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A wearable glove-based sensor is a portable and practical approach for onsite detection/monitoring of a variety of chemical threats. Herein, we report a flexible and sensitive wearable sensor fabricated on the nitrile glove fingertips by stencil-printing technique. The working electrodes were modified with multiwalled carbon nanotubes (MWCNTs)/poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) for sensitive and real-time analyses of hazardous or chemical treats, as picric acid (PA) explosive, diazepam (DZ) as drug-facilitated crimes and the emerging pollutant 4-nitrophenol (4-NP). The multi-sensing platform towards PA, 4-NP, and DZ offers the ability of in-situ qualitative and quantitative analyses of powder and liquid samples. A simple sampling by touching or swiping the fingertip sensor on the sample or surface under investigation using an ionic hydrogel combined with fast voltammetry measurement provides timely point-of-need analyses. The wearable glove-based sensor uses the square wave voltammetry (SWV) technique and exhibited excellent performance to detect PA, 4-NP, and DZ, resulting in limits of detection (LOD) of 0.24 μM, 0.35 μM, 0.06 μM, respectively, in a wide concentration range (from 0.5 μM to 100 μM). Also, we obtained excellent manufacturing reproducibility with relative standard deviations (RSD) in the range of 3.65%-4.61% using 7 different wearable devices (n = 7) and stability in the range of 4.86%-6.61% using different electrodes stored for 10 days at room temperature (n = 10), demonstrating the excellent sensor-to-sensor reproducibility and stability for reliable in-field measurements. The stretchable sensor presented great mechanical robustness, supporting up to 80 bending or stretching deformation cycles without significant voltammetric changes. Collectively, our wearable glove-based sensor may be employed for analyses of chemical contaminants of concern, such as explosives (PA), drugs (DZ), and emerging pollutants (4-NP), helping in environmental and public safety control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To explore the exposure level of pesticides and veterinary drugs in an aquaculture environment and its impact on the ecological environment, this study took the aquaculture environment in Shanghai as an example, and samples of water, sediment, and inputs from 40 major aquaculture farms were collected from July to September 2022. The types and contents of pesticides and veterinary drugs were screened using high-performance liquid chromatography-electrostatic field orbital ion trap mass spectrometry, and the risk quotient (RQ) method was used to assess the ecological risk of pesticide contamination in water and sediment. The results showed that 13 drugs were screened out from 204 samples (72 samples of water, 72 samples of mud, and 60 samples of input), namely, chlorpromazine, carbendazim, thiophanate, diazepam, florfenicol, simazine, amantidine, diazepam, trimethoprim, ciprofloxacin, ofloxacin, mebendazole, and enrofloxacin. Among them, 12 species were found in water samples with concentrations ranging from 0.016 μg·L -1 to 2.084 μg·L -1 . The concentrations of seven species in the mud samples ranged from 0.018 μg·kg -1 to 23.101 μg·kg -1 . The results showed that there were four types of inputs, ranging from 1.979 μg·kg -1 to 101.940 μg·kg -1 . Seven drugs were found in both water and sediment. The risk quotient (RQ) results showed that there were some high and middle risks in both water and sediment samples of aquaculture farms, and the ecological risks of carbendazim were the highest in both water and sediment samples of aquaculture farms; the RQ values were 3.848 and 1.580, respectively, indicating high risk. It is suggested to strengthen the control and management of exogenous pesticides and veterinary drugs in aquaculture environments to protect the ecosystem health of the aquaculture environment.

Drug-impaired driver detection is a critical element of traffic safety. However, shifting drug use patterns over time and geography may limit the long-term reliability of assay-based screening tools. In this work, we compare qualitative results from the Abbott SoToxa® oral fluid (OF) screening device to Quantisal™ OF and whole blood. Our objective was to examine these three qualitative toxicological approaches, scope applicability of OF collection at the roadside, and compare them with a previous analysis of SoToxa® in Wisconsin. OF specimens were screened with the SoToxa® for six drugs or drug classes including amphetamine, benzodiazepines, cocaine, methamphetamine, opioids and tetrahydrocannabinol (THC). OF and blood specimens were collected from 106 participants. Quantisal™ OF and blood specimens were screened for drugs on ultra-performance liquid chromatography coupled to quadrupole time-of-flight high-resolution mass spectrometry (UPLC-QToF-HRMS) using a data-independent acquisition mode. UPLC-QToF-HRMS data were compared to comprehensive spectral libraries, and drugs were qualitatively identified. Drug Recognition Expert evaluations were performed, and face sheets submitted for 21 participants in this work. In general, the SoToxa® results were consistent with the combined qualitative results observed in Quantisal™ OF specimens and whole blood specimens. Limitations were uncovered for benzodiazepines, opioids and THC. The SoToxa® benzodiazepine assay has high cutoff concentrations for diazepam and clonazepam, limiting its sensitivity and positive predictive value when considering these drugs. SoToxa® opioid screening did not detect fentanyl, which is increasingly prevalent among drug users. Finally, ∆9-THC and its major metabolite 11-nor-9-carboxy-∆9-THC are lipophilic, limiting partitioning into OF. Despite these limitations, the SoToxa® instrument may be useful in assisting law enforcement with identifying individuals driving under the influence of drugs and establishing probable cause at roadside for making impaired driving arrests. Furthermore, Quantisal™ OF may be useful as screening specimens due to their ease of collection and results consistent with whole blood., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The use of vitreous humor (VH) in forensic casework has been growing in the last years due to numerous advantages. Several compounds can be evaluated in this matrix, including benzodiazepines whose determination is essential due to their great availability and potential of dependance and misuse. Postmortem toxicological analyses are required to determine the influence of benzodiazepines in deaths. However, most of the analytical methods which determine these drugs in VH are laborious and time consuming. This article describes a simple method based on protein precipitation for the determination of eight benzodiazepines in VH samples. Samples were prepared through a protein precipitation method and analyzed by liquid chromatography tandem mass spectrometry. Solvent choice and sample and solvent volumes for precipitation were optimized using chemometric approaches. The method was validated for selectivity, lower limit of quantification (LLOQ), linearity, carryover, precision, bias, matrix effect and dilution integrity. In order to verify the applicability, 62 vitreous humor samples were analyzed. LLOQs were 1 ng/mL and calibration curves were linear from 1 to 25 ng/mL (r2 > 0,99) for all analytes. Bias, precision and dilution integrity results were satisfactory according to proper guidelines. Ionization suppression was significant with values ranging from 8 to 37%. Two samples from real cases were positive for diazepam with the following concentrations: 6.80 ng/mL and 47.68 ng/mL, approximately 10 times lower than those found in peripheral blood. The procedure described here can be used as a straightforward and low cost method for the quantitation of multiple benzodiazepines in VH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Background: Urine is conventionally used as a specimen to document diazepam-related crimes; however, few reports have described the pharmacokinetics of diazepam and its metabolites in urine., Objective: This study aimed to investigate the pharmacokinetics of diazepam and its metabolites, including glucuronide compounds, in the urine of Chinese participants., Methods: A total of 28 volunteers were recruited and each participant ingested 5 mg of diazepam orally. Ten milliliters of urine were collected from each participant at post-consumption timepoints of prior (zero), 1, 2, 4, 8, 12, and 24 h and 2, 3, 6, 12, and 15 days. All samples were extracted by solid-phase extraction and analyzed using high-performance liquid chromatography-tandem mass spectrometry. Diazepam and its main metabolites, except for temazepam, were detected in the urine of volunteers. Pharmacokinetic parameters were analyzed using the pharmacokinetic software DAS according to the non-compartment model., Results: Urinary diazepam peaked at 2.38 ng/mL (C max ) and 1.93 h (T max ). The urinary metabolite nordiazepam peaked at 1.17 ng/mL and 100.21 h; temazepam glucuronide (TG) peaked at 145.61 ng/mL and 41.14 h; and oxazepam glucuronide (OG) peaked at 101.57 ng/mL and 165.86 h. The elimination half-life (t ½z ) and clearance (CLz/F) for diazepam were 119.58 h and 65.77 L/h, respectively. The t ½z of the metabolites nordiazepam, TG, and OG was 310.58 h, 200.17 h, and 536.44 h, respectively. Finally, this study found that both diazepam and its main metabolites in urine were detectable for at least 15 days, although there were individual differences., Conclusion: The results regarding diazepam pharmacokinetics in urine would be of great help in forensic science and drug screening., (© 2022. The Author(s).)

