Quantitation of hexahydrocannabinol (HHC) and metabolites in blood from DUID-cases.

Hexahydrocannabinol (HHC) was first reported in the EU in May 2022. HHC has three chiral carbon atoms, but only (6aR,9R,10aR)-HHC (9R-HHC) and (6aR,9S,10aR)-HHC (9S-HHC) have been encountered in HHC products. The goal of this study was to develop and validate a method for the quantitative analysis of 9R-HHC, 9S-HHC, 11-OH-9R-HHC, 9R-HHC-COOH, 9S-HHC-COOH, and 8-OH-9R-HHC. In addition, an objective was to investigate the immunochemical cross reactivity. Blood samples from DUID-cases screened positive for cannabis using ELISA and confirmed negative for tetrahydrocannabinol (THC), 11-hydroxy-THC, and THC-COOH were reanalyzed with a newly validated HHC method to investigate the presence of HHC and metabolites. The LC-MS/MS method was validated for matrix effects, lower limit of quantification (LLOQ), calibration model, precision, bias, and autosampler stability. Cross reactivity on an ELISA method was investigated separately for 9R-HHC-COOH and 9S-HHC-COOH at a concentration range between 5-200 ng/mL. The cross reactivity was found to be 120% for 9R-HHC-COOH and 48% for 9S-HHC-COOH. In the LCMSMS method 9R-HHC-COOH, 9S-HHC-COOH, and 11-OH-9R-HHC showed matrix effects less than 25% at both concentrations while 8-OH-9R-HHC, 9R-HHC, and 9S-HHC matrix effects exceeded 25% at both concentrations but showed good precision (<10% for both inter and between day) and low bias (<6%) in the further validation. The LLOQ was investigated and established at 0.2 ng/mL for all analytes except the carboxylated metabolites that had an LLOQ of 2.0 ng/mL. The upper limit of quantification was 20 and 200 ng/ml respectively. Reanalysis of cases (N=145) confirmed HHC and metabolites in 32 cases (22%). It was determined that the major metabolite in blood after administration of HHC was 9R-HHC-COOH followed by 11-OH-9R-HHC and that presumptive positive cases are caught by the routine ELISA screening for cannabis.

Human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene.

Following isotonitazene scheduling in 2019, the availability of alternative 2-benzylbenzimidazole opioids (nitazenes) on the global drug market increased, resulting in many fatalities worldwide. Nitazenes are potent µ-opioid receptor agonists with strong narcotic/analgesic effects, and their concentrations in biological matrices are low, making the detection of metabolite biomarkers of consumption crucial to document use in clinical and forensic settings. However, there is little to no data on the metabolism of the most recently available nitazenes, especially desnitro-analogues. The aim of the research was to assess isotonitazene, metonitazene, etodesnitazene, and metodesnitazene human metabolism and identify specific metabolite biomarkers of consumption. The four analogues were incubated with 10-donor-pooled human hepatocytes, and the incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry and data mining with Compound Discoverer (Thermo Scientific); the analysis was supported by in silico metabolite predictions with GLORYx open-access software. Metabolites were identified in postmortem blood and/or urine samples from two metonitazene-positive and three etodesnitazene-positive cases following the same workflow, with and without glucuronide hydrolysis in urine, to confirm in vitro results. Twelve, nine, twenty-two, and ten metabolites were identified for isotonitazene, metonitazene, etodesnitazene, and metodesnitazene, respectively. The main transformations were N-deethylation at the N,N-diethylethanamine side chain, O-dealkylation, and further O-glucuronidation. In vitro and autopsy results were consistent, demonstrating the efficacy of the 10-donor-pooled human hepatocyte model to predict human metabolism. We suggest the parent and the corresponding O-dealkyl- and N-deethyl-O-dealkyl metabolites as biomarkers of exposure in urine after glucuronide hydrolysis, and the corresponding N-deethyl metabolite as additional biomarker in blood.

Characterization of neurotransmitter inhibition for seven cathinones by a proprietary fluorescent dye method.

