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.

Clinical Toxicological Follow-Up Analysis of a Suicide Attempt Using Chloroquine.

Chloroquine, a drug approved for the treatment of malaria, is frequently used to commit suicide. We report about a suicide attempt by ingesting a high dose of chloroquine in combination with other drugs. Findings of the emergency toxicology screening of blood and urine and those of the follow-up analyses in blood are discussed. Systematic toxicological analysis approaches revealed the presence of chloroquine, butylscopolamine, cafedrine, diazepam, lorazepam, metoclopramide, nordazepam, norephedrine and 11-nor-9-carboxy-∆9-tetrahydroxycannabinol in blood and/or urine of the subject. Suicide due to a combination of chloroquine and benzodiazepines is known as the so-called "Kusch method" in German-speaking countries. The initial chloroquine plasma concentration was determined to be 9.6 mg/L after precipitation and analysis by liquid chromatography-high-resolution tandem mass spectrometry. The analytical procedure was developed ad hoc and validated in accordance with international recommendations. Clinical toxicological follow-up analyses in blood were performed over a period of 3 weeks. The chloroquine concentration remained above the therapeutic range (up to 0.5 mg/L) for 2 weeks and dropped to 0.3 mg/L after 3 weeks. Furthermore, monodesethylchloroquine (MDCQ) and bisdesethylchloroquine (BDCQ) were determined to be the most abundant metabolites in plasma. Within 3 weeks, the area ratios of MDCQ and chloroquine increased 4-fold (from 0.090 to 0.350), and those of BDCQ and chloroquine increased 100-fold (from 0.002 to 0.218). This information may help to estimate the chloroquine excretion progress in the future.

Environmental concentrations of a delorazepam-based drug impact on embryonic development of non-target Xenopus laevis.

Benzodiazepines, psychotropics drugs used for treating sleep disorders, anxiety and epilepsy, represent a major class of emerging water pollutants. As occurs for other pharmaceutical residues, they are not efficiently degraded during sewage treatment and persist in effluent waters. Bioaccumulation is already reported in fish and small crustaceans, but the impact and consequences on other "non-target" aquatic species are still unclear and nowadays of great interest. In this study, we investigated the effects of a pharmaceutical preparation containing the benzodiazepine delorazepam on the embryogenesis of Xenopus laevis, amphibian model species, taxa at high risk of exposure to water contaminants. Environmental (1 µg/L) and two higher (5 and 10 µg/L) concentrations were tested on tadpoles up to stage 45/46. Results demonstrate that delorazepam interferes with embryo development and that the effects are prevalently dose-dependent. Delorazepam reduces vitality by decreasing heart rate and motility, induces marked cephalic and abdominal edema, as well as intestinal and retinal defects. At the molecular level, delorazepam increases ROS production, modifies the expression of some master developmental genes and pro-inflammatory cytokines. The resulting stress condition significantly affects embryos' development and threatens their survival. Similar effects should be expected as well in embryos belonging to other aquatic species that have not been yet considered targets for these pharmaceutical residues.

Physiologically Based Pharmacokinetic and Pharmacodynamic Modeling of Diazepam: Unbound Interstitial Brain Concentrations Correspond to Clinical End Points.