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., Competing Interests: Conflict of Interest There are no financial or other relations that could lead to a conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Toxicology investigation on human's buried dead bodies is a rare and challenging task in the forensic field. As requested by the Judicial Authority, this work aimed to verify testimonial evidence that emerged during a criminal investigation involving multiple murder cases. The statements indicated an improper medical administration of one or more alleged drugs (propofol, morphine, diazepam, and midazolam) which presumably caused the deaths. Since the supposed crimes took place several years before, the task of the present work was to obtain results to support the charges. The analyses involved 18 biological samples taken from four exhumed bodies, three of which were female and one male, each buried in a different date and mode. Each sample was treated with specific purification and extraction techniques (LLE - SPE) after the addition of the deuterated analogs of the searched analytes (propofol-d17, morphine-d3, diazepam-d5, midazolam-d4) as internal standards. Afterwards, the extracts were subjected to qualitative analysis by gas chromatography-mass spectrometry-Electron Impact (GC/MS - EI), both in full scan and SIM mode. Propofol, morphine, and diazepam were identified in the corpses. It supports testimonials that were administered just before the deaths occurred., Competing Interests: Conflit of interest The authors state that they have no conflit of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We report a case in which a tapentadol acute intoxication was suspected as the cause of death of a 39-year-old man: approximately two days after death, cardiac and femoral blood, as well as urine, bile, gastric content and chest hair, were collected during the autopsy. Tapentadol was detected before and after hydrolysis in femoral (530 ng/mL unconjugated and 1570 ng/mL conjugated) and cardiac (680 ng/mL unconjugated and 3440 ng/mL conjugated) blood, and additionally in bile (3200 ng/mL), urine (9300 ng/mL), chest hair (2850 pg/mg) and gastric content. LC-QTOF screening analysis confirmed the presence of five different tapentadol metabolites (tapentadol-O-glucuronide, tapentadol-O-sulfate, N-desmethyltapentadol, N-desmethyltapentadol-glucuronide and N-desmethyltapentadol-O-sulfate), in urine, bile, cardiac and femoral blood. Positivity of body hairs allowed us to conclude that the man had used tapentadol in the last weeks/months. Autopsy and toxicological results (also positive for clotiapine, diazepam and chlordesmethyldiazepam) suggested that tapentadol could have caused, even at low concentrations, a severe respiratory depression, which contributed to the death of the subject. This is one of the few cases in literature where tapentadol was detected in blood, together with its metabolites, and the only one in which the parent drug was identified in hairs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Etizolam is a benzodiazepine analogue that is approved for use in Japan, Italy and India but has recently appeared as a nonapproved product on the illicit drug market in Europe and North America. Etizolam was identified in a crystalline material seized at a Kentucky racetrack, raising concerns that this drug may have been used in racing. The aim of this study was to characterize the metabolism and excretion of etizolam in horses to generate information on its disposition and to incorporate the correct urinary and serum target analytes into anti-doping screening procedures. Etizolam was administered both intravenous and orally at a dose of 0.1 mg/kg of body weight to three horses using a two-way crossover design. Pre-administration and post-administration serum and urine samples were collected and experiments conducted to identify potential metabolites in these samples. Additionally, in vitro metabolism studies using horse liver S9 were undertaken to complement the in vivo metabolism studies. Numerous metabolites were id1entified in both serum and urine in additional to parent drug, with α-hydroxy-etizolam producing the most abundant analytical signal (in terms of signal intensity and duration of detection) of the identified metabolites in both matrices. Therefore, α-hydroxy-etizolam is considered to be the most appropriate analyte for detection for anti-doping purposes. Analytical methods were developed and validated and then applied to post-administration samples to generate concentrations of etizolam and its major metabolites in serum and urine, resulting in excretion profiles that can be used to guide approaches to detecting the use of the drug., (© 2020 John Wiley & Sons Ltd.)

The major objective of this work was to develop a portable, disposable, cost-effective, and reliable POC solid-state electrochemical sensor based on potentiometric transduction to detect benzodiazepine abuse, mainly diazepam (DZP), in biological fluids. To achieve that, microfabricated Cu electrodes on a printed circuit board modified with the conducting polymer poly(3-octylthiophene) (POT) have been employed as a substrate. This polymer was introduced to enhance the stability of the potential drift (0.9 mV/h) and improve the limit of detection (0.126 nmol mL -1 ). Nernstian potentiometric response was achieved for DZP over the concentration range 1.0 × 10 -2 to 5.0 × 10 -7  mol L -1 with a slope of 55.0 ± 0.4 mV/decade and E 0 ~ 478.9 ± 0.9. Intrinsic merits of the proposed sensor include rapid response time (11 ± 2 s) and long life time (3 months). In order to enhance the selectivity of the potentiometric sensor towards the target drug and minimize any false positive results, calix[4]arene (CX4) was impregnated as an ionophore within the PVC plastic ion-sensing membrane. The performance of the POC sensors was assessed using electrochemical methods of analysis and electrochemical impedance spectroscopy as a surface characterization tool. The studied sensors were applied to the potentiometric determination of DZP in different biological fluids (plasma, urine, saliva, and human milk) in the presence of its metabolite with an average recovery of 100.9 ± 1.3%, 99.4 ± 1.0%, 101.8 ± 1.2%, and 99.0 ± 2.0%, respectively. Graphical abstract.

A method was developed for the determination of diazepam in aquatic products by pass-through solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The analyte was extracted with acetonitrile directly and purified on a Prime HLB solid phase extraction column (60 mg/3 mL). The separation was performed on an Acquity UPLC BEH C 18 column (100 mm×2.1 mm, 1.7 μm)using methanol-0.1% (v/v) formic acid aqueous solution as the mobile phase in gradient elution mode. Qualitative analysis was performed in the multiple reaction monitoring (MRM) mode. The analyte was quantified by matrix-matched external standard curves. The results showed good linear relationship in the range of 0.1-10 ng/mL, and the correlation coefficient ( r 2 ) was greater than 0.99. The spiked recoveries of diazepam were 88.2%-101.1% at the spiked levels of 1.5, 3.0 and 15.0 μg/kg, and both the intra-and inter-day precisions were less than 10%. The developed method is simple, rapid and accurate, and it can meet the requirements for diazepam determination in aquatic product samples.