Many new psychoactive substances (NPS) are stimulants, and information about their potency and abuse potential is often lacking. To start addressing this need, a method measuring the inhibition of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, and NET) by stimulant drugs was developed. The use of a proprietary fluorescent dye mixture and three cell lines (CHO-K1, HEK 293, and MDCK), each expressing a single transporter, allowed for a semiautomated, one-pot determination of inhibition in a 384-well format. The method was validated using well characterized stimulants, including cocaine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), α-PVP, and fluoxetine and performed similarly to other methods. Seven synthetic cathinones all showed highest potency for DAT inhibition, followed by NET and SERT. The rank potency for DAT inhibition IC50 (nM) was MPHP (4.53) > 4Cl-α-PVP (8.05) > 3F-α-PVP (12.7) > α-PiHP (13.4) > N-ethylpentylone (16.9) > N-ethylhexedrone (44.5) > 4-methylpentedrone (261). All but 4-methylpentedrone were more potent than amphetamine (257) and cocaine (111). The DAT/SERT inhibition ratio for the cathinones was in the range from 5.02 for 4-methylpentedrone to >3730 for α-PiHP, compared to 1.64 for cocaine and >4030 for α-PVP. All seven substances had inhibition profiles similar to those of potent stimulants with high abuse potential.

Rilmazafone: A designer benzodiazepine pro-drug involved in fatal intoxications.

Rilmazafone is a pro-drug that can be prescribed in Japan to treat insomnia. Rilmazafone metabolizes into active compounds by a ring closure resulting in a triazolo benzodiazepine structure similar to alprazolam. In mid-2022, the National Board of Forensic Medicine in Sweden were requested to investigate two separate deaths with the suspected use of pagoclone. Packages labeled "Pagoclone" were found at each scene that was suspected to contain rilmazafone based on website information. During screening by high resolution mass spectrometry, rilmazafone metabolites were presumptively identified. Due to the lack of reference material for the active metabolites, the metabolites were synthesized in house and quantification of the compounds identified in the two autopsy cases was prompted. In Case 1, femoral blood concentrations of 7.9, 65 and 170 ng/g of the metabolites rilmazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included the medications haloperidol, alimemazine, fluoxetine, olanzapine and acetaminophen. In Case 2, femoral blood concentrations of 1.7, 1.4 and 70 ng/g of rimazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included loperamide, alimemazine and pregabalin. The intake of rilmazafone was determined as the cause of death in Case 1 and contributed in the Case 2.

Intralipid as a matrix additive for evaluating hyperlipidemic postmortem blood.

Postmortem whole blood samples can differ greatly in quality where hyperlipemia is a frequent variable that can influence the results of analytical methods. The aim of this study was to investigate the influence of lipemia on postmortem analysis as well as demonstrate the usage of Intralipid in comparison to pooled postmortem lipids as matrix additives for meaningful evaluation and validation of hyperlipidemic postmortem samples. Hyperlipidemic blood samples were simulated by adding different concentrations of Intralipid or pooled authentic postmortem lipids to bovine whole blood. The hyperlipidemic blood samples were spiked with 14 benzodiazepines and five sedative and antianxiety drugs (alprazolam, clonazepam, 7-aminoclonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, hydroxyzine, lorazepam, midazolam, nitrazepam, 7-aminonitrazepam, nordazepam, oxazepam, propiomazine, dihydropropiomazine, temazepam, triazolam, zolpidem and zopiclone). Samples were prepared with liquid-liquid extraction followed by ultra-high performance liquid chromatography-mass spectrometry. The effects of lipemia on the recovery of analytes and internal standards (ISs) were evaluated to determine the effect of, and any differences between, the two additives. Lipemia was found to cause major interference when quantifying the analytes. For most analytes, the ISs could compensate for analyte losses. However, the most hydrophilic analytes (7-amino metabolites), together with the most lipophilic analytes (propiomazine and dihydropropiomazine), were greatly affected by lipemia (<50% recovery), and the IS could not compensate for analyte losses. In general, lower analyte recoveries were observed for samples with Intralipid as a lipemic additive in comparison to those containing pooled postmortem lipids. Both Intralipid and pooled postmortem lipids showed marked effects on the analytical results. Intralipid gave a good indication of the effects of lipemia and could be a useful tool for making a meaningful evaluation of hyperlipidemic postmortem samples during the method development and validation.

Using in vitro receptor activity studies of synthetic cannabinoids to support the risk assessment of new psychoactive substances - A Swedish strategy to protect public health from harm.

