Deposition of diazepam and its metabolites in hair following a single dose of diazepam.

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.

Entomotoxicology in burnt bodies: a case of maternal filicide-suicide by fire.

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.

A comparative study of primary secondary amino (PSA) and multi-walled carbon nanotubes (MWCNTs) as QuEChERS absorbents for the rapid determination of diazepam and its major metabolites in fish samples by high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry.

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.

Occurrence of Z-drugs, benzodiazepines, and ketamine in wastewater in the United States and Mexico during the Covid-19 pandemic.

Z-drugs, benzodiazepines and ketamine are classes of psychotropic drugs prescribed for treating anxiety, sleep disorders and depression with known side effects including an elevated risk of addiction and substance misuse. These drugs have a strong potential for misuse, which has escalated over the years and was hypothesized here to have been exacerbated during the COVID-19 pandemic. Wastewater-based epidemiology (WBE) constitutes a fast, easy, and relatively inexpensive approach to epidemiological surveys for understanding the incidence and frequency of uses of these drugs. In this study, we analyzed wastewater (n = 376) from 50 cities across the United States and Mexico from July to October 2020 to estimate drug use rates during a pandemic event. Both time and flow proportional composite and grab samples of untreated municipal wastewater were analyzed using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry to determine loadings of alprazolam, clonazepam, diazepam, ketamine, lorazepam, nordiazepam, temazepam, zolpidem, and zaleplon in raw wastewater. Simultaneously, prescription data of the aforementioned drugs were extracted from the Medicaid database from 2019 to 2021. Results showed high detection frequencies of ketamine (90 %), lorazepam (87 %), clonazepam (76 %) and temazepam (73 %) across both Mexico and United States and comparatively lower detection frequencies for zaleplon (22 %), zolpidem (9 %), nordiazepam (<1 %), diazepam (<1 %), and alprazolam (<1 %) during the pandemic. Average mass consumption rates, estimated using WBE and reported in units of mg/day/1000 persons, ranged between 62 (temazepam) and 1100 (clonazepam) in the United States. Results obtained from the Medicaid database also showed a significant change (p < 0.05) in the prescription volume between the first quarter of 2019 (before the pandemic) and the first quarter of 2021 (pandemic event) for alprazolam, clonazepam and lorazepam. Study results include the first detections of zaleplon and zolpidem in wastewater from North America.

Pharmacokinetics of Diazepam and Its Metabolites in Urine of Chinese Participants.

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 (Cmax) and 1.93 h (Tmax). 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 (t½z) and clearance (CLz/F) for diazepam were 119.58 h and 65.77 L/h, respectively. The t½z 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.

A case report on fatal intoxication by tapentadol: Study of distribution and metabolism.

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.

Evaluation of the diagnostic performance of an oral fluid screening test device for substance abuse at traffic controls.

INTRODUCTION: The aim of this study was to evaluate the analytical performance of the Kite Biotechnology Oral fluid (OF) screening test device, which is used for roadside screening of cannabis, opiates, amphetamines, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), cocaine and benzodiazepines by comparing samples with matched plasma samples, analysed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) for confirmation. METHODS: OF and plasma samples were obtained simultaneously from a total of 100 subjects. OF samples were analysed by OF screening test based on immunochromatography. The OF screening test cut-off values were 50 ng/mL for amphetamines (d-amphetamine) and methamphetamine/MDMA (d-methamphetamine), 30 ng/mL for cocaine (benzoylecgonine), 40 ng/mL for opiates (morphine), 20 ng/mL for benzodiazepines (nordazepam), and 25 ng/mL for cannabis (Δ9-tetrahydrocannabinol). LC-MS/MS method validation was performed according to the CLSI C62-A recommendations with the following parameters: matrix effect, lower limit of quantification (LLOQ), linearity, intra-day and inter-day precision and accuracy. RESULTS: The overall specificity, accuracy and negative predictive values (NPV) were acceptable and met the DRUID standard of >80%. The OF screening test device showed good sensitivity for cocaine, amphetamines and opiates, whereas it indicated poor sensitivity for methamphetamine/MDMA (66.7%) and failed to detect cannabis and benzodiazepines. CONCLUSION: The present study is the first report to evaluate the Kite Biotechnology OF screening test device. The diagnostic performance of the OF screening test device was acceptable for opiates, cocaine and amphetamines, but it was insufficient for methamphetamine/MDMA, benzodiazepines and cannabis because of sensitivity issues.

An Experimental Pharmacokinetics Study of Diazepam and Its Metabolites in Oral Fluid of Chinese Population.

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.

Demoxepam Derivatization and GC-MS Analysis Produces Erroneous Nordiazepam and Oxazepam Results.