Benzodiazepines induce a series of clinical effects by modulating subtypes of γ-aminobutyric acid type A receptors in the central nervous system. The brain concentration-time profiles of diazepam that correspond to these effects are unknown, but can be estimated with physiologically based pharmacokinetic (PBPK) modeling. In this study, a PBPK model for the 1,4-benzodiazepines diazepam and nordiazepam was developed from plasma concentration-time courses with PK-Sim software to predict brain concentrations. The PBPK model simulations accurately parallel plasma concentrations from both an internal model training data set and an external data set for both intravenous and peroral diazepam administrations. It was determined that the unbound interstitial brain concentration-time profiles correlated with diazepam pharmacodynamic end points. With a 30-mg intravenous diazepam dose, the peak unbound interstitial brain concentration from this model is 160 nM at 2 minutes and 28.9 nM at 120 minutes. Peak potentiation of recombinant γ-aminobutyric acid type A receptors composed of α1ß2γ2s, α2ß2γ2s, and α5ß2γ2s subunit combinations that are involved in diazepam clinical endpoints is 108%, 139%, and 186%, respectively, with this intravenous dose. With 10-mg peroral administrations of diazepam delivered every 24 hours, steady-state peak and trough unbound interstitial brain diazepam concentrations are 22.3 ± 7.5 and 9.3 ± 3.5 nM. Nordiazepam unbound interstitial brain concentration is 36.1 nM at equilibrium with this diazepam dosing schedule. Pharmacodynamic models coupled to the diazepam unbound interstitial brain concentrations from the PBPK analysis account for electroencephalographic drug effect, change in 13- to 30-Hz electroencephalographic activity, amnesia incidence, and sedation score time courses from human subjects.

Rapid Extraction and Qualitative Screening of 30 Drugs in Oral Fluid at Concentrations Recommended for the Investigation of DUID Cases.

A rapid, simple extraction method followed by qualitative screening using liquid chromatography-tandem mass spectrometry (LC-MS-MS) for drugs in oral fluid is presented. The decision points were selected to be at, or lower, than those recommended as Tier I compounds by the National Safety Council's Alcohol, Drugs and Impairment Division for toxicological investigation of driving under the influence of drug (DUID) cases and were also at, or lower, than those recommended by Substance Abuse and Mental Health Service Administration and the Department of Transportation for Federal workplace drug testing programs. The method included 30 drugs: delta-9-tetrahydrocannabinol, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine, benzoylecgonine, carisoprodol, meprobamate, zolpidem, alprazolam, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, lorazepam, oxazepam, temazepam, codeine, morphine, 6-acetylmorphine, buprenorphine, fentanyl, hydrocodone, hydromorphone, oxycodone, oxymorphone, methadone, tramadol and phencyclidine. Phencyclidine was included because it is in the Federal workplace program even though it is considered a Tier II drug for DUID cases. A liquid-liquid extraction method using isopropanol, hexane and ethyl acetate to extract drugs from the oral fluid-buffer mix collected in a Quantisal™ device, followed by LC-MS-MS screening, was developed and validated according to ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Interference studies, limit of detection, precision at the decision point, ionization suppression/enhancement and processed sample stability were determined for each drug. The method was successfully applied to proficiency specimens and routine samples received in the laboratory.

TSDC and DSC Investigation on the Molecular Mobility in the Amorphous Solid State and in the Glass Transformation Region of Two Benzodiazepine Derivatives: Diazepam and Nordazepam.

In this work we study the molecular mobility in the amorphous solid state and in the glass transformation region of two compounds, diazepam and nordazepam; these are two benzodiazepines, a family of psychotropic drugs with sedative, anxiolytic and muscle-relaxing properties. The experimental techniques used are thermostimulated currents (TSC) and differential scanning calorimetry (DSC). TSC is a time-dependent technique recognized for its high resolving power; the use of this technique in the depolarization and polarization modes (TSDC and TSPC respectively), provides results that confirm and complement results of dielectric relaxation spectroscopy (DRS) published recently. On the other hand, the variation with the heating rate of the temperature position of the DSC glass transition signal also allowed the estimation of the activation energy at Tg and of the dynamic fragility of the two glass formers.

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.

Kinetic disposition of diazepam and its metabolites after intravenous administration of diazepam in the horse: Relevance for doping control.

