction and determination by liquid chromatography coupled to mass spectrometry. The limit of detection for bromazepam and lorazepam was 4.9 ± 1.0 ng L −1 and, for carbamazepine, clonazepam and diazepam was 6.1 ± 1.5 ng L −1 . The limit of quantification was 30.0 ± 1.1 ng L −1 , for bromazepam, clonazepam and lorazepam; for carbamazepine was 50.0 ± 1.8 ng L −1 and was 40.0 ± 1.0 ng L −1 for diazepam. The mean concentrations in the Emergency and General effluent treated currents were as follows: for bromazepam, 195 ± 6 ng L −1 and 137 ± 7 ng L −1 ; for carbamazepine, 590 ± 6 ng L −1 and 461 ± 10 ng L −1 ; for diazepam, 645 ± 1 ng L −1 and 571 ± 10 ng L −1 ; for lorazepam, 96 ± 7 ng L −1 and 42 ± 4 ng L −1 ; and for clonazepam, 134 ± 10 ng L −1 and 57 ± 10 ng L −1 . A preliminary risk assessment was conducted: carbamazepine and diazepam require considerable attention owing to their environmental toxicity. The occurrence of these psychoactive-drugs and the environmental risks that they pose demonstrated the need for a more efficient treatment system. As far we are aware, there have been no comparable studies to this on the hazards of hospital effluents in Brazil, and very few that have carried out a risk assessment of psycho-active drugs in hospital effluent in general.

A standard additions-dilution solid-state electrochemical method for the determination of psychoactive 1,4-benzodiazepine and antidepressants drugs used as adulterants in commercial slimming herbal formulations is described and compared with conventional standard addition method. The proposed method, based on the voltammetry of microparticles approach, permits quantify, via standard additions methodology, 1,4-benzodiazepine and antidepressants drugs in phytotherapeutic formulations with no need of sample dissolution using dilution with a reference electroactive compound. The method was used to measure 1,4-benzobenzodiazepines (clonazepam, flurazepam, alprazolam, midazolam, bromazepam, chlordiazepoxide, lorazepam and diazepam) and antidepressants (bupropion, sertraline, paroxetine and fluoxetine) in slimming formulations that have been commercialized in Brazil.

The purpose of this work was to develop a rapid, sensitive and validated HPLC method for the separation and analysis of a Bromazepam, Medazepam and Midazolam mixture. The three benzodiazepine compounds were separated on a reversed-phase C18 column at 50 °C using a mobile phase containing 25% acetonitrile, 45% methanol and 30% ammonium acetate (0.05 M). The pH was adjusted to pH=9 by the addition of ammonia solution (35%, w/w). The samples were detected using a UV detector at 240 nm. The validation study of the method included the effect of temperature, flow rate, ratio of the components of the mobile phase and the pH of the mobile phase on the efficiency of separation. The linear range of Bromazepam and Midazolam was between 0.12 and 0.18 mg/mL, while that of Medazepam was between 0.08 and 0.12 mg/mL. The relative standard deviation for precision was less than 2%. The linearity, selectivity, accuracy and robustness of the developed method showed acceptable values. The method was applied to the analysis of the samples of raw material of the three compounds under study, and the percentage of recoveries was 99.89%±1.06. It was also applied to the analysis of samples of pharmaceutical preparations of those compounds and spiked serum samples. Recoveries from serum samples ranged between 91.5% and 99.0%. The developed method is suitable for quality control of Bromazepam, Medazepam and Midazolam in their mixtures and in pharmaceutical preparations (tablets, capsules, ampoules). It can also be used to determine their concentrations in serum. Keywords: Benzodiazepine, Bromazepam, Medazepam, Midazolam, HPLC, Serum

A simple and rapid LC/MS method with direct injection analysis was developed and validated for the identification and quantification of ten benzodiazepines (flunitrazepam, nordiazepam, diazepam, 7-aminoflunitrazepam, flurazepam, bromazepam, midazolam, alprazolam, temazepam and oxazepam) in human urine using diazepam-d5 as internal standard (IS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as diluted urine samples were directly injected into LC/MS system. Electrospray ionization in positive mode using selected ion monitoring was chosen for the identification and quantification of the analytes. The linear range was 50-1000 ng/mL for each analyte, with square correlation coefficient (r(2))≥0.981. Interday and intraday errors were found to be ≤5.72%. The LC/MS method was applied at ten real samples found initially to be positive and negative, using immunoassay technique. Finally the results were confirmed with GC/MS. The method demonstrates simplicity and fast sample preparation, accuracy and specificity of the analytes which make it suitable for replacement of immunoassay screening in urine avoiding thus false negative/false positive results. Using this method, laboratories may overcome the problem of high cost instrumentation such as LC-MS/MS by providing similar sensitivity and specificity with other methods.