In the past 15 years, close to 1000 of new psychoactive substances (NPS) have been reported in Europe and globally. At the time of identification, data on safety, toxicity and carcinogenic potential of many NPS are not available or very limited. To work more efficiently, a strategy and collaboration between the Public Health Agency of Sweden (PHAS) and the National Board of Forensic Medicine was established involving in vitro receptor activity assays to demonstrate neurological activity of NPS. This report summarizes the first results on the synthetic cannabinoid receptor agonists (SCRAs), and subsequent actions taken by PHAS. A total of 18 potential SCRAs were selected by PHAS for in vitro pharmacological characterization. 17 compounds could be acquired and investigated for their activity on the human cannabinoid-1 (CB1) receptors expressed together with the AequoScreen system in CHO-K1 cells. Dose-response curves were established using eight different concentrations in triplicates at three occasions with JWH-018 as reference. For the MDMB-4en-PINACA, MMB-022, ACHMINACA, ADB-BUTINACA, 5F-CUMYL-PeGACLONE, 5C-AKB48, NM-2201, 5F-CUMYL-PINACA, JWH-022, 5Cl-AB-PINACA, MPhP-2201, 5F-AKB57 the half maximal effective concentration values ranged from 2.2 nM (5F-CUMYL-PINACA) to 171 nM (MMB-022). EG-018 and 3,5-AB-CHMFUPPYCA were none-active. The results contributed to 14 of these compounds being scheduled as narcotics in Sweden. In conclusion, many of the emerging SCRAs are potent activators of the CB1 receptor in vitro, although some lack activity or are partial agonists. The new strategy proved useful when data on psychoactive effects of the SCRAs under investigation were not available or limited.

Systematic In Vitro Metabolic Profiling of the OXIZID Synthetic Cannabinoids BZO-4en-POXIZID, BZO-POXIZID, 5F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID.

A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography-high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and improve detection.

In vitro metabolite identification of acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl using LC-QTOF-HRMS together with synthesized references.

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5 µM) with cryopreserved hepatocytes for up to 5 h in vitro. Metabolites were analyzed with liquid chromatography-quadrupole time of flight-high-resolution mass spectrometry (LC-QTOF-HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-phenylpropanoylfentanyl were mainly metabolized through N-dealkylation (forming nor-metabolites) and 3-fluoro-methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono-/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3-phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3-phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the ß position of the phenylpropanoyl moiety was highly favored; ß-OH-phenylpropanoylfentanyl was the most abundant metabolite after the nor-metabolite. Both metabolites have the potential to serve as biomarkers for 3-phenylpropanoylfentanyl. The nor-metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-fluoro-methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3-fluoro-methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement with findings in previous studies of similar fentanyl analogs.

Chiral separation and quantitation of methylphenidate, ethylphenidate, and ritalinic acid in blood using supercritical fluid chromatography.

Supercritical fluid chromatography (SFC) is a technique that analyzes compounds that are temperature-labile, have moderately low weight, or are chiral compounds. Methylphenidate (MPH) is a chiral compound with two chiral centers. MPH has two chiral metabolites, ethylphenidate (EPH) and ritalinic acid (RA). MPH is sold as a racemic mixture. The d-enantiomer of threo-MPH is responsible for medicinal effects. Due to the differing effects of the enantiomers, it is important to analyze the enantiomers individually to better understand their effects. This method utilizes SFCand solid-phase extraction (SPE) to separate and analyze the enantiomers of MPH, EPH, and RA in postmortem blood. The objective of this method was to assess a unique approach with SFC for enantiomeric separation of MPH, EPH, and RA. A SPE method was developed and optimized to isolate the analytes in blood and validated as fit-for-purpose following international guidelines. The linear range for MPH and EPH was 0.25-25 and 10-1000 ng/mL for RA in blood. Bias was -8.6% to 0.8%, and precision was within 15.4% for all analytes. Following method validation, this technique was applied to the analysis of 49 authentic samples previously analyzed with an achiral method. Quantitative results for RA were comparable to achiral technique, whereas there was loss of MPH and EPH over time. The l:d enantiomer ratio was calculated, and MPH demonstrated greater abundance of the d-enantiomer. This is the first known method to separate and quantify the enantiomers of all three analytes utilizing SFC and SPE.