Diazepam abuse is widespread all over the word, leading to an increasing number of forensic cases such as suicide, drug-driving and robbery, but relevant studies are limited regarding the extraction of diazepam and its metabolites in oral fluid. This study aimed to investigate the pharmacokinetics of diazepam and its metabolites in oral fluid after a single oral dose in healthy volunteers. There was a total of 28 volunteers, and each ingested 5 mg diazepam orally, then ~2 mL oral fluid were collected from each participant at post-consumption time-points of prior (zero), 1, 2, 4, 8, 12, 24 h and 2, 3, 6, 12 and 15 days, respectively. All samples were extracted with solid-phase extraction and analyzed with high-performance liquid chromatography-tandem mass spectrometry method, and diazepam and nordazepam were detected in the oral fluid of volunteers. Pharmacokinetics of diazepam in oral fluid conformed to a two-compartment model, and k01_HL, k12_HL, k10_HL were 0.7 ± 1.1, 31.4 ± 68.5, 12.1 ± 11.6 h, respectively, nordazepam conformed to an one-compartment model, and k01_HL, k10_HL were 41.5 ± 44.8, 282.3 ± 365.5 h, respectively. Both diazepam and nordazepam could be detected continuously for 15 days, although there were individual differences, and the results regarding diazepam detecting in oral fluid will be of much help in forensic science and drug screening filed., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The organ distribution of 3-fluorophenmetrazine (3-FPM), pyrazolam, diclazepam as well as its main metabolites delorazepam, lormetazepam and lorazepam, was investigated. A solid phase extraction (SPE) and a QuEChERS (acronym for quick, easy, cheap, effective, rugged and safe) - approach were used for the extraction of the analytes from human tissues, body fluids and stomach contents. The detection was performed on a liquid chromatography-tandem mass spectrometry system (LCMS/MS). The analytes of interest were detected in all body fluids and tissues. Results showed femoral blood concentrations of 10 μg/L for 3-FPM, 28 μg/L for pyrazolam, 1 μg/L for diclazepam, 100 μg/L for delorazepam, 6 μg/L for lormetazepam, and 22 μg/L for lorazepam. Tissues (muscle, kidney and liver) and bile exhibited higher concentrations of the mentioned analytes than in blood. Additional positive findings in femoral blood were for 2-fluoroamphetamine (2-FA, approx. 89 μg/L), 2-flourometamphetamine (2-FMA, hint), methiopropamine (approx. 2.2 μg/L), amphetamine (approx. 21 μg/L) and caffeine (positive). Delorazepam showed the highest ratio of heart (C) and femoral blood (P) concentration (C/P ratio = 2.5), supported by the concentrations detected in psoas muscle (430 μg/kg) and stomach content (approx. 210 μg/L, absolute 84 μg). The C/P ratio indicates that delorazepam displays susceptibility for post-mortem redistribution (PMR), supported by the findings in muscle tissue. 3-FPM, pyrazolam, diclazepam, lorazepam and lormetazepam did apparently not exhibit any PMR. The cause of death, in conjunction with autopsy findings was concluded as a positional asphyxia promoted by poly-drug intoxication by arising from designer benzodiazepines and the presence of synthetic stimulants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The development of analytical methods capable of determining micropollutants is essential for quality control of drinking water. Benzodiazepines, a class of pharmaceuticals with anxiolytic properties, have received increasing attention as micropollutants. The purpose of this study was to develop an analytical method for determination of three benzodiazepine drugs (bromazepam, clonazepam and diazepam) in surface water. For the extraction of the matrix analytes, SPE cartridges (C18, 500 mg/3 mL) were used. The method was validated according to the quality criteria of the USEPA 8000D Validation Guide. The developed and validated method showed recovery values between 57 and 100%, RSD < 20% and R 2  > 0.9949. LD ranged between 2.70 and 5.00 ng L -1 for bromazepam and clonazepam respectively whereas LQ was 0.01 μg L -1 for all analytes. The matrix affected the signal intensity of clonazepam thus evidencing the matrix effect by analysis statistic (F test).

Benzodiazepines are commonly seen in samples submitted for drug testing of patients, people involved in child welfare cases, work-place drug testing, as well as in drug-facilitated assaults. Limited previous experimental studies are available regarding the excretion of benzodiazepines in urine and oral fluid. The aim of this study was to investigate the concentrations of diazepam and alprazolam in oral fluid and urine for up to 2 weeks after ingestion of a single oral dose in healthy volunteers. A total of 11 healthy volunteers ingested 10 mg diazepam at the start of the study and 0.5 mg alprazolam on Day 3 of the study. A total number of 10 oral fluid samples and 17 urine samples were collected from each participant. The samples were analyzed by liquid chromatography with tandem mass spectroscopy and ultra-high performance liquid chromatography tandem mass spectrometry methods. The median detection time was 252 h for the longest detected diazepam metabolite in urine (oxazepam, range 203-322) and 132 h in oral fluid (N-desmethyldiazepam, range 109-136). For alprazolam, the median detection time was 36 h (metabolite α-OH-alprazolam, range 26-61) in urine and 26 h (alprazolam, range 4-37) in oral fluid. These results show that detection times are only 36 h for alprazolam in urine after intake of a single therapeutic oral dose. For diazepam in urine, detection times were 11 days. Detection times were generally shorter in oral fluid compared to urine. The results could be helpful in the interpretation of diazepam or alprazolam findings in drug testing cases involving urine or oral fluid., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Diazepam (DZP) is often found in source water and drinking water at dozens of nanograms per liter levels. The transformation of DZP in water chlorination disinfection process has aroused new concern because the toxic disinfection byproducts (DBPs) might be produced. However, the DBPs of DZP have not been fully identified, and their occurrence levels in drinking water have not been reported. In our chlorination experiment, five emerging DBPs of diazepam: (5-chloro-2-(methylamino) phenyl) (phenyl)methanone (BP-246), 6-chloro-1-methyl-4-phenylquinazolin-2(1H)-one (BP-271), N-(2-benzoyl-4,6-dichlorophenyl)formamide (BP-294), methyl-(2-benzoyl-4-chlorophenyl) (methyl)carbamate (BP-304 (1)) and 6-chloro-4-methoxy-1-methyl-4-phenyl-1,4-dihydro2H -benzo[d][1,3]oxazin-2-one (BP-304 (2)), were tentatively identified by high-resolution mass spectrometry and further characterized by nuclear magnetic resonance spectroscopy. We developed a trace analytical method for the analysis of these five DBPs in drinking water based on solid-phase extraction (SPE) followed liquid chromatography coupled with electrospray ionization tandem mass spectrometric detection. Ultrahigh sensitivities were achieved with limits of detection as low as 7 pg per liter. The recoveries at different spiking levels were all higher than 80% except for that of BP-246. Four of the DBPs and DZP were detected in real drinking water samples at concentrations ranging from several to dozens of nanograms per liter with relatively high detection frequencies. This is the first report on the existence of DZP-DBPs in drinking water. The method and results will be useful for further studies on the occurrence, toxicity, human exposure and control measures of these DBPs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The introduction of monolithic rods and core-shell particles as new morphologies of packing materials different from the conventional totally porous particles resulted in a leap forward for performance in LC. Meanwhile, environmental safety has become increasingly important in many areas, especially in industry and research laboratories. Objective: This study compared the efficiencies of commercially available columns of different lengths and diameters when greener chromatographic conditions were utilized. The main purpose of this study is to help practitioners select the most appropriate stationary phase for faster and greener analysis. Methods: The three types of stationary phases were compared in terms of separation efficiency, number of theoretical plates, peak shape, selectivity, resolution, analysis time, mobile phase consideration, and permeability using six drug molecules. Results: Results indicated that core-shell and monolithic stationary phases had superiority over the conventional totally porous particles in terms of efficiency and speed of analysis. Monolithic rods had lower column backpressure and higher permeability, so they are more suitable for higher mobile phase flow rates and viscosities. However, core-shell particles provided enhanced peak shapes and number of theoretical plates. Conclusions: The choice will depend on the main purpose of analysis and the composition of the mobile phase. Compromise must be made to obtain the best trade-off between separation efficiency and analysis speed. Highlights: This study is the first to consider green chromatography concepts for the selection of the best stationary phase of new morphologies.