Demoxepam, when derivatized by silylation and analyzed using gas chromatography-mass spectrometry (GC-MS), produces artifacts which are falsely identified as nordiazepam and oxazepam. Demoxepam was analyzed unextracted at various concentrations, using different derivatization procedures, and on different GC-MS systems. Oxazepam and nordiazepam were consistently identified in neat demoxepam samples, despite the changing variables. Under certain conditions, oxazepam was identified as low as 50 ng/mL derivatized demoxepam, and nordiazepam identified as low as 500 ng/mL derivatized demoxepam. The analysis of underivatized demoxepam resulted in nordiazepam detection at levels ≥2,500 ng/mL, whereas oxazepam was not detectable at or below 10,000 ng/mL demoxepam. Isolating the derivatization procedures and GC-MS analyses demonstrates that these processes are responsible for any degradation or rearrangement reactions which are taking place. Laboratories which follow similar procedures for benzodiazepine confirmations should consider these findings when interpreting analytical data from chlordiazepoxide cases.

Organ distribution of diclazepam, pyrazolam and 3-fluorophenmetrazine.

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.

Cardiac and peripheral blood similarities in the comparison of nordiazepam and bromazepam blood concentrations.

Concomitant heart and peripheral blood determinations were performed on 40 fatal cases involving nordiazepam (20 cases) and bromazepam (20 cases). The heart blood concentration for the two drugs (588 ng/mL for nordiazepam and 802 ng/mL for bromazepam) does not differ from the corresponding peripheral blood concentration (587 ng/mL for nordiazepam and 883 ng/mL for bromazepam). The mean ratios for the heart and peripheral blood concentrations were 0.95 for nordiazepam and 0.86 for bromazepam. No postmortem redistribution was observed for these two benzodiazepines. The authors thus suggest that corresponding heart blood can be proposed in the quantitative analysis of these drugs when peripheral blood is unavailable. The present study also shows the stability of the two drugs after a year of storage.

Pentobarbital in the context of possible suicides: Analysis of a Case.

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.

Methane negative chemical ionization analysis of 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones.

The methane negative chemical ionization (NCI) mass spectra of the medically important 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones generally consisted solely of M- and (M-H)- ions. Attempts to find the location of the H lost in the generation of the (M-H)- ion were unsuccessful, although many possibilities were eliminated. A Hammett correlation analysis of the relative sensitivities of a series of 7-substituted benzodiazepines suggested that the initial ionization takes place at the 4,5-imine bond. For certain benzodiazepines, the (M-H)- ion generated by methane NCI was 20 times more intense than the MH+ ion generated by methane positive chemical ionization (PCI). By using NCI, a sensitive and simple GC-MS assay for nordiazepam was developed that can quantitate this important metabolite of many of the clinically used benzodiazepines in the blood and brain of rats.

Long-lasting anticonvulsant effects of diazepam in different mouse strains: correlations with brain concentrations and receptor occupancy.

There were marked strain differences in the duration of the protective effects of diazepam against the convulsant actions of penylenetetrazole and picrotoxin in mice. In no case was significant protection found at 12 h or longer, regardless of whether the incidence of or the latencies to myoclonus or tonic-clonic convulsions were considered. These behavioural differences could not be explained simply in terms of strain differences in benzodiazepine metabolism or in percent of receptor occupancy, as determined by the fractional displacement of 3H-flunitrazepam binding in vivo. It is suggested that there might be strain differences in the percent of receptor occupancy needed in order to produce an anticonvulsant effect.

Automation of toxicological screenings on a Hewlett Packard Chemstation GC-MS system.

OBJECTIVES: To optimize the interpretation of GC-MS toxicological screenings (i.e., to facilitate ion specific queries, create custom reports specifically adapted to each confirmation procedure, and eliminate redundant and/or inaccurate data on library search reports). DESIGN AND METHODS: The MS Chemstation software of the Hewlett Packard 5972 is constructed in a modular way. We made extensive modifications to two modules, the data analysis and the report modules, using the built-in MS Chemstation macro language. RESULTS: Ion specific queries were automated for over 60 commonly encountered analytes. Custom reports were created for the confirmation of positive drugs-of-abuse immunoassay results. With the incorporation of decision support rules into the data processing and the reporting phases, we obtained sensitive, accurate, and concise reports. CONCLUSIONS: The MS Chemstation software can be tailored to the needs of each individual application. The incorporation of a rule-based decision support system enhances the quality of the GC-MS toxicological screenings and results in faster, easier, and more reliable processing.

Application of microdialysis to study the in vitro metabolism of drugs in liver microsomes.