International audience; The antipsychotic cyamemazine is a potent serotonin 5-HT(2A) receptor (5-HT(2AR)) antagonist. A positron emission tomography (PET) study in human patients showed that therapeutic doses of cyamemazine produce near saturation of 5-HT(2AR) occupancy in the frontal cortex, whereas dopamine D(2) occupancy remained below the level for motor side effects observed with typical antipsychotics. Recently, numerous studies have revealed the involvement of 5-HT(2AR) in the pathophysiology of anxiety and a double-blind, randomized clinical trial showed similar efficacy of cyamemazine and bromazepam in reducing the anxiety associated with benzodiazepine withdrawal. Therefore, we reviewed the above articles about 5-HT(2AR) and anxiety in order to understand better the anxiolytic mechanisms of cyamemazine in benzodiazepine withdrawal. The 5-HT(2AR) is the most abundant serotonin receptor subtype in the cortex. Non-pharmacological studies with antisense oligodeoxynucleotides and genetically modified mice clearly showed that cortical 5-HT(2AR) signaling positively modulates anxiety-like behavior. With a few exceptions, most other studies reviewed here further support this view. Therefore, the anxiolytic efficacy of cyamemazine in benzodiazepine withdrawal can be due to a 5-HT(2AR) antagonistic activity at the cortical level.

Phenazepam [7-bromo-5-(2-chlorophenyl)-1, 3-dihydro-2H-1,4-benzodiazepine-2-one], sometimes called fenazepam (Fig. 1A), is part of the 1,4-benzodiazepine group of benzodiazepines along with drugs such as diazepam, nordazepam, oxazepam and temazepam. It is however one of very few of the 1,4-benzodiazepine group to contain a bromine atom, along with the active form of gidazepam, another soviet-developed benzodiazepine5 and bromazepam. As with other benzodiazepines, it is used clinically for its anxiolytic, anticonvulsant, muscle-relaxing and sedating properties,6 and is available as 0.5-mg and 1-mg tablets,6 injectable solutions (0.1%, 0.3%) or transdermal patches (Phenopercuten).7 Clinically the dose of phenazepam that is given depends on what condition is being treated but does not exceed 10 mg day−1, and is usually not more than 5 mg day−1. Illicitly, phenazepam has been reported being sold as a powder, tablets, in the USA, spiked in lysergic acid diethylamide (LSD) mimic blotters8 and 9 and in New Zealand phenazepam has been found in a synthetic cannabinoid mix called ‘Kronic’.10 Reported recreational doses of phenazepam are around 2–10 mg, but sometimes more.8

Benzodiazepine derivatives are prescribed in large quantities globally and are potentially new emerging environmental contaminants. Unfortunately, a dearth of data exists concerning occurrence, persistence and fate in the environment. This paper redresses this by reviewing existing literature, assessing the occurrence of selected benzodiazepine anxiolytics (diazepam, oxazepam and bromazepam) in wastewater influent and effluent and surface water from Slovenia, evaluating their removal during water treatment and identifying the transformation products formed during water treatment. Their occurrence was monitored in hospital effluent, river water and in wastewater treatment plant influent and effluent. The study reveals the presence of benzodiazepine derivatives in all samples with the highest amounts in hospital effluents: 111 ng L −1 , 158 ng L −1 and 72 ng L −1 for diazepam, bromazepam and oxazepam, respectively. Removal efficiencies with respect to biological treatment of diazepam were 16–18% (oxic), 18–32% (anoxic → oxic), 53–76% (oxic → anoxic) and 83% (oxic → anoxic → oxic → anoxic cascade bioreactors), while the removal oxazepam was 20–24% under anoxic conditions. Coupled biological and photochemical treatment followed by the adsorption to activated carbon resulted in a removal efficiency of 99.99%. Results reveal the recalcitrant nature of benzodiazepine derivatives and suggest that only combinational treatment is sufficient to remove them. In addition, eight novel diazepam and four novel oxazepam transformation products are reported.