In horses, the benzodiazepine diazepam (DIA) is used as sedative for pre-medication or as an anxiolytic to facilitate horse examinations. As the sedative effects can also be abused for doping purposes, DIA is prohibited in equine sports. DIA is extensively metabolized to several active metabolites such as nordazepam, temazepam and oxazepam (OXA). For veterinarians, taking into account the detection times of DIA and its active metabolites is needed for minimizing the risk of an anti-doping rule violation. Therefore, a pharmacokinetic study on 6 horses was conducted using a single intravenous (IV) dose of 0.2 mg/kg DIA Plasma and urine samples were collected at specified intervals until 16 and 26 days post-administration, respectively. Samples were analysed by a sensitive liquid chromatography-electrospray ionization/tandem mass spectrometry method. DIA showed a triphasic elimination pattern in the horse. The mean plasma clearance of DIA was 5.9 ml/min/kg, and the plasma elimination half-life in the terminal phase was 19.9 h. Applying the Toutain model approach, an effective plasma concentration of DIA was estimated at 24 ng/ml, and irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were computed to 0.047 and 0.1 ng/ml, respectively. The detection time according to the European Horserace Scientific Liaison Committee (EHSLC), that is the time for which observed DIA plasma concentrations of all investigated horses were below the IPC was 10 days. Using Monte Carlo Simulations, it was estimated that concentrations of DIA in plasma would fall below the IPC 18 days after the DIA administration for 90% of horses. However, in the present study, a single administration of DIA could be detected for 24 days in urine via the presence of OXA, its dominant metabolite.

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.

Comparative Physical Study of Three Pharmaceutically Active Benzodiazepine Derivatives: Crystalline versus Amorphous State and Crystallization Tendency.

Chemical derivatization and amorphization are two possible strategies to improve the solubility and bioavailability of drugs, which is a key issue for the pharmaceutical industry. In this contribution, we explore whether both strategies can be combined by studying how small differences in the molecular structure of three related pharmaceutical compounds affect their crystalline structure and melting point (Tm), the relaxation dynamics in the amorphous phase, and the glass transition temperature (Tg), as well as the tendency toward recrystallization. Three benzodiazepine derivatives of almost same molecular mass and structure (Diazepam, Nordazepam and Tetrazepam) were chosen as model compounds. Nordazepam is the only one that displays N-H···O hydrogen bonds both in crystalline and amorphous phases, which leads to a significantly higher Tm (by 70-80 K) and Tg (by 30-40 K) compared to those of Tetrazepam and Diazepam (which display similar values of characteristic temperatures). The relaxation dynamics in the amorphous phase, as determined experimentally using broadband dielectric spectroscopy, is dominated by a structural relaxation and a Johari-Goldstein secondary relaxation, both of which scale with the reduced temperature T/Tg. The kinetic fragility index is very low and virtually the same (mp ≈ 32) in all three compounds. Two more secondary relaxations are observed in the glass state: the slower of the two has virtually the same relaxation time and activation energy in all three compounds, and is assigned to the inter-enantiomer conversion dynamics of the flexible diazepine heterocycle between isoenergetic P and M conformations. We tentatively assign the fastest secondary relaxation, present only in Diazepam and Tetrazepam, to the rigid rotation of the fused diazepine-benzene double ring relative to the six-membered carbon ring. Such motion appears to be largely hindered in glassy Nordazepam, possibly due to the presence of the hydrogen bonds. Supercooled liquid Tetrazepam and Nordazepam are observed to crystallize into their stable crystalline form with an Avrami exponent close to unity indicating unidimensional growth with only sporadic nucleation, which allows a direct assessment of the crystal growth rate. Despite the very similar growth mode, the two derivatives exhibit very different kinetics for a fixed value of the reduced temperature and thus of the structural relaxation time, with Nordazepam displaying slower growth kinetics. Diazepam does not instead display any tendency toward recrystallization over short periods of time (even close to Tm). Both these observations in three very similar diazepine derivatives provide direct evidence that the kinetics of recrystallization of amorphous pharmaceuticals is not a universal function, at least in the supercooled liquid phase, of the structural or the conformational relaxation dynamics, and it is not simply correlated with related parameters such as the kinetic fragility or activation barrier of the structural relaxation. Only the crystal growth rate, and not the nucleation rate, shows a correlation with the presence or absence of hydrogen bonding.