Urinary Pharmacokinetics of Immediate and Controlled Release Oxycodone and its Phase I and II Metabolites Using LC-MS-MS.

Oxycodone (OC) is a schedule II semisynthetic opioid in the USA that is prescribed for its analgesic effects and has a high potential for abuse. Prescriptions for OC vary based on the dosage and formulation, immediate release (IR) and controlled release (CR). Monitoring OC metabolites is beneficial for forensic casework. The limited studies that involve pharmacokinetics of the urinary excretion of OC metabolites leave a knowledge gap regarding the excretion of conjugated and minor metabolites, pharmacokinetic differences by formulation, and the impact of CYP2D6 activity on the metabolism and excretion of OC. The objectives of this study were to compare urinary excretion of phase I and II metabolites by formulation and investigate if ratio changes over time could be used to predict the time of intake. Subjects (n = 7) received a single 10 mg IR tablet of Oxycodone Actavis. A few weeks later the same subjects received a single 10 mg CR tablet of Oxycodone Actavis. During each setting, urine was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 9, 10, 12, 14, 24, 48 and 72 h. Urine samples (100 µL) were diluted with 900 µL internal standard mixture and analyzed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD using a previously validated method. The CYP2D6 phenotypes were categorized as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM). Comparisons between IR and CR were performed using two-tailed paired t-test at a significance level of P = 0.05. The metabolite ratios showed a general increase over time. Four metabolite to parent ratios were used to predict the time of intake showing that predictions were best at the early time points.

Retrospective identification of new psychoactive substances in patient samples submitted for clinical drug analysis.

New psychoactive substances (NPS) are life threatening through unpredictable toxicity and limited analytical options for clinicians. We present the retrospective identification of NPS in raw data from a liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based multidrug panel analysis on 14 367 clinical oral fluid samples requested during 2019 mainly by psychiatric and addiction care clinics. Retrospectively analysed NPS included 48 notified originally in 2019 by the European Union Early Warning System (EU EWS) and 28 frequently reported in Sweden. Of 88 included NPS, 34 (mitragynine, flualprazolam, 3F/4F-α-P(i)HP, etizolam, 4F-MDMB-BINACA, cyproheptadine, 5F-MDMB-PICA, isotonitazene, isohexedrone, MDPEP, N-ethylpentedrone, tianeptine, flubromazolam, 4'-methylhexedrone, α-P(i)HP, eutylone, mephedrone, N-ethylhexedrone, 5F-MDMB-PINACA, ADB-BUTINACA, 3-methoxy PCP, 4F-furanylfentanyl, 4F-isobuturylfentanyl, acrylfentanyl, furanylfentanyl, clonazolam, norfludiazepam, 3F-phenmetrazine, 3-MMC, 4-methylpentedrone, BMDP, ethylphenidate, methylone and α-PVP) were identified as 219 findings in 84 patients. Eight NPS notified in 2019 were identified, five before EWS release. NPS occurred in 1.20% of all samples and 1.53% of samples containing traditional drugs, and in 1.87% of all patients and 2.88% of patients using traditional drugs. NPS use was more common in men and polydrug users. Legal (not scheduled) NPS were more used than comparable illegal ones. Retrospective identification could be useful when prioritizing NPS for clinical routine analysis and when studying NPS epidemiology.

Fast and Sensitive Method for the Determination of 17 Designer Benzodiazepines in Hair by Liquid Chromatography-Tandem Mass Spectrometry.