There is growing interest in zebrafish as a model organism in behavioral pharmacology research. Several anxiety behaviors have been characterized in zebrafish, but the effect of anxiolytic drugs on these parameters has been scarcely studied. The purpose of this work was to assess the predictive validity of acute treatment with anxiolytic drugs on behavioral parameters of anxiety. In the first task we simultaneously observed behavior of adult zebrafish on four parameters: height in the tank, locomotion, color, and shoal cohesion. The second task was the assessment of light/dark preference for 5min. The benzodiazepines clonazepam, bromazepam, diazepam, and a moderate dose of ethanol significantly reduced shoal cohesion. Buspirone specifically increased zebrafish exploration of higher portions of the tank. In the light/dark task, all benzodiazepines, buspirone, and ethanol increased time spent in the light compartment. After treatment with anxiolytics, fish typically spent more than 60s and rarely less than 40s in the light compartment whereas controls (n=45) spent 33.3±14.4s and always less than 60s in the light compartment. Propranolol had no clear effects in these tasks. These results suggest that light/dark preference in zebrafish is a practical, low-cost, and sensitive screening task for anxiolytic drugs. Height in the tank and shoal cohesion seem to be useful behavioral parameters in discriminating different classes of these drugs.

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.

Benzodiazepines have wide-spread used in pharmacotherapy for their anxiolytic, myorelaxant, hypnotic, amnesic and anticonvulsive properties. Despite benzodiazepines are used in clinics over 50 years, they have not been surprisingly tested for capability to induce major drug-metabolizing cytochromes P450. In the current study, we have examined the potency of Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Medazepam, Midazolam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam to induce CYP1A2 and CYP3A4 in primary cultures of human hepatocytes. Benzodiazepines were tested in therapeutic concentrations and in concentrations corresponding to their plasma levels in intoxicated patients. We found weak but significant induction of CYP3A4 mRNA by Midazolam and Medazepam, while other benzodiazepines did not induce CYP3A4 expression. None of the tested compounds induced CYP1A2 mRNA in three independent human hepatocytes cultures. In addition, employing gene reporter assays with transiently transfected hepatocarcinoma cells, we found that tested benzodiazepines did not activate aryl hydrocarbon receptor (AhR), whereas Midazolam and Medazepam slightly activated pregnane X receptor (PXR). Consistently, two-hybrid mammalian assay using hybrid fusion plasmids GAL4-PXR ligand-binding domain (LBD) and VP16-SRC-1-receptor-interacting domain (RID) confirmed PXR activation by Midazolam and Medazepam. In conclusion, Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam can be considered as safe drugs in term of their inability to induce PXR- and AhR-dependent cytochrome P450 enzymes CYP1A2 and CYP3A4. Medazepam and Midazolam slightly activated pregnane X receptor and displayed weak potency to induce CYP3A4 mRNA in human hepatocytes.

The goal of the present study was to explore the dynamics of the gamma band using the coherence of the quantitative electroencephalography (qEEG) in a sensorimotor integration task and the influence of the neuromodulator bromazepam on the band behavior. Our hypothesis is that the needs of the typewriting task will demand the coupling of different brain areas, and that the gamma band will promote the binding of information. It is also expected that the neuromodulator will modify this coupling. The sample was composed of 39 healthy subjects. We used a randomized double-blind design and divided subjects into three groups: placebo ( n = 13), bromazepam 3 mg ( n = 13) and bromazepam 6 mg ( n = 13). The two-way ANOVA analysis demonstrated a main effect for the factors condition (i.e., C4–CZ electrode pair) and moment (i.e., C3–CZ, C3–C4 and C4–CZ pairs of electrodes). We propose that the gamma band plays an important role in the binding among several brain areas in complex motor tasks and that each hemisphere is influenced in a different manner by the neuromodulator.

