Benzodiazepine Concentrations Research Articles

B-276 Evaluation of the Roche Benzodiazepines II Immunoassay for Urine Drug Testing in Clinical Specimens

Abstract Background Benzodiazepines are one of the most commonly prescribed medications in the United States and are frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated metabolites excreted in urine. False negative results are especially prevalent with lorazepam, which is almost exclusively excreted as lorazepam glucuronide. Some clinical laboratories have addressed this problem with the addition of beta-glucuronidase to enhance assay sensitivity as a laboratory developed test (LDT). Roche Diagnostics recently received FDA clearance to offer a benzodiazepine immunoassay that includes beta-glucuronidase. Methods Performance characteristics of two FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 urine specimens. These immunoassays were directly compared to an LC-MS/MS benzodiazepine LDT to determine clinical sensitivity and specificity. Cross-reactivity of all three immunoassays were compared and evaluated based on the measured benzodiazepine concentrations determined by the LC-MS/MS LDT. Cross-reactivity for 7-aminoclonazepam, lorazepam, and lorazepam glucuronide were assessed using drug-free urine spiked with reference materials (Cerilliant) at concentrations ranging from 100 to 1000 ng/mL. The cutoff for positive results was set at 1000 mAbs, corresponding to 200 ng/mL of the nordiazepam calibrator. Results The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all three assays. Cross-reactivity of the Benzodiazepines II assay was greater across the range of benzodiazepine concentrations tested in comparison to the other immunoassays. In particular, the Benzodiazepines II assay was the most sensitive for 7-aminoclonazepam, as it was the only immunoassay out of the three that detected the presence of specimens containing this metabolite alone. Cross-reactivity analysis shows that both 7-aminoclonazepam and lorazepam yielded positive results when tested using the Benzodiazepines II assay at a concentration of 300 ng/ml. 7-aminoclonazepam tested positive at 800 ng/ml with the LDT and Benzodiazepines Plus assays, whereas lorazepam tested positive at 600 ng/ml with these assays. Additionally, the LDT and Benzodiazepines II assays detected lorazepam glucuronide at concentrations of 400 ng/ml and 250 ng/ml, respectively. However, the Benzodiazepines Plus assay yielded negative results for all concentrations of lorazepam glucuronide tested (up to 1000 ng/mL). Conclusions A comprehensive evaluation of these three immunoassays demonstrates that the Benzodiazepines II immunoassay has increased clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The inclusion of a beta-glucuronidase greatly improved the sensitivity of the Benzodiazepines II and LDT immunoassays for lorazepam, which is primarily excreted as a glucuronide metabolite in urine.

Prevalence and blood concentrations of benzodiazepines and opioids in opioid-positive death investigations in Ontario, Canada, from 2017 to 2021.

The aim of this study was to investigate the incidence of benzodiazepines in opioid-positive death investigations, including trends in frequency and combination of drugs, as well as demographic data and blood concentrations, where available. Additionally, naloxone concentrations in polysubstance compared to opioid-only cases were analyzed. This was a retrospective study that consisted of all post-mortem toxicology cases in Ontario, Canada, from January 01, 2017, to December 31, 2021, with an opioid finding in any analyzed autopsy specimen. There were 11,033 death investigations identified. The overall rate of benzodiazepine co-involvement was 54.5%. Males accounted for the majority of cases (71%), and the most affected age group was 30- to 39-year-olds. The most frequently detected opioid was fentanyl and the most frequently detected benzodiazepine was etizolam, which was also the most frequently observed opioid/benzodiazepine combination. Findings related to differences in concentrations of opioids when naloxone was also present were mostly non-significant, except for methadone. The rate of benzodiazepine detection with opioids grew faster than opioid detections overall, potentially due to the increasingly toxic drug supply. Detection of novel psychoactive drugs fluctuated more unpredictably than opioids and benzodiazepines associated with clinical use. These findings can help inform policy decisions by public health agencies in exploring harm reduction efforts, for example, education and drug-checking services.