One of the most common methods of maternal filicide is by fire. In this case study, a 40-year-old female and her children were found completely burned in a burnt out car. All bodies showed a degree of destruction by fire consisting to a level 3 of the Crow-Glassman Scale (CGS) and early stage of insect activity. Toxicological analyses were performed on soft tissues and body fluids still available. The results were positive for diazepam and its metabolites only for children with blood concentrations consistent with therapeutic doses of benzodiazepines. Home video surveillance cameras confirmed sedation prior to death recording the mother while administering some drops of sedative drugs in a soft drink to the children just a couple of hours before setting fire to the car. Based on autopsy findings, all victims were still alive at the time of fire. The cause of death was determined as carbon monoxide poisoning and fatal thermal injuries by fire. This case study has a special focus on the entomotoxicology and the potential role of insects in death investigations of burnt bodies, supposed to be an inadequate substratum for insect colonization. It demonstrates that in burnt bodies, arthropod colonization can be quite immediate after fire is extinguished. Toxicological analyses performed on larvae actively feeding on the children's bodies were positive for diazepam and its metabolites in small amount compared with blood concentrations, whereas the larvae collected from the mother's body were totally negative. These data, according to the autopsy findings and the toxicological results from the victim's blood and tissues, supported the suspect of a non-lethal sedation prior to death, which is a common behaviour in maternal filicide.

We describe herein the fabrication of an electrochemical microfluidic paper based device (EμPAD) for the detection of diazepam, a sedative, anxiety-relieving and muscle-relaxing drug. To achieve it, silica coated gold nanorods (Si@GNRs) were synthesized and drop casted on an electrochemical microfluidic paper based device (EμPAD) for the detection of diazepam. The synthesized composites were characterized by recording its images in scanning electron microscope (SEM) and transmission electron microscope (TEM). The experimental results confirmed that Si@GNRs had good electrocatalytic activity towards diazepam. The modified paper based electrode showed a stable electrochemical response for diazepam in the concentration range of 3.5 nM to 3.5 mM. EμPAD offers many advantageous features such as facile approach, economical and have potential for commercialization. Si@GNRs modified EμPAD was also employed for determination of diazepam in spiked human urine samples. Reported facile lab paper approach integrated with Si@GNRs could be well applied for the determination of serum metabolites., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Occurrence of diazepam and its metabolites, nordiazepam, temazepam, and oxazepam in the water environment in Beijing was investigated. Samples were collected from four rivers flowing through the city and from all the thirteen sewage treatment plants in the urban area. Average influent concentrations of diazepman, temazepam, and oxazepam in 2013 summer ranged from 0.9 to 7.1, 1.5 to 3.4, and 2.9 to 12.4 ng L -1 , respectively, whereas nordiazepam concentrations were below quantification limit on the majority of sampling dates. No significant seasonal variation in influent concentrations was observed. Removal during treatment was low for diazepman (<50%), temazepam (<20%), and oxazepam (<20%), consistent with previous findings reported in the literature. Wastewater-based epidemiology approach was applied to back-calculate population size-normalized diazepam consumption (using temazepam as biomarker) in Beijing, which was found to be at least 3.8 times more of the national average. Diazepam, temazepam, and oxazepam were widely detected in surface waters, with concentrations greater than concentrations in sewage influents at many sampling points, strongly indicating direct discharge of wastewater of high diazepam concentrations into the surface waters in the city.

Pentobarbital is a barbiturate, acting as a central nervous system depressant (CNS), being used for its anticonvulsant, sedative, hypnotic and anaesthetic properties. Barbiturates were replaced by benzodiazepines, leading to a decrease in poisoning cases with these compounds. However, pentobarbital is still used in many countries as an anaesthetic in veterinary medicine. Due to its properties, this compound is sought after by people who wish to commit suicide, acquiring it on the black market. The authors present an unusual fatal pentobarbital intoxication case, in a 37 years-old male salesperson, with no known connection with the veterinary field, being more difficult to obtain this compound. Toxicological results in cardiac blood revealed the presence of pentobarbital (111mg/L), ethanol (0.94g/L), diazepam (33ng/mL), nordiazepam (50ng/mL), oxazepam (3.3ng/mL), temazepam (5.3ng/mL), and metoclopramide. No illicit drugs were detected. Pentobarbital analysis in urine and gastric content was also positive, as well as its presence in the glass powder and in the bottle residue sent to the laboratory. In the present case, it was possible to conclude that the death was a suicide due to pentobarbital intoxication in association with other depressants of the CNS (benzodiazepines and ethanol). It is important to search pentobarbital in routine toxicological analyses, since it is one of the drugs most frequently mentioned by entities defending "painless death", advising the simultaneous use of metoclopramide for emesis avoidance., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Administration sets are delivery tools for the direct application of drugs into the body and are composed of a spike, a drip chamber, tubes, Luer adapters (connectors), a needle cover for protection, and other accessories. Drug sorption to tubes of administration sets is a critical issue in terms of safety and efficacy. Although drug sorption is an important factor in the quality of an administration set, there are no standard evaluation methods for the regulation of drug sorption to the tubes. Here, we describe an evaluation protocol for drug sorption to tubes of administration sets. Tubes made of polyvinyl chloride (PVC)- and non-PVC-based polymeric materials were cut to 1 m in length. Diazepam and tacrolimus were used as model drugs. In the kinetic sorption study, we selected the drug concentration and flow rate based on the clinical usage of these drugs. After the dilution of each drug in a glass bottle, the diluted drug solution was delivered through tubes of administration sets using a pump. Samples were collected in amber vials at appropriate time points and the drugs were analyzed using high-performance liquid chromatography. Drug concentrations and sorption levels to tubes of the administration sets were calculated. Acceptable criteria to ensure the quality of administration sets are recommended.