In recent years, identification and analysis of designer benzodiazepines have become a challenge in forensic toxicology. These substances are analogs of the classic benzodiazepines, but their pharmacology is not well known, and many of them have been associated with overdoses and deaths. As a result, there has been a surge in efforts to develop analytical methods to determine these compounds in different biological samples. Our aim was to develop and validate a fast, sensitive and specific method for determining 17 designer benzodiazepines (adinazolam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, N-desmethylclobazam, nifoxipam, nitrazolam, meclonazepam, pyrazolam and zolazepam) in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized, and a 20 mg aliquot was incubated in methanol in an ultrasound bath (1 h, 25°C). The supernatant was evaporated and reconstituted in 200 µL of mobile phase, and the extracts were filtered (nano-filter vials) before injection into LC-MS-MS. All analytes were eluted from the chromatographic column in 8 min, and two multiple-reaction monitoring (MRM) transitions were used to identify each compound. The limits of quantification were 5 or 25 pg/mg depending on the analyte, and the calibration functions were linear to 200 pg/mg. Imprecision was <19.2% (n = 15), and bias was from -13.7 to 18.3% (n = 15). All the analytes yielded high extraction efficiencies >70% and displayed ion suppression between -62.8% and -23.9% (n = 10). The method was applied to 19 authentic cases. Five samples were positive for flualprazolam ( 200 pg/mg) and/or etizolam (47.4-88.5 pg/mg). In conclusion, the present validated method has proven to be fast, sensitive, specific and capable of determining 17 designer benzodiazepines in hair using LC-MS-MS.

Postmortem Metabolomics Reveal Acylcarnitines as Potential Biomarkers for Fatal Oxycodone-Related Intoxication.

Postmortem metabolomics has recently been suggested as a potential tool for discovering new biological markers able to assist in death investigations. Interpretation of oxycodone concentrations in postmortem cases is complicated, as oxycodone tolerance leads to overlapping concentrations for oxycodone intoxications versus non-intoxications. The primary aim of this study was to use postmortem metabolomics to identify potential endogenous biomarkers that discriminate between oxycodone-related intoxications and non-intoxications. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry data from 934 postmortem femoral blood samples, including oxycodone intoxications and controls positive and negative for oxycodone, were used in this study. Data were processed and evaluated with XCMS and SIMCA. A clear trend in group separation was observed between intoxications and controls, with a model sensitivity and specificity of 80% and 76%. Approximately halved levels of short-, medium-, and long-chain acylcarnitines were observed for oxycodone intoxications in comparison with controls (p < 0.001). These biochemical changes seem to relate to the toxicological effects of oxycodone and potentially acylcarnitines constituting a biologically relevant biomarker for opioid poisonings. More studies are needed in order to elucidate the potential of acylcarnitines as biomarker for oxycodone toxicity and their relation to CNS-depressant effects.

Characterization of recent non-fentanyl synthetic opioids via three different in vitro µ-opioid receptor activation assays.

New synthetic opioids (NSOs) are one of the fastest growing groups of new psychoactive substances. Amid this dynamic landscape, insight into the pharmacology of NSOs is important to estimate the harm potential of newly emerging drugs. In this work, we determined the µ-opioid receptor (MOR) affinity and activation potential of seven poorly characterized non-fentanyl NSOs (N-ethyl-U-47700, 3,4-difluoro-U-47700, U-47931E/bromadoline, 2,4-difluoro-U-48800, U-62066/spiradoline, 2F-viminol, ketobemidone) and a panel of nine reference opioids. MOR affinity was determined via [3H]-DAMGO binding in rat brain tissue homogenates, and was found to correlate well with different functional parameters. MOR activation potential was studied at different levels of receptor signaling using three distinct assays (NanoBiT® MOR-ß-arrestin2/mini-Gαi and AequoScreen®). The most active compounds were ketobemidone (EC50 32.8-528 nM; Emax 105-271%, relative to hydromorphone) and N-ethyl-U-47700 (EC50 241-767 nM; Emax 139-247%). The same opioids showed the strongest MOR affinity. As most of the other NSOs only weakly activated MOR in the three assays (EC50 values in the high nM-µM range), they likely do not pose a high overdose risk. 2F-viminol (EC50 2.2-4.5 µM; Emax 21.2-61.5%) and U-47931E/bromadoline (EC50 0.55-2.9 µM; Emax 52.8-85.9%) were partial agonists compared to hydromorphone, and maximum receptor activation was not reached for 2,4-difluoro-U-48800 (EC50 > 22 µM). We further highlight the importance of considering specific assay characteristics upon interpretation of potencies, efficacies and biased agonism. As absolute values may greatly differ between assays with varying experimental set-ups, a comparison of functional parameters to those of well-characterized reference agonists is considered the most informative.

Oxycodone-Related Deaths: The Significance of Pharmacokinetic and Pharmacodynamic Drug Interactions.