Water contamination by delorazepam induces epigenetic defects in the embryos of the clawed frog Xenopus laevis

Delorazepam, a derivative of diazepam, is a psychotropic drug belonging to the benzodiazepine class. Used as a nervous-system inhibitor, it treats anxiety, insomnia, and epilepsy, but is also associated with misuse and abuse. Nowadays benzodiazepines are considered emerging pollutants: conventional wastewater treatment plants indeed are unable to eliminate these compounds. Consequently, they persist in the environment and bioaccumulate in non-target aquatic organisms with consequences still not fully clear. To collect more information, we investigated the possible epigenetic activity of delorazepam, at three concentrations (1, 5 and 10 μg/L) using Xenopus laevis embryos as a model. Analyses demonstrated a significant increase in genomic DNA methylation and differential methylation of the promoters of some early developmental genes (otx2, sox3, sox9, pax6, rax1, foxf1, and myod1). Moreover, studies on gene expression highlighted an unbalancing in apoptosis/proliferation pathways and an aberrant expression of DNA-repair genes. Results are alarming considering the growing trend of benzodiazepine concentrations in superficial waters, especially after the peak occurred as a consequence of the pandemic COVID-19, and the fact that benzodiazepine GABA-A receptors are highly conserved and present in all aquatic organisms.

Comparative Study of Postmortem Concentrations of Benzodiazepines and Z-Hypnotics in Several Different Matrices.

Benzodiazepines and z-hypnotics are detected in the majority of fatal overdose cases in Norway, often in combination with other drugs of abuse, and their concentrations in peripheral blood (PB) might be important to elucidate the cause of death. In some forensic autopsies, PB is however not available. The aim of the present study was to compare concentrations of benzodiazepines and z-hypnotics in five alternative matrices to assess whether these concentrations are comparable to concentrations in PB. A total of 109 forensic autopsy cases were included. PB, cardiac blood (CB), pericardial fluid (PF), psoas muscle (PM), lateral vastus muscle (LVM) and vitreous humor (VH) from each case were analyzed using ultra high performance liquid chromatography--tandem mass spectrometry. We were able to detect clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, nitrazepam, 7-aminonitrazepam, diazepam, nordiazepam, oxazepam, alprazolam, midazolam, zopiclone and zolpidem in all the analyzed matrices. Concentrations measured in VH were generally much lower than those of PB for all compounds except zopiclone. 7-Amino metabolite concentrations were high compared to the parent compounds, although less so for the muscle samples. Concentrations of the parent nitrobenzodiazepines in muscles were higher than those in PB, but for the other compounds, concentrations in muscle showed good correspondence with PB. Both CB and PF were viable alternative matrices for PB, although a larger variation and a tendency for higher concentrations in PF were observed. This study shows that CB, PM, LVM and PF can give comparable concentrations to PB for benzodiazepines and z-hypnotics, while VH was less suitable. The concentrations in alternative matrices must, however, be interpreted carefully.

Review of findings in a case series of NPS benzodiazepines in UK deaths

Review the other drugs present and circumstances in fatalities where NPS benzodiazepines have been detected in a case series from the UK. Determine the most frequent NPS benzodiazepines involved in deaths and concentrations where possible. Benzodiazepine-type New Psychoactive Substances (NPS) have been increasingly detected in toxicology casework worldwide. These NPS benzodiazepines are potentially more potent than traditional benzodiazepines and may present an increased risk to the user, especially if other CNS depressant drugs are taken. Also, concentration data are sometimes lacking that may be useful in determining additional toxicological significance to the cause of death. As part of routine post-mortem toxicology analysis, NPS benzodiazepines have been detected, and where able, measured in blood by LC-MS-MS. Data from 35 cases involving such drugs are presented, including other drug and alcohol findings as well as the context of the death (e.g. drug related death or alternative cause). The number and frequency of different NPS benzodiazepines has been compiled along with concentrations (where available) as well as other drug findings collated by drug type. Across the 35 fatalities, 7 different NPS benzodiazepines were detected. In decreasing order of frequency; flubromazolam (13 cases), bromazolam (12), etizolam (9), flualprazolam (5), flubromazepam (1), phenazepam (1) and bromazepam (1). 80% of cases had only one NPS benzodiazepine present with 20% of cases having two NPS benzodiazepines. All fatalities were either a drug related death–83% (including cases where other drugs were present that may have been more significant than the NPS benzodiazepines) or involved an alternative cause of death–17% (including hanging, hit by a train or other medical cause). Other drugs or alcohol were detected in all cases; traditional benzodiazepines (e.g. diazepam, lorazepam, clonazepam, alprazolam) were the most common (28 detections), followed by cocaine and antidepressants (21), methadone (16), heroin/morphine (15), pregabalin (14), codeine (12), other opioids (12), alcohol and cannabis (8), zopiclone (5), ibuprofen/paracetamol (4), ketamine, antipsychotics and anticonvulsants (3), antihistamines, amphetamines and erectile dysfunction drugs (2). In terms of concentrations (where measured), flualprazolam concentrations ranged 8.7-26 ng/mL (median 20 ng/mL, n = 5), flubromazolam concentrations ranged 2.9-45 ng/mL (median 13 ng/mL, n = 11) and etizolam concentrations ranged 4.6-95 ng/mL (median 37.5 ng/mL, n = 6). As drugs with CNS depressant capabilities, NPS benzodiazepines present a risk of toxicity to users, especially if other CNS depressant drugs are co-used. This risk is increased due to the availability of these drugs often as adulterants or in falsified traditional benzodiazepines (such as diazepam and alprazolam), whereby the user may not be aware of the constituent drugs in any product consumed and this was suspected to have occurred in some of the cases featured along with use of traditional benzodiazepines as well. The review shows that poly-drug use is a particular issue with these cases; principally traditional benzodiazepines, cocaine, heroin/morphine, antidepressants, methadone and other opiates and opioids. The concentrations of NPS benzodiazepines found, where measured, provide some data for toxicologists if investigating post-mortem casework and indicate a need for sensitive qualitative and quantitative techniques. The concentrations also overlap with available data for other (non-fatal) case types such as driving under the influence so “fatal” ranges do not seem to apply. NPS benzodiazepines have been detected in a number of drug related and non-drug related deaths in the UK supporting their prevalence worldwide. Toxicologists should ensure the necessary analytical capabilities to detect what could be low concentrations despite presenting potential toxicological significance to cause of death.