The aim of this study was to investigate the influence of bromazepam on EEG and the motor learning process when healthy subjects were submitted to a typewriting task. We investigated bromazepam due to its abuse by various populations and its prevalent clinical use among older individuals which are more sensitive to the negative effects of long half-life benzodiazepines. A randomized double-blind design was used with subjects divided into three groups: placebo (n=13), bromazepam 3mg (n=13) and bromazepam 6 mg (n=13). EEG data comprising theta, alpha and beta bands was recorded before, during and after the motor task. Our results showed a lower relative power value in the theta band in the Br 6 mg group when compared with PL. We also observed a reduction in relative power in the beta band in the Br 3mg and Br 6 mg when compared with PL group. These findings suggest that Br can contribute to a reduced working memory load in areas related to attention processes. On the other hand, it produces a higher cortical activation in areas associated with sensory integration. Such areas are responsible for accomplishing the motor learning task. The results are an example of the usefulness of integrating electrophysiological data, sensorimotor activity and a pharmacological approach to aid in our understanding of cerebral changes produced by external agents.

Reversed-phase liquid chromatography was coupled to a multi-detection system composed of ultraviolet (UV) detection, evaporative laser scattering detection (ELSD) and inductively coupled plasma mass spectrometry (ICP-MS). By applying the principle of post-column solvent compensation, the organic modifier content was kept constant in ELSD and ICP-MS under gradient elution. Chlorine ( 35 Cl), bromine ( 79 Br and 81 Br) and sulfur ( 34 S) were monitored in several pharmaceutical compounds. The limit of quantitation (LOQ) was 80 ng/mL for chlorine (chlorpropamide) and 2 ng/mL for bromine (bromazepam). Calibration graphs were linear from 1.0 μg/mL to 100 μg/mL for chlorpropamide ( r 2 0.990) and from 10 ng/mL to 500 ng/mL for bromazepam ( r 2 0.996). The low LOQ value for bromine allows to quantify bromine in pharmaceutical samples below the 0.05% level of the active pharmaceutical ingredient.

In this work the applicability of micellar elektrokinetic capillary chromatography (MECC) for the determination of benzodiazepines (BZD) has been studied. The applied method was used for the simultaneous separation of 8 BZDs (alprazolam, bromazepam, chlordiazepoxide, diazepam, flunitrazepam, medazepam, oxazepam, nitrazepam), and also for the study of stability in acidic medium. A fast and reliable method has been developed; using a separation buffer composed of sodium tetraborate 25 mM (pH 9.5), SDS (50 mM) and methanol (at least 12%) as an organic modifier.

To investigate the effects of bromazepam on motor performance and electroencephalographic activity (qEEG) in healthy subjects, during the process of learning a typewriting task, with a focused attention demand. A randomized double-blind model was used to allocate subjects in one of the following conditions: placebo (n=13), bromazepam 3 mg (n=13) or bromazepam 6 mg (n=13). Forty minutes after treatment administration, subjects were submitted to the motor task. EEG activity was recorded simultaneously. The analyzed variables were: number of errors and execution time, which were extracted from each block of the typewriting task, and mean relative power values in the beta band (13-35 Hz), extracted from the qEEG. A significantly lower number of typing errors was observed in both bromazepam conditions (Br 3 mg and Br 6 mg) when compared to the placebo. There was no difference between the two bromazepam conditions. For the execution time variable, a better performance was observed in the Br 3 mg condition, but with no statistical significance. The highest degree of cortical activation during the task was observed in Br 3 mg and Br 6 mg when compared to placebo. The medication's anxiolytic effect intensifies the attentional focus over predictable events occurring in reduced perceptual fields. The qEEG's accentuated response in pre-motor and primary motor areas suggests a greater effort directed to the most relevant aspects of the task. In short, the doses employed (3 and 6 mg) seem to enhance the learning of motor tasks that involve focused attention, such as typewriting.

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.