A multivariate curve resolution - alternating least squares (MCR-ALS) analysis was used to quantify diazepam (DZP) in thirty commercial liquid formulations. MCR calibration was run on the UV spectrophotometric data of the commercial DZP samples over the range 200-400nm, allowing the resolution of the drug signal and then the excipients contained in all the formulations. A single model MCR for the determination of the drug in all samples was then built through the adoption of the correlation constraint. This model was optimized by an appropriate selection of the most useful wavelength ranges and then validated on external samples. DZP concentrations in the pharmaceutical formulations were measured by HPLC-DAD analysis. The performance of the MCR model was compared with that from application of classical partial least squares regression (PLSR). The results, in terms of error of prediction, were very satisfactory, reaching a relative error below of 1.66% against 2.56%, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Defining a method development methodology for achiral drug impurity profiling in SFC requires a number of steps. Initially, diverse stationary phases are characterized and a small number of orthogonal or dissimilar phases are selected for further method development. In this paper, we focus on a next step which is the investigation of the modifier composition on chromatographic selectivity. A solvent-triangle based approach is used in which blends of organic solvents, mainly ethanol (EtOH), propanol (PrOH), acetonitrile (ACN) and tetrahydrofuran (THF) mixed with methanol (MeOH) are tested as modifiers on six dissimilar stationary phases. The tested modifier blends were composed to have equal eluotropic strengths as calculated on bare silica. The modifier leads to minor changes in terms of elution order, retention and mixture resolution. However, varying only the modifier composition on a given stationary phase does not lead to the creation of dissimilar systems. Therefore the modifier composition is an optimization parameter, with the stationary phase being the factor determining most the selectivity of a given mixture in achiral SFC., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10 mg of diazepam. Hair was collected from all volunteers 1 month after exposure, and also 2 months post-exposure from men and 10 months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5 pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10 pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2 months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10 months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair.

Purpose: Plastic materials such as polyurethane (PUR), polyethylene (PE), polypropylene (PP) and polyvinyl chloride (PVC) are widely used in double-lumen extension tubing. The purposes of our study were to 1) compare in vitro drug delivery through the double extension tubes available on the market 2) assess the plastic properties of PUR in infusion devices and their impact on drug delivery., Methods: The study compared eight double-lumen extension tubes in PUR, co-extruded (PE/PVC) plastic and plasticised PVC from different manufacturers. Isosorbide dinitrate and diazepam were used as model compounds to evaluate their sorption on the internal surface of the infusion device. Control experiments were performed using norepinephrine known not to absorb to plastics. Drug concentrations delivered at the egress of extension tubes were determined over time by an analytical spectrophotometric UV-Vis method. The main characteristics of plastics were also determined., Results: Significant differences in the sorption phenomenon were observed among the eight double-lumen extension tubes and between pairs of extension tubes. Mean concentrations of isosorbide dinitrate delivered at the egress of double-lumen extension tubes after a 150-minute infusion (mean values ± standard deviation in percentage of the initial concentrations in the prepared syringes) ranged between 80.53 ± 1.66 (one of the PUR tubes) and 92.84 ± 2.73 (PE/PVC tube). The same parameters measured during diazepam infusion ranged between 48.58 ± 2.88 (one of the PUR tubes) and 85.06 ± 3.94 (PE/PVC tube). The double-lumen extension tubes in PUR were either thermosetting (resin) or thermoplastic according to reference., Conclusions: Clinicians must be aware of potential drug interactions with extension tube materials and so must consider their nature as well as the sterilisation method used before selecting an infusion device.

Pharmaceutical drugs are continuously discharged into the aquatic environment primarily through wastewater discharge; therefore, their possible effects on wildlife is a reason of concern. Diazepam is a widely prescribed benzodiazepine drug used to treat insomnia and anxiety disorders, and it has been found in wastewater effluents worldwide. The present study tested the effects of diazepam on fecundity and the reproductive behavior of the fathead minnow, Pimephales promelas, a fish that exhibits male parental care. Sexually mature fathead minnows were housed at a ratio of one male and two females per tank and exposed to nominal (measured) concentrations of 0, 0.1 (0.14 ± 0.06), 1.0 (1.04 ± 0.15), 10 (13.4 ± 1.5) µg L(-1) for 21 days. Fish receiving the low diazepam treatment had significantly larger clutches than fish receiving the highest concentration but neither were different from controls. Diazepam exposure was not associated with a significant change in fertilization rate, hatchability or time to hatch, but a trend toward a higher number of eggs/day was observed in fish exposed to the low diazepam concentration relative to those exposed to the medium concentration. There were no significant differences in any of the behaviors analyzed when responses were averaged over time. The results showed that exposure to diazepam at concentrations as high as 13 µg L(-1) did not significantly impact the reproductive behavior of fathead minnow., (© 2014 Wiley Periodicals, Inc.)

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 μL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alternative specimens have been occasionally considered as substitutes for whole blood for postmortem toxicology testing. We studied the applicability of vitreous humor, and evaluated whether it would be suitable to replace (or augment) whole blood for routine drug screening. Results showed that from 51 autopsy cases, we were able to identify an aggregate of 209 findings in whole blood compared with 169 in vitreous. The total number of compounds identified was 71 for whole blood and 60 for vitreous humor. Quantitative analysis showed that whole-blood concentrations of trazodone were several fold higher than vitreous humor concentrations (1.42 ± 0.57 vs. 0.15 ± 0.05 mg/L, respectively) and similar results were also obtained for diazepam (0.37 ± 0.06 vs. 0.13 ± 0.01, respectively). For other drugs such as oxycodone, hydrocodone and doxylamine, a trend suggesting higher concentrations in vitreous humor vs. whole blood was observed; however, this was not significant. Our results are consistent with the limited work of other investigators, and suggest that vitreous humor could be an appropriate matrix for drug screening in postmortem toxicology., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This is the first application based on electron ionization (EI) using a Direct-EI LC interface and MS/MS to detect unequivocally target compounds in a very small real sample. The determination and quantification of benzodiazepines in very small residues of beverages, collected at the scene of drug-facilitated crimes are mandatory in legal procedures. A specific and sensitive analytical instrumentation is needed, involving little or no sample preparation. Here, a direct flow injection analysis of alcoholic beverages spiked with commercially available drugs containing diazepam and flunitrazepam is presented. The method proposed is very fast and requires neither sample preparation nor chromatographic separation. Linearity (R(2) ) was between 0.9977 and 0.9992; LOD and LOQ spanned from 0.01 to 0.02 ng/μL and from 0.1 to 0.5 ng/μL, respectively; intra- and interday repeatabilities were between 1 and 8%. No matrix effects were observed from the comparison of the linear regression curves obtained in real fortified samples and in pure ethanol. Vodka, whisky, and white wine specimens were fortified with commercial drugs, Valium(®) and Rohypnol(®) , at two different concentrations (20 and 50 ng/μL) to simulate the typical amounts found in adulterated real samples and analyzed to demonstrate the method applicability to forensic analyses., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oxcarbazepine (OXC), a second-generation antiepileptic drug, undergoes rapid reduction with formation of the active metabolite 10,11-dihydro-10-hydroxy-carbazepine (MHD) in vivo. In this study, a method for simultaneous determination of OXC and MHD in rat plasma using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS-MS) was developed and validated. Under given chromatographic conditions, OXC, MHD and internal standard diazepam were separated well and quantified by electrospray positive ionization mass spectrometry in the multiple reaction monitoring transitions mode. The method validation demonstrated good linearity over the range of 10-2,000 ng/mL for OXC and 5-1,000 ng/mL for MHD. The lower limit of quantification was 5 ng/mL for OXC and 2.5 ng/mL for MHD, respectively. The method was successfully applied to the evaluation of the pharmacokinetics of OXC and MHD in rats, with or without pretreatment by ketoconazole (KET) and voriconazole (VOR). Statistics indicated that KET and VOR significantly affected the disposition of OXC and MHD in vivo, whereas VOR predominantly interfered with the disposition of MHD. This method is suitable for pharmacokinetic study in small animals., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: A simple and fast modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method is presented for the determination of diazepam and its three major metabolites, nordiazepam, temazepam and oxazepam (benzodiazepines) in fish samples by liquid chromatography-electrospray ionisation-tandem mass spectrometry., Results: Muscle tissues were extracted with acetonitrile, and then cleaned with primary secondary amino (PSA) adsorbents. The cleanup effect of PSA was compared with that of multi-walled carbon nanotubes (MWCNTs) in term of extraction efficiency. The better results were obtained when PSA was used. The chromatography separation was achieved within 5.0 min on a C18 column. The limit of detection was 0.5 µg kg(-1) and the limit of quantification was 2.5 µg kg(-1). Average recoveries of diazepam and its main metabolites were in the range of 88.5-110.1%, with a relative standard deviation lower than 10.0%., Conclusion: The proposed method for fish samples gives good recoveries, linearity, precision and accuracy., (© 2015 Society of Chemical Industry.)