Deaths related to the combination of pregabalin and other drugs of abuse: About two cases

The number of deaths involving pregabalin has dramatically increased in recent years. In the majority of the cases described in France, pregabalin is associated with opioids, mainly methadone, as well as benzodiazepines and other psychoactive drugs, notably antidepressants and antipsychotics. Association with alcohol, cannabis and psychostimulants is also mentioned in the literature (Evoy KE et al. Drugs 2021;81:125–56). In this context, pregabalin concentrations are usually high, above therapeutic concentrations, in order to achieve euphoric effects, to enhance the effects of other substances or to improve sociability (Hofmann M & Besson M. Psychiatry Res 2021;35:114193). The authors present two cases of unusual deaths, a few days apart, for which toxicological analyses, showing low concentrations of pregabalin, buprenorphine, clonazepam and psychostimulants, are in favor of new patterns of use. Case 1: a 30-year-old man with a known addiction to pregabalin and cocaine, was seen by a witness snorting buprenorphine. He was found dead two hours later. Case 2: A young homeless man is found dead in the street in the early hours of the morning. No information is available on his identity and medical history. In both cases, the autopsy performed within 48 hours only show diffuse polyvisceral congestion with major cerebral and pulmonary oedema and the pathological examination did not allow to determine the cause of death. The usual toxicological samples, including femoral blood, with sodium fluoride as preservative, were collected and complete toxicological analyses were requested. Analyses were performed in femoral blood. Alcohols were measured by HS-GC-FID. Non-targeted screening and simultaneous quantification was performed by LC-HRMS. Specific detection of drugs of abuse (DOA) was done by LC-MS/MS. In case 1, analyses showed the presence of pregabalin (4900 ng/mL), buprenorphine (2.5 ng/mL), norbuprenorphine (2.5 ng/mL), 7-aminoclonazepam (14 ng/mL), diazepam (51 ng/mL), nordiazepam (137 ng/mL), oxazepam (7 ng/mL), cocaine (4 ng/mL), benzoylecgonine (177 ng/mL), ecgonine methylester (33 ng/mL) and THC-COOH (4 ng/mL). In case 2, results showed the presence of pregabalin (5700 ng/mL), buprenorphine (4.5 ng/mL), norbuprenorphine (0.5 ng/mL), 7-aminoclonazepam (23 ng/mL), MDMA (1140 ng/mL) and MDA (54 ng/mL). Alcohol testing was negative in both cases. In both cases, the concentrations of pregabalin, benzodiazepines and buprenorphine are far below the toxic concentrations. While the risk of death when buprenorphine is combined with benzodiazepines at therapeutic doses, particularly when buprenorphine is injected or absorbed nasally (Sansone RA & Sansone LA. Innov clin NeuroSci 2015;12:32–6), is well described in the literature, the potential role of pregabalin remains to be determined. Indeed, if the combination of gabapentinoids/opioids/other depressants is known to favor the occurrence of respiratory depression, it seems that the favoring factors are a high dose, an advanced age and co-morbidities (chronic pulmonary disease, renal insufficiency in particular) (Gomes T. et al. PLoS Med 2017;14:e1002396). Finally, if the epidemiological profile of the two victims is compatible with the data found in the literature concerning pregabalin misuse (young man, history of addiction for case n o 1), these results are in favor of new patterns of use.