In recent years, reports of drug-facilitated crimes (DFC) have been increasing. The drugs involved are sometimes difficult to detect, because of their low dosages and the long time ellapsed between alleged DFC and blood and urine sampling. In order to detect benzodiazepines and benzodiazepine-like hypnotics, we developed an approach for hair analysis by liquid chromatography-tandem mass spectrometry using a triple stage quadrupole with an electrospray ionization (LC-ESI-MS/MS). Separation was performed on an Uptisphere ODB C18 column using a gradient of 2mM formate buffer and acetonitrile. For the 23 compounds studied, detection limits are lower than 2 pg/mg, but a specific extraction procedure is needed for 7-amino metabolites. Over a 1-year period within the city limits of Paris and three suburbs, we tested blood and urine from victims of sexual assaults, robbery and battery in which psychoactive substances were suspected of being involved. Hair was collected 4-8 weeks after the alleged DFC. Over the 128 cases studied, results of simultaneous analysis of blood, urine, and hair allowed us to conclude that 23 cases were real DFC. In 18 cases, no conclusion was possible since no hair was sampled and/or results were negative. In 56 cases, victims were previously using narcotics, cannabis, and/or a prescribed drug, according to the compounds detected in hair strands. Thirty-one cases were not DFC cases. This study indicates that the prevalence of zolpidem and clonazepam is high, followed by bromazepam, nordazepam, and midazolam. Others benzodiazepines and analogs are rare. LC-ESI-MS/MS is a good tool for toxicological investigations of DFC. Testing blood, urine, and hair by this technique may reveal drug presence, even if it was administered at a single therapeutic dose. That may be helpful to prosecute perpretators or to exclude a drug-facilitated crime.

A procedure is presented for the screening of 17 benzodiazepines and hypnotics in oral fluid after collection with the Intercept(R) device by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon, zopiclone and zolpidem). The method involves extraction of 0.5 mL of oral fluid (previously stored in the Intercept blue buffer) treated with 0.5 mL of phosphate buffer (pH 8.4) in the presence of 5 ng diazepam-d(5) used as internal standard, with 3 mL of diethyl ether/methylene chloride (50/50) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.1 to 0.2 ng/mL. Linearity is observed from the limit of quantification of each compound to 20 ng/mL (r(2)>0.99). Coefficients of variation at 2 ng/mL, measured on 6 points range from 4 to 8% for all drugs, except zopiclone (34%). Extraction recovery, measured at the same concentration was higher than 90%. Ion suppression was evaluated for each compound and was lower than 10% for all drugs except zopiclone (93%). These results were found suitable to screen for 17 benzodiazepines in oral fluid and detect them at very low concentrations, making this method suitable for monitoring subjects under the influence.

Two novel analytical methodologies using capillary electrophoresis (CE) and reversed-phase high-performance liquid chromatography (RP-HPLC) for the determination of diazepam in commercial and simulated tablet formulations were developed and compared. The CE analysis was carried out in a bare fused-silica capillary with 75 microm i.d. and total length of 50 cm (28 cm to the detector) with a buffer solution containing 20 mmol L(-1) sodium tetraborate and 20 mmolL(-1) sodium dodecylsulfate (SDS), pH 9.23. The applied voltage was 20 kV and bromazepam was used as internal standard (IS). The RP-HPLC analysis was carried out in a LiChrospher((R)) 100 RP-18 (5 microm) column with a mobile phase constituted of methanol, acetonitrile and water (45:25:30) with a flow rate of 0.8 mL/min, using acetaminophen as IS. In both cases, detection was carried out by ultraviolet (UV) absorption at 242 nm. Under the optimized conditions, the CE retention times for the standard diazepam and bromazepam (IS) were 4.08 and 3.43 min, respectively, and the retention times of the RP-HPLC analysis for the standard diazepam and acetaminophen (IS) were 4.86 and 1.58 min, respectively. The resolution and efficiency for CE were 7.4 and 1.18 x 10(5)plates/m and for RP-HPLC, 7.5 and 1.76 x 10(4) plates/m. Analytical curves of peak area versus concentration presented correlation coefficients of 0.9996 for CE and 0.9994 for RP-HPLC. The limits of detection (LOD) and quantitation (LOQ) were 4.24 and 12.85 microg/mL for CE and 1.44 and 4.36 microg/mL for RP-HPLC. Relative standard deviations (R.S.D.) were 1.62 and 0.98% for CE and RP-HPLC, respectively. The percentage recovery determined with CE was 100.27+/-1.25 and with RP-HPLC was 101.12+/-2.48. Although both methodologies were shown to be suitable for the determination of diazepam in tablets, performing in a similar manner with regards to several aspects (linearity, recovery and specificity), CE provided a faster analysis and column efficiency whereas RP-HPLC presented a superior repeatability and sensitivity.