Postmortem Analysis of Benzodiazepines in Human Bone by Gas Chromatography-Mass Spectrometry.

A procedure based on gas chromatography-mass spectrometry was developed for the analysis of benzodiazepines (nordiazepam, oxazepam, lormetazepam, lorazepam, clonazepam, bromazepam and alprazolam) in postmortem human ribs. Powdered bone samples, including marrow remains inside, with the internal standard diazepam-d5 were subjected to enzymatic hydrolysis with 100 µL of ß-glucoronidase and were incubated in sodium hydroxide for 1 h in a 70°C oven. Samples underwent liquid phase extraction and ethyl acetate was used as eluent. Chromatography was performed on a fused silica capillary column and the selected-ion-monitoring mode was used for analytes determination. The method was validated in the range 0.1-0.5 ng/mg (depending on the benzodiazepine) to 100 ng/mg with average values of recovery, matrix effect and process efficiency ranged from 83.2 to 94.3%, from 97.3 to 102.1% and from 80.5 to 91.2%, respectively. The intra- and inter-day accuracy was <15%. The procedure was tested in rib specimens obtained during routine autopsies from 20 cases where these benzodiazepines were found in blood. Benzodiazepines were detected in the combined bone and marrow samples in 60% of cases. Lorazepam was detected in bone in the range of 0.3-0.7 ng/mg, nordiazepam at 1.3-4.2 ng/mg and oxazepam at 1.1-1.2 ng/mg. To our knowledge, this protocol for the simultaneous analysis of these benzodiazepines is the first performed and validated using human ribs.

LC-MS-MS Method for the Determination of Antidepressants and Benzodiazepines in Meconium.

An LC-MS-MS method for the determination of 14 benzodiazepines (BZDs) (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (ADs) (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25 ± 0.02 g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed phase, with a gradient of 0.1% formic acid in 2 mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of ADs and another for BZDs. Analytes were monitored by tandem mass spectrometry employing electrospray positive mode in MRM mode (2 transitions per compound). Method validation included: linearity [n = 5, limit of quantification (LOQ) to 400 ng/g], limits of detection (n = 6, 1-20 ng/g), LOQ (n = 9, 5-20 ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n = 15, 90.6-111.5%), imprecision (n = 15, 0-14.6%), matrix effect (n = 10, -73 to 194.9%), extraction efficiency (n = 6, 35.9-91.2%), process efficiency (n = 6, 20.1-188.2%), stability 72 h in the autosampler (n = 3, -8.5 to 9%) and freeze/thaw stability (n = 3, -1.2 to -47%). The method was applied to four meconium specimens, which were analyzed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC-MS-MS method allows a high throughput determination of the most common BZDs and ADs in meconium, which could be useful in clinical and forensic settings.

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.

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.

Validation of an LC-MS/MS Method for the Quantification of 13 Designer Benzodiazepines in Blood.

The misuse of designer benzodiazepines, as an alternative to prescription benzodiazepines and for drug-facilitated sexual assaults, has emerged as a growing threat, due in part to the ease of purchasing these drugs on the internet at low prices. Causing concern for safety is the lack of dosage information resulting in users self-medicating, often leading to unintended overdoses, coma or death at higher doses. With limited published data regarding the quantification of designer benzodiazepines in forensic cases, a method was validated for the determination of 13 designer benzodiazepines in postmortem blood, to add to the in-house method that already included a limited number of common designer benzodiazepines. The developed method included 3-hydroxyphenazepam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, meclonazepam, nifoxipam and pyrazolam in 0.5 mL postmortem blood using liquid chromatography-tandem mass spectrometry. The analytes were treated with solid phase extraction before undergoing separation on a C18 column and analyzed on the mass spectrometer in electrospray positive mode using multiple reaction monitoring. The linear range of the calibration curve was 1-200 ng/mL and up to 500 ng/mL for 3-hydroxyphenazepam, clobazam, flubromazepam and pyrazolam. The limits of detection and quantitation were 0.5 ng/mL (signal-to-noise ratio >3) and 1 ng/mL, respectively. The calculated bias, intra-day imprecision, relative standard deviation (RSD) and inter-day imprecision RSD were ±12%, 3-20% and 4-21%. Matrix effects ranged from -52% to 33% with RSD values ranging from 3-20%, indicating consistent effects throughout multiple sources. Recovery ranged from 35 to 90%, where only two compounds were <50%. Other parameters tested included carryover, stability, interference and dilution integrity, which all yielded acceptable results. With the application of this method to blood specimens from the New York City Office of Chief Medical Examiner, this validated method proved to be simple, reproducible, sensitive and robust.