Screening suspect pharmaceuticals for illicit designer benzodiazepines using raman, SERS, and FT-IR prior to comprehensive analysis using LC-MS

The emergence of illicit designer benzodiazepines with high dependency and no approved clinical use are of great US public health concern. Due to the increasing numbers of illicit designer benzodiazepines encountered in the US supply chain, there is a need to develop robust analytical methods that can rapidly detect these chemicals. Suspect counterfeit tablets, powders, or liquid formulations were first screened using Raman spectroscopy and surface-enhanced Raman scattering spectroscopy (SERS) for the presence of legal or illicit benzodiazepines, and then further analyzed using Fourier-transform infrared (FT-IR) spectroscopy and liquid chromatography with tandem mass spectrometric detection (LC-MS). Several microextraction procedures were developed and used to extract benzodiazepines from samples prior to SERS, FT-IR, and LC-MS analysis. Conventional Raman analyses using handheld Raman spectrometers afforded the ability to examine samples through enclosed plastic bags but were only able to detect high concentrations of various benzodiazepines in the suspect samples. The developed SERS methods were sufficient for detecting at least one benzodiazepine in the low-dose suspect samples, thereby allowing prioritization using other analytical tools that require more sample preparation and time-consuming analyses. The use of FT-IR spectroscopy coupled with extraction and spectral subtraction was found to be selective to multiple benzodiazepines and various excipients in the analyzed samples. This study demonstrated that the developed SERS and FT-IR procedures could be used in satellite laboratories to screen suspect packages at ports of entry and prioritize samples for additional laboratory-based analyses in an effort to prevent dangerous and illicit pharmaceutical products from reaching the US supply chain.

Concentrations of psychoactive substances in blood samples from non-fatal and fatal opioid overdoses.

AimThe primary aim was to compare concentrations of psychoactive substances in blood in non‐fatal and fatal opioid overdoses. The secondary aim was to assess the concentration levels of naloxone in blood in non‐fatal overdoses and the association between naloxone findings and concomitantly detected drugs.Method DesignCase‐control study.SettingNorway. Fatal overdoses from 2017 and non‐fatal overdoses from February 2018 to September 2019.CasesThirty‐one non‐fatal and 160 fatal opioid overdose cases. Data from the non‐fatal overdoses were collected from hospital records and blood samples, and data from the fatal overdoses were collected from autopsy reports. Concentrations of psychoactive substances (including ethanol) in blood samples were collected at the time of hospital admission for the non‐fatal overdoses and during autopsy for the fatal overdoses.ResultsThe median number of different substances detected was four for fatal and five for non‐fatal overdoses. The fatal overdoses had higher pooled concentrations of opioids (188 vs 57.2 ng/mL, P < .001), benzodiazepines (5467 vs 2051 ng/mL, P = .005) and amphetamines (581 vs 121 ng/mL, P < .001) than the non‐fatal overdoses. A linear relationship between naloxone and concomitant pooled opioid concentrations was found (95% confidence interval = 0.002‐0.135, P < .05).ConclusionThe total load of drug concentrations was associated with the fatal outcome of an overdose, while the number of drugs used, to a lesser extent, differentiated between those who survived and those who died from an overdose. Higher opioid concentrations were associated with treatment with higher naloxone doses.

Prevalence and concentrations of new designer stimulants, synthetic opioids, benzodiazepines, and hallucinogens in postmortem hair samples: A 13-year retrospective study.

Hair samples are frequently analyzed in order to characterize consumption patterns of drugs. However, the interpretation of new psychoactive substance (NPS) findings in hair remains difficult because of lacking data for comparison. In this study, selected postmortem hair samples (n = 1203) from 2008 to 2020 were reanalyzed for synthetic cathinones, piperazines, phenethylamines, hallucinogens, benzodiazepines and opioids to evaluate prevalence data and concentration ranges. Hair samples were extracted using a two-step extraction procedure and analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Overall NPSs were detected in 381 cases (31.6%). Many cases were tested positive for more than one NPS in the same time span. A variety of NPS with a large range of concentrations was observed. For better comparability and interpretation of positive cases in routine work, quantitation data for 13 NPS were calculated as percentiles. The most frequently detected NPS in this study were N-ethylamphetamine, α-pyrrolidinovalerophenone, mephedrone, benzedrone, metamfepramone, and 4-fluoroamphetamine. In conclusion, a high prevalence of these drugs was observed from postmortem hair samples. The results show a growing use of many different NPSs by mainly young drug-using adults. Consequently, NPS screening procedures should be included in forensic toxicology. Our quantitative data may support other toxicologists in their assessment of NPS hair concentrations.