The number of reports on drug facilitated crimes is increasing these last years. Apart from ethanol and cannabis, benzodiazepines (BZD) and analogs are the most common drugs reported to be used probably due to their amnesic and sedative properties. We have developed a rapid and sensitive method using LC–MS/MS triple stage quadrupole (TSQ) for the determination of single exposure to bromazepam (Lexomil®, 6 mg) and clonazepam (Rivotril®, 2 mg) in urine and hair of healthy volunteers. Chromatography was carried out on a Uptisphere ODB 5 μm, 2.1 mm × 150 mm column (Interchim) with a gradient of acetonitrile and formate 2 mM buffer, pH 3. Urine was extracted with Toxitube A® (Varian) and allowed the detection of bromazepam, 3-hydroxy-bromazepam, clonazepam and 7-Aminoclonazepam for more than 6 days. Head hair, collected 1 month after the exposure, was treated by incubation with Soerensen buffer pH 7.6, followed by liquid–liquid extraction with dichloromethane for common BZD. A specific pre-treatment for amino-BZD, with an incubation of 15 min at 95 °C in 0.1 N NaOH before liquid–liquid extraction with dichloromethane, gave better recoveries and repeatability. After single exposure, bromazepam was present in powdered hair at 28 pg/mg and 7-Aminoclonazepam at 22 pg/mg in the first 1-cm segment, while no clonazepam was detectable. This method was applied in two forensic cases. It allowed us to determine bromazepam in urine 3 days after the alleged offense and in cut head hair at a concentration of 6.7 pg/mg only in the 2-cm proximal segment. The other case showed the presence of clonazepam and 7-Aminoclonazepam in urine a few hours after the offense and the presence of 7-Aminoclonazepam at about 3.2 pg/mg in axillary hair 4 months later.

The aim of this study was to clarify the efficacy of antiepileptic drugs during a cluster of seizures in patients with convulsions with mild gastroenteritis (CwG). We retrospectively investigated the details of antiepileptic treatment in 110 consecutive episodes in 103 patients with CwG. The temporal course of the seizures and the use of antiepileptic drugs were investigated in each episode. Drugs were judged as effective when seizure cessation was achieved after administration of the drug. As the first drug, diazepam (DZP)/bromazepam (BZP) was effective in 38%, phenobarbital (PB) in 40%, and lidocaine (LD) in 100%. As the second drug, DZP/BZP was effective in 42%, PB in 69%, and LD in 100%. As the third drug, PB was effective in 70%. When the efficacy of the first doses of PB and LD were compared, the efficacy rate was significantly higher for LD than for PB (P = 0.047). In conclusion, LD was effective for the cessation of seizures in patients with CwG.

Newly developed solid contact ion-selective electrodes (SC-ISE) have been proposed for determining diazepam, bromazepam and clonazepam 1,4-benzodiazepines in pure forms and in pharmaceutical preparations. The electrodes are based on polyvinylchloride (PVC) membranes doped with drug-tetraphenyl borate (TPB) or drug-phosphotungestic acid (PTA) ion pair complexes as electroactive materials. The new electrodes proved highly selective with selectivity coefficients ranging from 10 −4 to 10 −6 and detection limits ranging from 0.1 to 0.63g/ml. Successful application of developed electrodes for drugs determinations in pharmaceutical preparations was obtained. Calibration graphs were linear over the ranges of 0.29–71.20, 0.79–31.62 and 0.32–31.57g/ml for diazepam, bromazepam and clonazepam, respectively with slopes of 50–65 mV per decade in the optimum pH range of 4–7. Relative average errors ≤1.72% and relative standard deviations ≤1.13% were obtained. Statistical student’s t-test and F-test showed insignificant systematic error between measured and real values and also between developed ion-selective electrodes methods and a standard HPLC method. © 2003 Elsevier B.V. All rights reserved.