Current methods for studying in vitro drug metabolism involve add-incubate-separate-measure approach. Separation of the desired analytes requires removal of protein which is typically accomplished by precipitation and centrifugation and extraction of the analytes into an organic phase. The analysis scheme then becomes more complex resulting in a decrease in precision and an increase in assay time. Microdialysis sampling circumvents these problems by allowing researchers to sample the reaction mixture periodically and obtain the complete metabolic profile. In the present study, microdialysis sampling was used to investigate Phase I metabolism of salicylic acid, diazepam and ibuprofen in rat liver microsomes. The major metabolites of these drugs were profiled by LC. Michaelis-Menten enzyme kinetic parameters, Km and Vmax were obtained for the formation of diazepam metabolites by both microdialysis and conventional microsomal incubations and were in good agreement with the values reported in the literature. This study shows that microdialysis has considerable promise as a sampling technique for in vitro drug metabolism studies. By making minor modifications to the instruments, microdialysis can be applied to other in vitro systems such as isolated hepatocytes to study the Phase II metabolism or tissue slices to study drug distribution.

Determination of benzodiazepines in human urine and plasma with solvent modified solid phase micro extraction and gas chromatography; rationalisation of method development using experimental design strategies.

Solid phase micro extraction (SPME) and gas chromatographic analysis was used for the analysis of several benzodiazepines (oxazepam, diazepam, nordiazepam, flunitrazepam and alprazolam) in human urine and plasma. Several factors likely to affect the analyte recovery were screened in a fractional factorial design in order to examine their effect on the extraction recovery. Parameters found significant in the screening were further investigated with the use of response surface methodology. The final conditions for extraction of benzodiazepines were as follows: Octanol was immobilised on a polyacrylate fibre for 4 min. The fibre was placed in the sample and extraction took place at pH 6.0 for 15 min. Urine samples were added to 0.3 g ml(-1) sodium chloride. In plasma, the extraction recovery was less than in urine and releasing the benzodiazepines from plasma proteins followed by protein precipitation was found necessary prior to sampling. The method was validated and found linear over the range of samples. The limits of detection in urine were determined to be in the range 0.01-0.45 micromol l(-1). The corresponding limits of detection in plasma were in the range 0.01-0.48 micromol l(-1). Finally, the method developed was applied to determine some benzodiazepines after administration of a single dose. This method offers sufficient enrichment for bioanalysis after a single dose of high dose benzodiazepines as diazepam, but for low dose benzodiazepines as flunitrazepam, further sensitivity is needed.

Radioimmunoassay for determination of prazepam and its metabolites.

The development of a radioimmunoassay for the analysis of prazepam and its metabolites is described. It includes the preparation of the immunogen, immunization and measurement of specificity and sensitivity.

Buprenorphine in drug-facilitated sexual abuse: a fatal case involving a 14-year-old boy.

The first case involving repetitive sexual abuse linked to the use of buprenorphine is reported. Under the tradename Subutex, buprenorphine is largely used for the substitution management of opiate-dependent individuals, but it can also be easily found on the black market. A 14-year-old boy was found dead at the home of a well-known sex offender of minors. At the autopsy, no particular morphological changes were noted, except for pulmonary and visceral congestion. There was no evidence of violence, and no needle marks were found by the pathologist. Toxicological analyses, as achieved by liquid chromatography-mass spectrometry, demonstrated both recent and repetitive buprenorphine exposure in combination with nordiazepam. Buprenorphine concentrations were 1.1 ng/mL and 23 pg/mg in blood and hair, respectively. The boy's death was attributed to accidental asphyxia in a facilitated repetitive sexual abuse situation due to the combination of buprenorphine and benzodiazepines, even at therapeutic concentrations. The use of buprenorphine as a sedative drug was not challenged by the perpetrator.

Feto-maternal concentrations of diazepam and W-demethyldiazepam after intra-amniotic diazepam injection.

Ten milligrams of diazepam were injected intraamniotically in 8 mothers prior to therapeutic abortion between 12 and 19 weeks. The diazepam concentrations in the maternal plasma were comparable to those found after the same intramuscular diazepam dose to the mother. The concentration of diazepam in the amniotic fluid 12 to 18 hours after the injection was no longer significantly higher than in the maternal plasma. The concentrations of diazepam in the fetal plasma, liver and brain were comparable to the concentrations resulting from a 10 mg intramuscular diazepam dose to the mother about 2 hours before legal abortion. The feto-maternal ratio of diazepam was of same magnitude as after the intramuscular application to the mother. The results indicate that the disappearance of diazepam from the amniotic fluid in this stage of pregnancy occurs extraplacentally, through the mambranes into the uterine circulation. In the treatment of a fetus with drugs having properties similar to diazepam, intra-amniotic administration is no better than intramuscular administration to the mother.