Blood concentrations of new psychoactive substances belonging to benzodiazepine class

There has been a global increase in the number and availability of new psychoactive substances (NPS) belonging to the benzodiazepine class. New benzodiazepines are much more dangerous to health and life than pharmaceutical benzodiazepines. These substances have been associated with many deaths. Interpreting own results usually requires a comparison to previously published cases; therefore, a referenced compilation of previously published concentration data would be useful. The data collection was based on a search of PubMed, Scopus, and Google search engine. All available data from articles and reports that measured new benzodiazepine concentrations in plasma, serum, or whole blood were included in the data analysis. The presented tables list the observed concentrations in fatal and nonfatal cases involving 13 NPS belonging to the benzodiazepine class. The interpretation of the concentrations of the NPS belonging to the benzodiazepine class is difficult. Concomitant use of benzodiazepines and other drugs as well as other factors often contribute to toxic effects or deaths in real cases. This report enables quick access to the source articles quantifying new benzodiazepines.

Benzodiazepine Concentrations in the Breast Milk and Plasma of Nursing Mothers: Estimation of Relative Infant Dose.

Objective: Benzodiazepines are common therapies for mental illness and insomnia, and are used during pregnancy and lactation. Although benzodiazepines have been shown to be transferred into breast milk, the amount transferred is small and compatible with breastfeeding. However, information is not available for all drugs. Therefore, we aimed to determine the milk to plasma (M/P) ratio and relative infant dose (RID), which are used as indicators of drug transfer to breast milk, to determine the safety of such drugs for lactating women and breastfeeding infants. Methods: The study comprised of 11 pregnant women who visited the obstetrics department of Hokkaido University Hospital (approval number: 017-0131) and Tenshi Hospital (approval number: 103) for childbirth. The samples were analyzed using liquid chromatography-tandem mass spectrometry, and the M/P ratio and RID were calculated. The condition of the mother and baby at 1 month after delivery was determined from the clinical information. The target benzodiazepines were alprazolam, brotizolam, clonazepam, clotiazepam, etizolam, ethyl loflazepate, flunitrazepam, and lorazepam. Results: For all drugs, the M/P ratios were <1 and remained constant over time. For drugs other than ethyl loflazepate, the RID values were <10%, which are considered safe; however, even with ethyl loflazepate, it was only slightly >10%. No abnormalities were found in breastfeeding infants whose mothers were receiving these medications. Conclusions: The RID results of this study suggest that drug exposure through breast milk is small; thus, maternal drug treatment and breastfeeding are compatible.

Post-mortem toxicological analyses of blood samples from 107 patients receiving opioid agonist treatment: substances detected and pooled opioid and benzodiazepine concentrations.

To present the substances and their concentrations detected post-mortem in patients receiving opioid agonist treatment (OAT) stratified by cause of death, estimate the pooled opioid and benzodiazepine concentrations using established conversion factors for blood concentrations from the Norwegian Road Traffic Act, and explore the association between drug-induced cause of death and the pooled opioid and benzodiazepine concentrations. Cross-sectional nationwide study. Norway. One hundred and seven patients who died during OAT (i.e. within 5 days after the last intake of OAT medication) between 1 January 2014 and 31 December 2015, with post-mortem femoral blood available for toxicology. Data were collected from hospital records, the Norwegian Cause of Death Registry and autopsy reports. Presence of alcohol and non-alcohol substances in the bloodstream at time of death, determined through records of toxicology of post-mortem femoral blood. A median of four substances was detected across the causes of death. At least one benzodiazepine was detected in 81 (76%) patients. The median pooled opioid concentration was significantly higher in drug-induced deaths compared with other causes of death (362 ng/mL versus 182 ng/mL, P < 0.001), in contrast to the pooled benzodiazepine concentration (5466 versus 5701 ng/mL, P = 0.353). The multivariate regression analysis showed that only increasing pooled opioid concentration (ng/ML) was associated with increased odds of a drug-induced cause of death (odds ratio, 1.003; 95% confidence interval: 1.001-1.006). In Norway, overall opioid concentration seems to play an important role in drug-induced deaths during opioid agonist treatment in patients prescribed methadone or buprenorphine. Patients prescribed buprenorphine tend to replace their agonist with full agonists, while patients prescribed methadone tend to have high opioid concentrations from methadone as the only opioid.

