Metabolism of ADB-FUBIATA in zebrafish and pooled human liver microsomes investigated by liquid chromatography-high-resolution mass spectrometry.

RATIONALE: ADB-FUBIATA is one of the most recently identified new psychoactive substance (NPS) of synthetic cannabinoids. The co-use of in vitro (human liver microsomes) and in vivo (zebrafish) models offers abundant metabolites and may give a deep insight into the metabolism of NPS. METHODS: In vivo and in vitro metabolic studies of new synthetic cannabinoid ADB-FUBIATA were carried out using zebrafish and pooled human liver microsome models. Metabilites were structurally characterized by liquid chromatography-high-resolution mass spectrometry. RESULTS: In total, 18 metabolites were discovered and identified in the pooled human liver microsomes and zebrafish, including seventeen phase I metabolites and one phase II metabolite. The main metabolic pathways of ADB-FUBIATA were hydroxylation, dehydrogenation, N-dealkylation, amide hydrolysis, glucuronidation, and combination thereof. CONCLUSION: Hydroxylated metabolites can be recommended as metabolic markers for ADB-FUBIATA because of the structural characteristics and high intensity. These metabolism characteristics of ADB-FUBIATA were useful for its further forensic or clinical related investigations.

Application of a simultaneous screening method for the detection of new psychoactive substances in various matrix samples using liquid chromatography/electrospray ionization tandem mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: Recently, new psychoactive substances (NPS) have emerged as a public health risk. Particularly, their chemical structures are modified to avoid detection. Synthetic NPS with effects similar to those of illegal drugs have been recently detected and synthesized worldwide, including MDMB-FUBINACA and APINAC, making it essential to rapidly and accurately detect NPS. METHODS: Fourteen NPS with similar structures were selected and their structures identified using 1 H and 13 C NMR spectroscopy. Additionally, we proposed the fragmentation pattern of each compound using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS). A simultaneous analytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was also developed and applied to real samples to detect the 14 NPS. The method was validated based on the specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, and stability according to international validation guidelines. RESULTS: The established method was used to screen 65 different matrix samples using LC/ESI-MS/MS. By comparing the calculated product ion ratios with those of standards, 2C-B in one of the real samples and 5F-MDMB-PICA in 20 samples were identified. For re-confirmation of detected compounds, the fragmentation pattern of each compound was compared with that of each standard using LC/QTOF-MS. CONCLUSIONS: In this study, LC/QTOF-MS data were used to elucidate the structures and fragmentation patterns of 14 NPS. A simultaneous method was developed using LC/ESI-MS/MS, which was applied to 65 real samples. The presented method and results can assist in ensuring the safety of public health from illegal adulteration.

Characterization of the metabolites of H3B-6545 in vitro and in vivo by using ultra-high performance liquid chromatography combined with electrospray ionization linear ion trap-orbitrap tandem mass spectrometry.

H3B-6545 is a selective ERα covalent antagonist, which has been demonstrated to be effective in anti-tumor. To fully understand its mechanism of action, it is necessary to investigate the in vitro and in vivo metabolic profiles. For in vitro metabolism, H3B-6545 (50 µM) was incubated with the hepatocytes of rat and human for 2 h. For in vivo metabolism H3B-6545 was orally administered to rats at a single dose of 10 mg/kg, and plasma, urine and fecal samples were then collected. All samples were analyzed by using ultra-high performance liquid chromatography combined with linear ion trap-orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) operated in positive ion mode. The structures of the metabolites were elucidated by comparing their MS and MS2 spectra with those of parent drug. A total of 11 metabolites, including a GSH adduct, were detected and structurally identified. M2, M7 and M8 were further unambiguously identified by using reference standards. Among these metabolites, M1, M5, M7 and M10 were newly found and reported for the first time. The metabolic pathways of H3B-6545 included deamination (M8 and M9), dealkylation (M2, M3 and M10), N-hydroxylation (M6), hydroxylation (M1 and M4), formation of amide derivatives (M5 and M7) and GSH conjugation (G1).

Assessment of synthetic cannabinoid FUB-AMB and its ester hydrolysis metabolite in human liver microsomes and human blood samples using UHPLC-MS/MS.

FUB-AMB, an indazole carboxamide synthetic cannabinoid recreational drug, was one of the compounds most frequently reported to governmental agencies worldwide between 2016 and 2019. It has been implicated in intoxications and fatalities, posing a risk to public health. In the current study, FUB-AMB was incubated with human liver microsomes (HLM) to assess its metabolic fate and stability and to determine if its major ester hydrolysis metabolite (M1) was present in 12 authentic forensic human blood samples from driving under the influence of drug cases and postmortem investigations using UHPLC-MS/MS. FUB-AMB was rapidly metabolized in HLM, generating M1 that was stable through a 120-min incubation period, a finding that indicates a potential long detection window in human biological samples. M1 was identified in all blood samples, and no parent drug was detected. The authors propose that M1 is a reliable marker for inclusion in laboratory blood screens for FUB-AMB; this metabolite may be pharmacologically active like its precursor FUB-AMB. M1 frequently appears in samples in which the parent drug is undetectable and can point to the causative agent. The results suggest that it is imperative that synthetic cannabinoid laboratory assay panels include metabolites, especially known or potential pharmacologically active metabolites, particularly for compounds with short half-lives.

Phase I metabolic profiling of the synthetic cannabinoids THJ-018 and THJ-2201 in human urine in comparison to human liver microsome and cytochrome P450 isoenzyme incubation.