BACKGROUND AND OBJECTIVES: Oxycodone is frequently prescribed as well as detected in postmortem cases. Concurrent use of pharmacodynamically or pharmacokinetically interacting drugs can cause adverse effects or even fatal intoxication. The aims of this study were to investigate differences in prescriptions for and toxicological findings of pharmacodynamically and pharmacokinetically interacting drugs in fatal oxycodone-related intoxications and other causes of death. We also aimed to investigate the differences in prevalence of oxycodone prescriptions, and the detected postmortem oxycodone concentrations between fatal oxycodone-related intoxications and other causes of death. METHODS: Forensic autopsy cases (2012-2018) where oxycodone was identified in femoral blood (n = 1236) were included. Medical history and prescription data were retrieved from national databases and linked to the forensic toxicology findings. RESULTS: Oxycodone-related deaths were found to have higher blood concentrations of oxycodone (median 0.30 µg/g vs. 0.05 µg/g) and were less likely to have a prescription for oxycodone (OR 0.62) compared to nonintoxication deaths. Pharmacodynamically interacting drugs were prescribed in 79% and found in blood in 81% of the cases. Pharmacokinetically interacting drugs were rarely prescribed (1%). Oxycodone-related deaths were more likely to have prescriptions for a pharmacodynamically interacting drug (OR 1.7) and more often have co-findings of one or multiple pharmacodynamically interacting drugs (OR 5.6). CONCLUSION: The results suggest that combined use of oxycodone and pharmacodynamically interacting drugs is associated with oxycodone-related death and that non-medical use of oxycodone is a potential risk factor for oxycodone-related intoxication.

Frequency of postmortem ethanol formation in blood, urine and vitreous humor - Improving diagnostic accuracy with the use of ethylsulphate and putrefactive alcohols.

PURPOSE: This study aimed to compare the frequency of postmortem ethanol formation in blood, urine and vitreous humor according to negative ethylsulphate (EtS) in blood or positive putrefactive alcohols (PA's) in either medium. Furthermore, it aimed to evaluate the interpretational value of calculated ethanol ratios in relation to EtS and PA results. METHODS: Blood ethanol positive forensic cases were included; one dataset consisting of 2504 cases with EtS analysed in blood and another dataset with 8001 cases where PA's were analysed. RESULTS: PA's were found in 24.4% of cases. EtS was negative in 15.3%, 9.4% and 7.4% of cases that were positive for ethanol in blood, urine and vitreous humor, respectively. In EtS negative cases, the concentrations of ethanol in blood, urine and vitreous humor were lower than 0.20 g/kg in 51.3%, 67.4% and 77.8%, respectively. It was 1.0 g/kg or higher in blood in 4.2% of cases. More EtS negative and PA positive cases were seen in central compared to peripheral blood. Ethanol ratios between urine or vitreous humor and blood were significantly lower in both EtS negative and PA positive cases, but large variations were observed. CONCLUSION: EtS and PA analysis improve the diagnostic accuracy of ethanol in postmortem cases. Postmortem ethanol formation in vitreous humor and urine were both more frequent than expected and we recommend the analysis of ethanol primarily in peripheral blood if available.

The metabolism of the synthetic cannabinoids ADB-BUTINACA and ADB-4en-PINACA and their detection in forensic toxicology casework and infused papers seized in prisons.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB-BUTINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-butyl-1H-indazole-3-carbox-amide). ADB-BUTINACA was first detected in a seizure in Sweden in 2019, and we report its detection in 13 routine Swedish forensic toxicology cases soon after. In January 2021, ADB-BUTINACA was detected in SCRA-infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA-infused papers tested between January and July 2021. In this work, ADB-BUTINACA was incubated with human hepatocytes (HHeps), and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono-hydroxylation on the n-butyl tail) and B16 (mono-hydroxylation on the indazole ring) are recommended biomarkers in blood, while metabolites B4 (dihydrodiol formation on the indazole core), B9, and B16 are suitable biomarkers in urine. ADB-4en-PINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-[pent-4-en-1-yl]-1H-indazole-3-carboxamide) was detected in Scottish prisons in December 2020, but, unlike ADB-BUTINACA, prevalence has remained low. ADB-4en-PINACA was incubated with HHeps, and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB-BUTINACA (EC50 , 11.5 nM and ADB-4en-PINACA (EC50 , 11.6 nM) are similar to that of MDMB-4en-PINACA (EC50 , 4.3 nM). A third tert-leucinamide SCRA, ADB-HEXINACA was also detected in prison samples and warrants further investigation.