Driving Impairment Cases Involving Etizolam and Flubromazolam.

This study describes 12 cases of drivers stopped for impaired driving, where a designer benzodiazepine was detected, specifically etizolam or flubromazolam. Etizolam was detected in three cases, with blood concentrations ranging from 40 to 330ng/mL. Two of these cases had low concentrations of methamphetamine and/or amphetamine, and in the third case tetrahydrocannabinol (THC) was detected. Flubromazolam was detected in nine cases; in all cases, at least one other drug was detected, with THC being the most prevalent. The mean blood concentration of flubromazolam was 16.3ng/mL and had a median concentration of 17.0ng/mL, ranging from 7.0 to 31ng/mL. The low concentrations of designer benzodiazepines that produce pharmacological effects may allow many of these drugs to go undetected using routine testing in laboratories; therefore, it is necessary to include these novel compounds within validated analytical methods to reduce the chance of reporting false negative results. The prevalence in which laboratories are detecting the presence of novel benzodiazepines in impaired drivers illustrates the increased threat to public safety. These case studies demonstrate the importance of investigating agencies and forensic laboratories to be vigilant in monitoring the emerging novel psychoactive substances in their region.

The distinctive assembly pattern of ε subunit in ternary α1β3ε and binary β3ε GABAA receptors

Among all GABAA receptor subunits, ε subunit is a more recent discovery. ε subunit-containing GABAA receptors exhibit spontaneous channel activity, rapid desensitisation, low sensitivity to Zn2+, smaller GABAmediated current amplitudes and an insensitivity to benzodiazepines, despite displaying an agonistic effect at higher benzodiazepine concentrations. The promiscuous role of the ε subunit, co-assembling with other subunits forming GABAARs, may add to the complexities of the pharmacological properties of GABAARs; however, these varying pharmacological responses can be used to distinguish varying subunit combinations of these receptors. Using two electrode voltage-clamped electrophysiology, we investigated the GABAA α1β3ε and β3ε receptors and explored the effects of different stoichiometries of these receptor subtypes by varying the relative ratios of α1/β3/ε (for α1β3ε receptors) and β3/ε (for β3ε receptors) subunit complementary RNA injections into Xenopus laevis oocytes. We discovered the existence of different populations of GABAA α1β3ε and β3ε receptors, due to subunit ratio variation, in which receptors formed at each injection ratio showed different level of GABA sensitivities, spontaneous current activities and Zn2+ mediated current inhibition. These unique pharmacological features are tightly associated with various subtypes of GABAA receptors contributed by the unique assembly pattern of ε subunit.

Benzodiazepines in the oral fluid of Spanish drivers

BackgroundDriving under the influence of alcohol, illicit drugs and certain medicines is not allowed worldwide. Roadside drug testing is considered an important tool for determining such behavior. In Spain, mandatory roadside oral fluid drug testing is carried out regularly. The aim of this study was to determine the prevalence of benzodiazepines and benzodiazepines in combination with other drugs in drivers, examine benzodiazepine concentrations in drivers, and analyze the association of these factors with age and sex.MethodsThis study assessed data on Spanish drivers with confirmed drug-positive results recorded by the Spanish National Traffic Agency (Dirección General de Tráfico) between 2011 and 2016, accounting for 179,645 tests and 65,244 confirmed drug-positive tests.ResultsBenzodiazepines were confirmed in 4.3% of all positive roadside drug tests. In most of those cases (97.1%), other substances were also detected, particularly cocaine (75.3%) and cannabis (64.0%). The frequency of benzodiazepine-positive drivers (OR, 1.094; 95% CI, 1.088–1.100) increased with age, while the frequency of drivers who tested positive for benzodiazepines in conjunction with other substances, compared with drivers who tested positive for benzodiazepines alone, decreased with age (OR, 0.903; 95% CI, 0.825–0.988). Nordiazepam (54.8%) and alprazolam (46.9%) were the most common benzodiazepines detected.ConclusionConcomitant use of benzodiazepines and other psychoactive substances was found to be a common behavior among drivers who tested positive on the road. It is important to raise awareness of all those involved in the consumption of driving-impairing substances (authorities, healthcare providers, patients and their families, etc.): roadside detection of driving-impairing substances is suggested, in addition to promoting the use of fewer driving-impairing medications and the provision of clear information to patients.