A simple micellar liquid chromatographic (MLC) procedure is reported for the determination of several benzodiazepines in serum: bromazepam, diazepam, flunitrazepam, halazepam, medazepam, nitrazepam, oxazepam and tetrazepam. The optimization studies have been made in C(18) and C(8) columns, using solutions containing sodium dodecyl sulphate (SDS) modified with butanol or pentanol as mobile phases. The method proposed for the determination of the benzodiazepines uses a hybrid micellar mobile phase of 0.06 M SDS-5% butanol-0.01 M phosphate buffer (pH 7) at 25 degrees C, and UV detection (230 nm) in a C(18) column. The serum samples were injected directly, without any pretreatment, and eluted in less than 22 min, in accordance with their relative polarities, as indicated by their octanol-water partition coefficients. The limits of detection (ng ml(-1)) were within the ranges of 2-6 and 4-18 for aqueous and serum samples, respectively. Repeatability and intermediate precision were tested for three different concentrations of the drugs, and RSD (%) was below 10 for most of the assays. The MLC results were compared with those obtained from a conventional HPLC method using methanol-water 5:5 (v/v) which requires a previous extraction procedure.

New, simple, low cost and sensitive ion-selective electrodes are described for the determination of some 1,4-benzodiazepines in their pharmaceutical preparations as well as in biological fluids. Drug-tetraphenylborate and drug-phosphotungstate ion pairs have been prepared and used as electroactive materials. Poly(vinyl chloride) (PVC), and poly(esterurethane)s (UPCLs, UPBA, and UPDEGA) are used as the matrix. The proposed sensors give rapid Nernstian responses for 10−4–10−6 M of the abovementioned drugs in a pH range of 3–7. The membranes developed have potential stability for up to 4 weeks and show high selectivity for the investigated drugs over many interfering ions. The electrodes are used for determining trace amounts of bromazepam, clonazepam and diazepam in their pharmaceutical preparations as well as in biological fluids. For pharmaceutical preparations, the relative standard deviation (RSD) values were in the range, 1.37–4.1, 0.7–2.52 and 0.3–1.2% for bromazepam, clonazepam and diazepam, respectively using the PVC based electrodes, while for the UPBA based electrodes, the (RSD) values were in the range 0.6–3.35, 0.7–2.45 and 0.6–2.41%. For biological samples, the RSD values were 0.64–1.88, 0.37–1.8 and 1.36–3.57% for bromazepam, clonazepam and diazepam, respectively. Comparison of the results obtained using the proposed electrodes with those found using a (HPLC) reference method showed that the ion-selective electrode technique is sensitive, reliable and can be used with very good accuracy and high % recovery without pretreatment procedures of the samples to minimize interfering matrix effects.

Time-of-flight mass spectrometry (TOF-MS) offers new perspectives for forensic toxicology. Qualitative and quantitative analyses of a mixture of three selected benzodiazepines (diazepam, nordazepam and bromazepam) were used to compare gas chromatography (GC-TOF-MS, quadrupole GC-MS, GC-ECD) and liquid chromatography (HPLC-DAD) data. Method validation parameters like LOD, LOQ, S/N-ratios reflect the capabilities of GC-TOF-MS. Five-point calibrations for bromazepam in human peripheral blood (50, 100, 160, 200, 300 ng/ml) using medazepam as internal standard (1000 ng/ml) were performed. The calibrations using GC-TOF-MS (using the fragments of m/z 236 and 288), GC-ECD (dual system) and HPLC-DAD (at 235 nm) all showed correlation coefficients close or superior to 0.99. Quadrupole GC-MS data was not used in the comparison of extracted samples due to the low sensitivity in the full scan mode. Two analyses of real cases concerning bromazepam are presented. In the first case, the presence or absence of bromazepam could not be established with both HPLC-DAD and GC-ECD due to background signals. The extracted ion chromatograms and spectrum traces after the analysis with the GC-TOF-MS could clearly excluded the presence of bromazepam. The second case illustrates the quantitation of bromazepam, where both HPLC-DAD and GC-ECD were unable to give satisfactory results, again due to interfering background signals. The analyses performed on the GC-TOF-MS-system demonstrated high sensitivity and also high selectivity due to the high quality of mass spectra obtained. The advantages of GC-TOF-MS make it a promising analytical technique for forensic toxicology.