The Quantification of Oxycodone and Its Phase I and II Metabolites in Urine.

The purpose of this research was to develop and validate an analytical method for the detection and quantification of noroxymorphone-3ß-D-glucuronide (NOMG), oxymorphone-3ß-D-glucuronide (NOMG), noroxymorphone (NOM), oxymorphone (OM), 6α-oxycodol (αOCL), 6ß-oxycodol (ßOCL), noroxycodone (NOC) and oxycodone (OC) in urine by liquid chromatography tandem mass spectrometry to be used in a human study. The method was validated according to the Academy Standards Board Standard Practices for Method Development in Forensic Toxicology. The method was then applied to a single-dose pilot study of a subject. Urine samples were collected from the subject after ingesting 10-mg OC as an immediate-release tablet. Additionally, urine specimens (n = 15) that had previously been confirmed positive for OC were analyzed using the validated method. The calibration range for NOMG and OMG was 0.05-10 µg/mL; for all other analytes, it was 0.015-10 µg/mL. Validation parameters such as bias, precision, carryover and dilution integrity, all met the validation criteria. After the method was validated, urine samples from the first subject in the controlled dose study were analyzed. It was observed that OC, NOC and OMG contained the highest concentrations and were present in either the 0.5 or 1 h void. NOC and OMG were detected until the 48 h collection, while OC was detectable till the 24 h collection. Time to reach maximum concentration (Tmax) in the urine was achieved within 1.5 h for OC and within 3 h for NOC and OMG. Maximum concentration (Cmax) in the urine for OC, NOC and OMG was 3.15, 2.0 and 1.56 µg/mg, respectively. OC concentrations in authentic urines ranged from 0.015 to 12 µg/mL. Ranges for NOMG and OMG were 0.054-9.7 µg/mL and 0.14-67 µg/mL, respectively. A comprehensive method for the quantification of NOMG, OMG, NOM, OM, αOCL, ßOCL, NOC and OC in urine was optimized and met the validation criteria. The concentrations of NOMG and OMG presented in this study provide the details needed in the forensic community to better comprehend OC pharmacokinetics.

Circumstances, Postmortem Findings, Blood Concentrations and Metabolism in a Series of Methoxyacetylfentanyl-Related Deaths.

Methoxyacetylfentanyl is one of many fentanyl analogs available as new psychoactive substances. It have been encountered in both the European Union and the United States, and existing literature suggest that methoxyacetylfentanyl is around 3- to 5-fold less potent than fentanyl. The aim of the present work was to combine case information with blood concentrations and abundance of urinary metabolites to investigate the importance of these parameters for toxicological interpretation. Quantification of methoxyacetylfentanyl in femoral blood was performed by LC--MS-MS and urinary metabolites were analyzed by LC--QTOF-MS with and without hydrolysis with ß-glucuronidase/arylsulfatase. For confirmation of identified metabolites, methoxyacetylfentanyl was incubated with hepatocytes for up to 5 hours and analyzed with the same method as the urine samples. In eleven postmortem cases (27 to 41 years old and including one female) methoxyacetylfentanyl was reported in femoral blood. The cause of death was intoxication by methoxyacetylfentanyl alone or in combination with other drugs in all but one case, where death was attributed to acute complications of an underlying heart disease but with possible contribution from methoxyacetylfentanyl. In total, 27 urinary metabolites were found, including eight glucuronides. Major biotransformations were O-demethylation, dealkylation to form the nor-metabolite, mono- and dihydroxylations of the phenethyl moiety, as well as combinations thereof. The most abundant metabolites in hydrolyzed urine included O-desmethyl-, O-desmethyl-phenethyl-hydroxy-, O-desmethyl-phenethyl-hydroxymethoxy- and nor-methoxyacetylfentanyl. Differences in the abundance of methoxyacetylfentanyl and its major metabolites could be interpreted to indicate fatal intoxications in abstinent or chronic users. We postulate that urinary concentrations of methoxyacetylfentanyl and two metabolites, in combination with the methoxyacetylfentanyl concentration in femoral blood, might be good indicators of the time between administration and death as well as prior use.