Concentrations of Antidepressants, Antipsychotics, and Benzodiazepines in Hair Samples from Postmortem Cases

Certain postmortem case constellations require intensive investigation of the pattern of drug use over a long period before death. Hair analysis of illicit drugs has been investigated intensively over past decades, but there is a lack of comprehensive data on hair concentrations for antidepressants, antipsychotics, and benzodiazepines. This study aimed to obtain data for these substances. A LC-MS/MS method was developed and validated for detection of 52 antidepressants, antipsychotics, benzodiazepines, and metabolites in hair. Hair samples from 442 postmortem cases at the Institute of Legal Medicine of the Charité-University Medicine Berlin were analyzed. Postmortem hair concentrations of 49 analytes were obtained in 420 of the cases. Hair sample segmentation was possible in 258 cases, and the segments were compared to see if the concentrations decreased or increased. Descriptive statistical data are presented for the segmented and non-segmented cases combined (n = 420) and only the segmented cases (n = 258). An overview of published data for the target substances in hair is given. Metabolite/parent drug ratios were investigated for 10 metabolite/parent drug pairs. Cases were identified that had positive findings in hair, blood, urine, and organ tissue. The comprehensive data on postmortem hair concentrations for antidepressants, antipsychotics, and benzodiazepines may help other investigators in their casework. Postmortem hair analysis results provide valuable information on the drug intake history before death. Pattern changes can indicate if drug intake stopped or increased before death. Results should be interpreted carefully and preferably include segmental analysis and metabolite/parent drug ratios to exclude possible contamination.

Chronic Exposure to Oxazepam Pollution Produces Tolerance to Anxiolytic Effects in Zebrafish (Danio rerio).

Environmental concentrations of the anxiolytic drug oxazepam have been found to disrupt antipredator behaviors of wild fish. Most experiments exposed fish for a week, while evidence from mammals suggests that chronic exposure to therapeutic concentrations of benzodiazepines (such as oxazepam) results in the development of tolerance to the anxiolytic effects. If tolerance can also develop in response to the low concentrations found in the aquatic environment, it could mitigate the negative effects of oxazepam pollution. In the current study, we exposed wild-caught zebrafish to oxazepam (∼7 μg L-1) for 7 or 28 days and evaluated behavioral and physiological parameters at both time points. Females showed reduced diving responses to conspecific alarm pheromone after 7 days, but not after 28 days, indicating that they had developed tolerance to the anxiolytic effects of the drug. Zebrafish males were not affected by this oxazepam concentration, in line with earlier results. Serotonin turnover (ratio 5-HIAA/5-HT) was reduced in exposed females and males after 28 days, indicating that brain neurochemistry had not normalized. Post-confinement cortisol concentrations and gene expression of corticotropin-releasing hormone (CRH) were not affected by oxazepam. We did not find evidence that chronically exposed fish had altered relative expression of GABAA receptor subunits, suggesting that some other still unknown mechanism caused the developed tolerance.

The correlation between concentrations of zolpidem and benzodiazepines in segmental hair samples and use patterns

The aim of this study was to investigate the correlation between histories of zolpidem and benzodiazepines use and their concentrations in hair as determined by segmental hair analysis, that is, by analyzing hair samples taken 0–1, 1–2, 2–3, 3–4, 4–5, and 5–6cm etc. and 0–3cm from the scalp, and whole hair. Of the 23 hair samples examined, 18 were collected from patients in a rehabilitation program and five were from patients that had taken zolpidem only once by prescription. All 23 patients provided written informed consent after reviewing the research plan, described their zolpidem and benzodiazepines use histories accurately, and provided hair samples, which were weighed, washed, cut into lengths of <1mm, and extracted in 100% methanol for 16h (diazepam-d5 was used as an internal standard). Extracts were evaporated under reduced pressure and reconstituted with aqueous methanol (1:1 v/v). These extracts (10μL) were analyzed by Liquid Chromatography/Tandem Mass Spectrometry (LC–MS/MS). The method used was validated by determining LOD, LOQ, calibration curves, intra- and inter-accuracies, precisions, matrix effects, process efficiencies, extraction efficiencies, and processed sample stabilities. Five hundred and ninety-five 1cm hair segments showed 61.59% positive probability and 86.71% negative probability of quality correlation between zolpidem and benzodiazepines use and concentrations in hair. Good qualitative correlations were observed between drug use and detection in hair. False positivity and false negativity were very low. Of the hair samples taken from patients in a rehabilitation program, subject nos. 4, 5, and 12 had correlation coefficients of 0.68, 0.54 and 0.71, respectively, for relationships between zolpidem use and concentration of zolpidem in hair. For the 5 patients taking only a single dose of zolpidem (10mg), the average zolpidem concentrations in hair were 20, 15 and 40pg/mg after 5, 30 and 60 days, respectively. This study shows a relationship between history of zolpidem and benzodiazepines use and their concentrations in 1cm hair segment.