The possible antiulcer potential of bromazepam was investigated in relation to its effect on the levels of central neurotransmitters in rats. Peptic ulcer was induced by cold-restraint stress, by immobilizing the animals in open wire restraint cages placed for 2 h at 4 degrees C. Bromazepam (1 and 2 mg x kg(-1), i.p.) was given as prophylactic regimens, either as a single (2 h before ulcer induction) or repeated (twice daily for 15 days) administration. Results revealed that single (1 mg x kg(-1)) and repeated (1 and 2 mg x kg(-1)) dose regimens of bromazepam succeeded in preventing gastric ulceration, without significant effects on the protein-bound hexose content of gastric mucus. Increases in gamma-aminobutyric acid (GABA) concentrations in almost all tested brain regions were observed in bromazepam-treated groups, as compared to the control stressed group. Cortical dopamine (D) concentrations were reduced following single (2 mg x kg(-1)) as well as repeated administration of bromazepam. Similarly, norepinephrine (NE) concentrations were decreased in the cerebral cortex and thalamus/hypothalamus by repeated doses of bromazepam. Cortical 5-hydroxytryptamine (5-HT) was elevated by single (1 mg x kg(-1)) and repeated (1 mg x kg(-1)) doses of the drug. It could be concluded that bromazepam affords a good gastroprotective potential against cold-restraint stress-induced gastric ulceration and the possible mechanisms might involve an increase in the inhibitory GABA and a suppression of the stimulatory NE and D in central regions, especially the cerebral cortex and/or thalamus/hypothalamus.

The transfer of benzodiazepine derivatives (diazepam, bromazepam, alprazolam, oxazepam, nitrazepam, clonazepam, chlordiazepoxide, flunitrazepam, midazolam and lorazepam) across the water ∣ 1,2-dichloroethane interface was studied using cyclic voltammetry. The partition coefficients of ionic species of benzodiazepines, log PXH+, were calculated from the transfer potentials measured at pH

The applications of LC-API-MS in routine forensic toxicological casework were presented. This technique has been used for routine determination of several groups of drugs: opiate agonists (like morphine, codeine, dihydrocodeine and their glucuronides, methadone, buprenorphine) cocaine and its metabolites (benzoylecgonine and ecgonine methyl ester), amphetamine and other psychoactive phenethylamines, like MDMA, MDE or MDA, benzodiazepine derivatives (flunitrazepam and metabolites, triazolam, bromazepam), hallucinogens (LSD, psilocybin, psilocin) and olanzapine, A common solid-phase extraction procedure for all drugs (with exception of LSD) has been developed. Among two ionization sources, atmospheric pressure chemical ionization appeared more universal and assured generally higher sensitivity. Only in the case of very polar drugs (e.g. psilocin or psilocybin) electrospray ionization was more sensitive. LC-API-MS became a very powerful and flexible method for dedicated analyses of substances of forensic interest. The use of this technique for general, broad applicable screening depends on the establishing of interlaboratory database of standardized mass spectra.

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepam, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n=42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n=14), flunitrazepam (19-148 pg/mg, n=31), lorazepam (31-49 pg/mg, n=4) and alprazolam (0.3-1.24 ng/mg, n=2). Bromazepam, diazepam and triazolam were not detected.

A 71-year-old man was found dead in a car into which exhaust fumes had been introduced. His wife who was in the same car recovered consciousness following hospitalization. She claimed that they had both attempted suicide by taking a large number of sleeping pills. Autopsy revealed no significant external injuries or medical disorders that would have led to the husband's death. The concentrations of alcohol and carbon-monoxide hemoglobin in his whole blood were 0.26 mg/ml and

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commercially available 2 or 5 microns particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C18 reversed-phase column (condition 1: 100 x 4.6 mm I.D., particle size 2 microns, TSK gel Super-ODS: conditon 2: 100 x 4.6 mm I.D., particle size 5 microns, Hypersil ODS-C18). The mobile phase was composed of methanol-5 mM NaH2PO4 (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (condition 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 microns was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.