Benzodiazepines (BDs) are used widely in clinical practice, due to their multiple pharmacological functions. In this study a dispersive nanomaterial-ultrasound assisted- microextraction (DNUM) method followed by high performance liquid chromatography (HPLC) was used for the preconcentration and determination of chlordiazepoxide and diazepam drugs from urine and plasma samples. Various parameters such as amount of adsorbent (mg: ZnS-AC), pH and ionic strength of sample solution, vortex and ultrasonic time (min), and desorption volume (mL) were investigated by fractional factorial design (FFD) and central composite design (CCD). Regression models and desirability functions (DF) were applied to find the best experimental conditions for providing the maximum extraction recovery (ER). Under the optimal conditions a linear calibration curve were obtained in the range of 0.005-10μgmL(-1) and 0.006-10μgmL(-1) for chlordiazepoxide and diazepam, respectively. To demonstrate the analytical performance, figures of merits of the proposed method in urine and plasma spiked with chlordiazepoxide and diazepam were investigated. The limits of detection of chlordiazepoxide and diazepam in urine and plasma were ranged from 0.0012 to 0.0015μgmL(-1), respectively., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study addresses the occurrence of carbamazepine and diazepam and their metabolites in the wastewater of the University Hospital (HUSM) of the Federal University of Santa Maria, RS-Brazil. Samples were collected from three sampling points of the sewage treatment system: point A ('emergency effluent'), point B ('general effluent') and point C ('water course-receptor'). Eight metabolites were identified: carbamazepine-10-11-epoxide, 10-dihydro-carbamazepine, 2-OH-carbamazepine, iminoquinone, acridone, nordiazepam, oxazepam and temazepam. The mean concentrations in the emergency, general effluent and water course-receptor were as follows: 433.0 ± 4.7, 349.0 ± 5.0 and 485.0 ± 5.6 ng L(-1), for carbamazepine and 550.0 ± 4.3, 441.0 ± 7.9 and 586.6 ± 9.3 ng L(-1), for diazepam, respectively. Liquid chromatography with electrospray ionization tandem mass spectrometry (LC-QqLIT-MS) proved to be a method fit-to-purpose. The determination of carbamazepine and diazepam, and the identification of active metabolites showing environmental persistence (carbamazepine-10-11-epoxide, nordiazepam and oxazepam) revealed the need for a more effective treatment of the HUSM effluent. As far as we know, no similar study has been carried out on the wastewater of Brazilian hospitals.

Background: Clonazepam, diazepam, and alprazolam are benzodiazepines with sedative, anticonvulsant, and anxiolytic effects, but their prevalence in drug abuse and drug overdoses has long been recognized. When detection times for psychoactive drugs in oral fluid are reported, they are most often based on therapeutic doses administered in clinical studies. Repeated ingestions of high doses, as seen after drug abuse, are however likely to cause positive samples for extended time periods. Findings of drugs of abuse in oral fluid collected from imprisoned persons might lead to negative sanctions, and the knowledge of detection times of these drugs is thus important to ensure correct interpretation. The aim of this study was to investigate the time window of detection for diazepam, clonazepam, and alprazolam in oral fluid from drug addicts admitted to detoxification., Methods: Twenty-five patients with a history of heavy drug abuse admitted to a detoxification ward were included. Oral fluid was collected daily in the morning and the evening and urine samples every morning for 10 days, using the Intercept device. Whole blood samples were collected if the patient accepted. The cutoff levels in oral fluid were 1.3 ng/mL for diazepam, N-desmethyldiazepam, and 7-aminoclonazepam and 1 ng/mL for clonazepam and alprazolam. In urine, the cutoff levels for quantifications were 30 ng/mL for alprazolam, alpha-OH-alprazolam, and 7-aminoclonazepam, 135 ng/mL for N-desmethyldizepam, and 150 ng/mL for 3-OH-diazepam and for all the compounds, the cutoff for the screening analyses were 200 ng/mL., Results: The maximum detection times for diazepam and N-desmethyldiazepam in oral fluid were 7 and 9 days, respectively. For clonazepam and 7-aminoclonazepam, the maximum detection times in oral fluid were 5 and 6 days, respectively. The maximum detection time for alprazolam in oral fluid was 2.5 days. New ingestions were not suspected in any of the cases, because the corresponding concentrations in urine were decreasing. Results from blood samples revealed that high doses of benzodiazepines had been ingested before admission, and explains the longer detection times in oral fluids than reported previously after intake of therapeutic doses of these drugs., Conclusions: This study has shown that oral fluid might be a viable alternative medium to urine when the abuse of benzodiazepines is suspected.