Differences in combinations and concentrations of drugs of abuse in fatal intoxication and driving under the influence cases

BackgroundIn toxicology, international classification systems focus on single intoxicants as the cause of death. It is, however, well known that very few drug related deaths are caused by a single substance and that information concerning the drug concentrations as well as the combinations of drugs are essential in order to ascertain the cause of death. The aim of the study was to assess whether those prone to fatal intoxications differ significantly from chronic drug users – in terms of demographics and drug exposure patterns. Material and methodsFatal psychoactive drug intoxications in Norway during 2012, where a forensic autopsy including toxicological analysis were performed, were included. Analytical findings in blood were compared with concentrations in blood from apprehended drivers under the influence of drugs and ethanol (DUID) during the same time period. The opioid and benzodiazepine concentrations were assessed as morphine and diazepam equivalents, respectively, in order to compare concentrations across the different groups. ResultsA total of 194 autopsy cases and 4811 DUID cases were included. Opioids were detected in around 90% of the drug intoxication cases, but in only 16% of the DUID cases. The number of substances detected in fatal intoxications was 4.9 compared to 2.6 in the DUID cases. The total opioid concentrations were significantly higher in the fatal intoxication cases compared to DUID cases (229ng/mL versus 56.9ng/mL morphine equivalents, respectively). Benzodiazepines were detected in 90% of the fatal cases. Only one fatal opioid mono-intoxication was found; a case with a very high methadone concentration (1238ng/mL). DiscussionMono-intoxication with heroin was not seen in any of the fatal intoxications in Norway, and single drug intoxications were rare (1.5%). Fatal intoxications were caused by a combination of drugs with significantly more substances as well as higher total drug concentrations among the fatal cases compared to the DUID cases. The combination of opioids and benzodiazepines seemed to represent an increased risk of death. ConclusionThe total load of drugs influence the degree of intoxication and the total concentration level must be considered, including the total number of substances. Our findings imply that international statistics regarding an opioid being the main intoxicant should have a shift in focus towards combinations of drugs (especially opioids and benzodiazepines) as a major risk factor for fatal drug overdoses.

Prevalence and concentrations of drugs in older suspected drugged drivers

ABSTRACTObjective: Older drivers are somewhat more likely to be involved in car crashes than middle-aged drivers but less likely to be involved than younger drivers. This study aimed to assess the extent of drug use in older suspected drunk and drugged drivers, with respect to which drugs were detected and at which concentrations.Methods: Blood samples from older suspected drunk and drugged drivers taken between February 1, 2012, and May 22, 2013, were identified from the database at the Norwegian Institute of Public Health and were retrospectively analyzed for a broad repertoire of drugs relevant for impairment. The prevalence of different drugs among the suspected drunk and drugged drivers was studied. Regarding drug concentrations, the findings in older drivers (>65 years) were compared to a reference group of apprehended drivers aged 20–40 years.Results: Four hundred and ten older suspected drunk and drugged drivers were included. Any drug (including ethanol) was detected in 92% of blood samples, and ethanol was detected in 81%. Benzodiazepines were found in 15% of the older drivers and z-hypnotics (zopiclone or zolpidem) were detected in 13%. The most frequent single legal drugs found in blood samples taken from older impaired drivers were zopiclone (9.8%) and diazepam (9.3%). Regarding drug combinations, older drivers used a mean number of 1.4 drugs, and 20% of ethanol-positive cases showed at least one other drug. High drug concentrations of clonazepam and diazepam were more frequently seen in the younger group.Conclusions: This study showed that drugs were detected in the vast majority of older drivers suspected for drunk or drugged driving. Ethanol was the most frequent drug detected, followed by zopiclone and diazepam. Older drivers combine drugs to a lesser degree than younger drivers, but their combination of ethanol and other drugs represents a considerable traffic risk. Lower concentrations of benzodiazepines are seen in older compared to younger drivers.