Acidic transformation of nordiazepam can affect recovery estimate during trace analysis of diazepam and nordiazepam in environmental water samples by liquid chromatography-tandem mass spectrometry.

In this study, a special interest was focused on the stability of diazepam and nordiazepam in aqueous samples at acidic and neutral pH. The aim of the study was to isolate and illustrate one of the many possible sources of error that can be encountered when developing and validating analytical methods. This can be of particular importance when developing multi-analyte methods where there is limited time to scrutinize the behavior of each analyte. A method was developed for the analysis of the benzodiazepines diazepam and nordiazepam in treated wastewater. The samples were extracted by solid phase extraction, using SPEC C18AR cartridges, and analyzed by the use of liquid chromatography, with a C18 stationary phase, coupled to tandem mass spectrometry. Environmental water samples are often acidified during storage to reduce the microbial degradation of the target compounds and to preserve the sample. In some cases, the samples are acidified before extraction. In this study, it was found that a chemical equilibrium between nordiazepam and a transformation product could cause inaccurately high extraction recovery values when the samples were stored at low sample pH. The stability of nordiazepam was shown to be low at pH 3. Within 12 days, 20% of the initial concentration of nordiazepam was transformed. Interestingly, the transformed nordiazepam was shown to be regenerated and reformed to nordiazepam during sample handling. At a sample pH of 7, diazepam and nordiazepam were stable for 12 days. It was concluded that great care must be taken when acidifying water samples containing nordiazepam during storage or extraction. The storage and the extraction should be conducted at neutral pH if no internal standard is used to compensate for degradation and conversion of nordiazepam. The developed method was validated in treated wastewater and applied for the quantification of diazepam and nordiazepam in treated wastewater samples.

The unmasking of hidden severe hyponatremia after long-term combination therapy in exacerbated bipolar patients: a case series.

Hyponatremia is occasionally unmasked in psychiatric patients during hospitalization after routine blood and urinary tests, and correlates in most cases with an inappropriate secretion of antidiuretic hormone, mainly due to iatrogenic factors. Only a few studies have regarded the combination of psychotropic drugs as triggers of chronic, asymptomatic hyponatremia in bipolar patients, who require to be hospitalized because of the exacerbation of their mental illness. We presented three clinical cases of patients affected by a long-term psychiatric disorder and under polypharmacotherapy for several months. After excluding other potential factors, we hypothesized that pharmacological treatment with a mood stabilizer (oxcarbazepine) associated with a benzodiazepine (delorazepam), a second-generation antipsychotic (olanzapine) or an antidepressant (fluvoxamine), triggered severe hyponatremia ([Na+] ≤125 mEq/L), serum hypo-osmolarity, and elevated inappropriate urine osmolarity added to more diluted sodium concentration. When we discontinued the treatment, clinical conditions of our patients improved, despite the previous administration of hypertonic saline jointly with water restriction. Psychiatrists should consider that bipolar patients on long-term polypharmacotherapy may present a higher risk of severe hyponatremia not clinically detectable. Consequently, routine laboratory tests should be periodically repeated as they represent the only available tool to unmask such electrolyte imbalances.