Hair specimen is necessary to complement blood and/or urine analyses as it permits differentiation of a single exposure from chronic use of a drug by segmentation of the hair for a stated growth period. Moreover, due to a frequent long delay between event and police declaration, hair can be the only solution for lack of corroborative evidence of a committed crime. With the exception of lower amount of biological material in children versus adults, there is no specific analytical problem when processing samples from children. The issue is the interpretation of the findings, with respect to the different pharmacological parameters. In some very young children, the interpretation can be complicated by potential in utero exposure. Twenty-four cases from daily practice have been reviewed. Children were less than 1 year old, hair was always longer than 4 cm and the corresponding mothers admitted having used drugs during pregnancy. Drugs involved include methadone, tramadol, diphenhydramine, diazepam, cannabis, heroin, amitriptyline and bromazepam. Analyses were achieved by hyphenated chromatographic validated procedures after hair decontamination and segmentation. The concentrations measured in the hair of children were lower than those observed in subjects using therapeutically (or illegally) these drugs. In that sense, the frequency of exposures appears as un-frequent (low level of exposure), with marked decrease in the more recent period. However, the parents denied any administration in all cases and there was no reason to suspect re-exposure after delivery and no clinical problem during the period between delivery and hair collection during regular visits to the physician was noticed. The pattern of drug distribution was similar in all these cases, low concentrations in the proximal segments and highest concentration in the distal segment (last segment). When considering the concentration in the distal segment as the 100% of the response (highest concentration), after analysis of 4 segments (irrespective of the length of the segment but longer than 1cm), it was observed the following pattern: proximal segment, 5-35% of the response; segment 2, 15-50% of the response; segment 3, 25-60% of the response; and distal segment, 100% of the response. It is proposed to consider 100% in utero contribution to the final interpretation when the ratio concentration of the proximal segment to the concentration of the distal segment is lower than 0.5. This can be applied only when the child is under 1 year old and the hair shaft length is at least 4 cm (to achieve suitable segmentation). It is important, when using this cut-off to have at least 3 or 4 segments to be able to observe the variation in drug concentrations, whatever the length of each segment (>1cm)., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

A novel liquid chromatography-tandem mass spectrometry method was validated for identification and quantification of diazepam, flunitrazepam and metabolites in reinforced clostridial medium (RCM), a complex matrix used to provide the nutrients required for bacterial growth. The method was designed for subsequent use in the investigation of gastrointestinal bacteria as a potential source of postmortem alteration of drugs of abuse and respective metabolite concentrations. A literature review yielded no experimental means or model for the extraction and analysis of samples from RCM or similar bacterial medium. Development and validation of a new experimental method were therefore critical. In future work, this method could be adapted and extended to similar organic compounds of interest. The calibration curves extended from 0.100 to 500 ng/mL. Analyte recoveries ranged from 95 to 119% and matrix effects from 97 to 119%. Bias was ≤±17.6%, within-run precision ≤12.2%, and between-run precision ≤11.7% across all concentration levels. The limits of detection and quantitation ranged from 0.100 to 1 ng/mL. Dilution integrity was maintained for 1:2 and 1:5 dilutions. Analytes were stable through two freeze-thaw cycles and processed samples for 48 h. Method robustness was evaluated by changes in buffer composition and column temperature as well as samples prepared by an alternate analyst., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The objective of the present study was to produce a generic monoclonal antibody for immunoassay of residues of benzodiazepine drugs in swine tissues. Clonazepam was used to synthesize a hapten that was coupled to bovine serum albumin as an immunogen for the production of monoclonal antibody. Results showed that the obtained monoclonal antibody was able to recognize five benzodiazepine drugs simultaneously (clonazepam, flunitrazepam nitrazepam, diazepam, and oxazepam). The cross-reactivities were in the range of 24-100% and the limits of detection were in the range of 0.2-1.5 ng mL(-1) depending on the drug. Then a competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of five benzodiazepines in swine tissues (muscle, liver and kidney). The recoveries of five analytes from the fortified blank samples were in the range of 74.5-96.5% with coefficients of variation lower than 16.7%. Therefore, this immunoassay could be used as a rapid and simple method for the screening of residues of five benzodiazepine drugs in animal-derived foods.

Designer benzodiazepines, first offered in online shops selling 'research chemicals' in 2012, provide an attractive alternative to prescription-only benzodiazepines as they are readily available over the Internet at a low price. However, as data regarding pharmacokinetic parameters, metabolism, and detectability in biological fluids are limited, they present a challenge for forensic laboratories. Most recently, diclazepam (other names: 2-chlorodiazepam, Ro 5-3448 or 7-chloro-5-(2-chlorophenyl)-1-methyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one) emerged as a new compound in this class of drugs. In this paper, this new designer benzodiazepine was characterized utilizing nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS) as well as liquid chromatography tandem mass spectrometry (LC-MS/MS) techniques. Furthermore, a self-experiment was performed to gain preliminary data on pharmacokinetic properties and to identify the main metabolites. For this purpose, one tablet of diclazepam (declared amount: 1 mg) was ingested by one of the authors, and serum as well as urine samples were collected for 14 and 21 days, respectively. Based on this study, diclazepam has an approximate elimination half-life of 42 h and is metabolized into the pharmacologically active benzodiazepines delorazepam, lorazepam, and lormetazepam which can be detected in urine for 6, 19, and 11 days, respectively, when applying the presented LC-MS/MS method. In serum, the consumption could be proven between 99 h post-intake targeting the parent compound and up to 10 days targeting the metabolite delorazepam. As immunochemical assays are applied for screening purposes quite often, detectability using this technique was assessed, especially since detection of low-dosed benzodiazepines can be sometimes problematic. However, only one of the utilized immunochemical assays was capable of detecting the intake of one tablet diclazepam, and the positive results were restricted to a few urine samples showing relatively high creatinine concentrations., (Copyright © 2014 John Wiley & Sons, Ltd.)

In drug-facilitated sexual assault (DFSA) cases, drug residues may be detected in beverage remains found in cups or glasses known to have been used by the victims. In this small naturalistic study, the possibility of beverages being contaminated, either by drug concentrated oral fluid or by oral cavity contained tablets, was investigated. Analysis of residues from cups containing soft drinks was performed by immunoassay and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Beverage with both added tablets and spiked oral fluid was investigated, as well as simulation of swallowing tablets. Only the residues from added tablets were positive with immunoassay, while drugs were detectable in all cups using more sensitive UPLC-MS/MS. In conclusion, the possibility of detecting drug residues in beverages due to a contamination, from either drug concentrated oral fluid or oral cavity contained tablets at a time of consumption, should be kept in mind when performing sensitive analysis., (© 2013 American Academy of Forensic Sciences.)

A rapid and sensitive flow-injection chemiluminescence (FI-CL) method is described for the determination of diazepam based on its reaction with N-bromosuccinimide (NBS) in alkaline medium in the presence of dichlorofluorescein (DCF) as an effective energy-transfer agent. Under optimum conditions, the proposed method allowed the measurement of diazepam over the range of 2.0 × 10(-6) to 2.0 × 10(-4) mol/L with a detection limit of 5.0 × 10(-7) mol/L. The relative standard deviation for 11 parallel measurements of 2.0 × 10(-5) mol/L diazepam was 2.1%. The method was applied satisfactorily for the determination of diazepam in pharmaceutical preparations, and the results agree well with those obtained by spectrophotometry. The use of the proposed system for the determination of diazepam in urine and plasma samples was also tested. The possible mechanism of the chemiluminescence reaction is discussed briefly., (Copyright © 2012 John Wiley & Sons, Ltd.)

We report the case of a man who died twelve hours after ingesting powdered iboga root, commonly taken for its stimulant and hallucinogenic properties. Ibogaine and ibogamine were quantified in the powder ingested and the victim's body fluids by GC-MS/MS after liquid-liquid extraction (Toxi-tubes A(®)). The concentrations of ibogaine measured in the blood samples taken at the scene and in the peripheral blood, urine, and gastric fluid samples taken during the autopsy were 0.65, 1.27, 1.7, and 53.5 μg/mL, while the iboga content in the powder was 7.2%. Moreover, systematic toxicological analyses of biological samples showed the presence of diazepam and methadone in therapeutic concentrations. Death was attributed to the ingestion of a substantial quantity of iboga in the context of simultaneous methadone and diazepam consumption., (© 2013 American Academy of Forensic Sciences.)

Reuse of treated wastewater to irrigate agricultural crops is increasing in many arid and semi-arid areas around the world. The presence of numerous pharmaceutical and personal care products (PPCPs) in treated wastewater and their potential transfer into food produce such as vegetables poses an unknown human health risk. The goal of this study was to identify PPCPs that have a comparatively high potential for plant uptake and translocation. A total of 20 frequently-occurring PPCPs were compared for their accumulation into four staple vegetables (lettuce, spinach, cucumber, and pepper) grown in nutrient solutions containing PPCPs at 0.5 or 5μgL(-1). Triclocarban, fluoxetine, triclosan, and diazepam were found at high levels in roots, while meprobamate, primidone, carbamazepine, dilantin, and diuron exhibited more active translocation from roots to leaves. Root uptake of neutral PPCPs was positively correlated with the pH adjusted log Kow(i.e., log Dow), and was likely driven by chemical adsorption onto the root surfaces. In contrast, translocation from roots to leaves was negatively related to log Dow, suggesting hydrophilicity-regulated transport via xylems. Compounds preferentially sorbed to roots should be further evaluated for their uptake in tuber vegetables (e.g., carrot, radish) under field conditions, while those easily translocated into leaves (e.g., carbamazepine, dilantin) merit focused consideration for leafy and other vegetables (e.g., lettuce, cucumber). However, estimation of dietary intake by humans suggested the implied risks from exposure to PPCPs via wastewater irrigation to be negligible., (© 2013.)

Unlabelled: Pregnant women are exposed to benzodiazepines for therapeutic purposes during gestation. The goal of this study was to evaluate prenatal exposure to benzodiazepines. Time of exposure during course of pregnancy is a significant aspect of fetal exposure to drugs. Benzodiazepine concentration assay in hair of mothers and newborns exposed prenatally to these drugs was performed in the studies. Development, validation and evaluation of benzodiazepine determination method in mothers and their newborns enables assessment of health risks for the child and implementation of adequate therapeutic procedures. We used A LC-ESI-MS/MS method that allowed determination of diazepam (the main benzodiazepine used by pregnant women was diazepam) and its metabolites (nordazepam, oxazepam) in hair of mothers and newborns. LOQ 10 pg/mg of hair was used in the study., Results: concentration of nordazepam was higher than parent drug (diazepam) and higher in newborns' hair when compared to mothers'. The mean concentrations of diazepam in mothers' hair were 31.6±36.0 and 34.1±42.4 pg/mg in the second and third trimester of pregnancy respectively. The mean concentration of diazepam in newborns' hair was higher and reached levels of 53.3±36.5 pg/mg. The mean concentration of nordazepam in the mothers' hair corresponding to the second and third trimester was 52.9±48.1 and 89.9±122.8 pg/mg, respectively. Nordazepam in the newborns' hair was detected at the mean level of 108.1±144.2 pg/mg. It was concluded that diazepam and nordazepam are permanently incorporated into the hair structure. Presence of diazepam and its metabolites in newborn's hair confirms that these benzodiazepines permeate placental barrier. Segmental analysis of mothers' hair enabled the assessment of drug administration time. Diazepam and its metabolites determined in hair of newborns may serve as biomarkers of prenatal exposure to these drugs. The performed LC-MS/MS analysis was accurate enough to determine even low concentrations of benzodiazepines, at the level of few pg/mg of hair. Levels of diazepam detected in hair of newborns were higher than levels determined in mothers. This may confirm the fact, that fetus's ability to metabolize diazepam is scarce. Nordazepam was found in higher concentrations in hair of newborns than in hair of mothers, which may suggest that it is cumulated in child's organism. Other metabolites of diazepam--oxazepam and temazepam--were detected in very few cases, in low concentrations.

Using an in-house forensic toxicology database, we selected 1000 cases of driving under the influence of drugs (DUIDs) over a 12-month period if diazepam (D) and nordiazepam (ND) were both present in the blood samples. Quantitative analysis of D and ND in blood was done by solvent extraction (butyl acetate) and capillary column gas chromatography (GC) with a nitrogen-phosphorous (N-P) detector. The limits of quantitation of this analytical method for D and ND in blood were 0.05 mg/L. The correlation between D and ND concentrations in blood was statistically significant (r = .58, P < .001), as expected for a parent drug and its primary metabolite. However, the frequency distributions were markedly skewed to the right with mean (median) and highest concentrations of 0.37 (0.20) and 6.1 mg/L for D and 0.39 (0.20) and 5.6 mg/L for ND. The mean (median) total concentration (D + ND) was 0.76 mg/L (0.50 mg/L), and the concentration ratios D/ND and ND/D were 1.29 (median 0.95) and 1.41 (median 1.06), respectively. In 90 cases (9%), the concentration of D in blood exceeded 0.83 mg/L, which corresponds to an upper therapeutic limit in plasma (∼1.5 mg/L), assuming a plasma/blood distribution ratio of 1.8:1.

Human remains found in aquatic contexts are frequently recovered incomplete and badly decomposed, and therefore present a challenge for medico-legal institutes as their possibilities of analysis for identification and investigation of cause and manner of death are limited. This article aims to demonstrate the potential of forensic examination and analyses (DNA, toxicology, diatoms and entomology) on a set of bones recovered from a river in Strasbourg and the possibility to trigger identification of the victim and circumstances of death despite the state of decomposition and incompleteness of remains., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Recently, an essential oil of selected quality produced from the flowering tops of Lavandula angustifolia Mill. by steam distillation (Silexan) has been approved in Germany for the treatment of restlessness in case of anxious mood. Based on the observed clinical effects, it has been speculated that lavender oil may exert benzodiazepine-like action including the known dependence and abuse potential of this class of drugs. Although no evidence for such an activity was generated during the long-standing medicinal use of lavender oil, further preclinical investigations were now conducted to evaluate this potential side effect in more detail. Twelve adult, male, Sprague-Dawley rats were trained to discriminate the benzodiazepine drug diazepam (2 mg/kg i.p.) from saline using a two-lever operant procedure. After approximately 40 training sessions the majority of rats learned the discrimination and pre-treatment with ascending doses of diazepam (0.3-2 mg/kg i.p.) produced a dose related generalization to the diazepam cue. In these same animals Silexan was administered to see if animals recognized the drug as "diazepam-like" i.e. generalized to diazepam or "saline-like". Silexan tested at doses 3-30 mg/kg i.p. produced almost exclusively (>90%) saline-like responding. Also there was no effect of Silexan on response rate, i.e. rate of lever pressing, at any dose suggesting that the test article is well tolerated and does not exert a sedating effect. In sum, Silexan has no diazepam-like interoceptive property in adult, male rats. This suggests that Silexan does not share the potential of benzodiazepines to induce the development of tolerance, dependence and addiction., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

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.