Despite the increasing relevance of synthetic cannabinoids as one of the most important classes within "New Psychoactive Substances", there is still a lack of knowledge concerning their metabolism in humans. Due to the extensive metabolism of synthetic cannabinoids, metabolites are necessarily the best target analytes in urine, posing additional challenges to forensic analysis. The aims of this study were to identify appropriate urinary targets indicating intake of THJ-018 or THJ-2201 as well as to elucidate the most important cytochrome P450 isoenzymes within the metabolism of THJ-018 and THJ-2201 in vitro. For this purpose, the in vitro metabolism of THJ-018 and THJ-2201 was initially established using pooled human liver microsomes. The results obtained were compared to previously published in vitro results as well as to the results of the metabolic profiles from selected recombinant cytochrome P450 isoenzymes and from 23 urine samples from forensic cases. LC-HRMS was used to conduct product ion scans and to examine the metabolite spectra. For THJ-018, 17 different metabolite groups containing 33 different metabolites and isomers were detected after microsomal incubation, with the major metabolic pathways being monohydroxylation at the pentyl chain and of the naphthyl moiety as well as dihydroxylation of both residues. For THJ-2201, 19 different metabolite groups and 46 different metabolites and isomers were observed. The major metabolic pathways were monohydroxylation at the naphthyl moiety and oxidative defluorination. Significant contribution to the in vitro metabolism of THJ-018 and THJ-2201 originated from CYP2B6, CYP2C19, CYP3A4, and CYP3A5. As several cytochrome P450 isoenzymes are involved in the metabolism of these synthetic cannabinoids, a co-consumption with other drugs is unlikely to have an impact on their metabolism.

UPLC-HR-MS/MS-based determination study on the metabolism of four synthetic cannabinoids, ADB-FUBICA, AB-FUBICA, AB-BICA and ADB-BICA, by human liver microsomes.

Since 2012, several cannabimimetic indazole and indole derivatives with valine amino acid amide residue have emerged in the illicit drug market, and have gradually replaced the old generations of synthetic cannabinoids (SCs) with naphthyl or adamantine groups. Among them, ADB-FUBICA [N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide], AB-FUBICA [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide], AB-BICA [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-benzyl-1H-indole-3-carboxamide] and ADB-BICA [N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-benzyl-1H-indole-3-carboxamide] were detected in China recently, but unfortunately no information about their in vitro human metabolism is available. Therefore, biomonitoring studies to screen their consumption lack any information about the potential biomarkers (e.g. metabolites) to target. To bridge this gap, we investigated their phase I metabolism by incubating with human liver microsomes, and the metabolites were identified by ultra-performance liquid chromatography-high resolution-tandem mass spectrometry. Metabolites generated by N-dealkylation and hydroxylation on the 1-amino-alkyl moiety were found to be predominant for all these four substances, and others which underwent hydroxylation, amide hydrolysis and dehydrogenation were also observed in our investigation. Based on our research, we recommend that the N-dealkylation and hydroxylation metabolites are suitable and appropriate analytical markers for monitoring their intake.

Distinguishing Intake of New Synthetic Cannabinoids ADB-PINACA and 5F-ADB-PINACA with Human Hepatocyte Metabolites and High-Resolution Mass Spectrometry.

BACKGROUND: ADB-PINACA and its 5-fluoropentyl analog 5F-ADB-PINACA are among the most potent synthetic cannabinoids tested to date, with several severe intoxication cases. ADB-PINACA and 5F-ADB-PINACA have a different legal status, depending on the country. Synthetic cannabinoid metabolites predominate in urine, making detection of specific metabolites the most reliable way for proving intake in clinical and forensic specimens. However, there are currently no data on ADB-PINACA and 5F-PINACA metabolism. The substitution of a single fluorine atom distinguishes the 2 molecules, which may share common major metabolites. For some legal applications, distinguishing between ADB-PINACA and 5F-PINACA intake is critical. For this reason, we determined the human metabolic fate of the 2 analogs. METHODS: ADB-PINACA and 5F-PINACA were incubated for 3 h with pooled cryopreserved human hepatocytes, followed by liquid chromatography-high-resolution mass spectrometry analysis. Data were processed with Compound Discoverer. RESULTS: We identified 19 and 12 major ADB-PINACA and 5F-ADB-PINACA metabolites, respectively. Major metabolic reactions included pentyl hydroxylation, hydroxylation followed by oxidation (ketone formation), and glucuronidation of ADB-PINACA, and oxidative defluorination followed by carboxylation of 5F-ADB-PINACA. CONCLUSIONS: We recommend ADB-PINACA ketopentyl and hydroxypentyl, and ADB-PINACA 5-hydroxypentyl and pentanoic acid, as optimal markers for ADB-PINACA and 5F-ADB-PINACA intake, respectively. Since the 2 compounds present positional isomers as the primary metabolites, monitoring unique product ions and optimized chromatographic conditions are required for a clear distinction between ADB-PINACA and 5F-ADB-PINACA intake.

Human mass balance study and metabolite profiling of 14C-niraparib, a novel poly(ADP-Ribose) polymerase (PARP)-1 and PARP-2 inhibitor, in patients with advanced cancer.

Niraparib is an investigational oral, once daily, selective poly(ADP-Ribose) polymerase (PARP)-1 and PARP-2 inhibitor. In the pivotal Phase 3 NOVA/ENGOT/OV16 study, niraparib met its primary endpoint of improving progression-free survival (PFS) for adult patients with recurrent, platinum sensitive, ovarian, fallopian tube, or primary peritoneal cancer in complete or partial response to platinum-based chemotherapy. Significant improvements in PFS were seen in all patient cohorts regardless of biomarker status. This study evaluates the absorption, metabolism and excretion (AME) of 14C-niraparib, administered to six patients as a single oral dose of 300 mg with a radioactivity of 100 µCi. Total radioactivity (TRA) in whole blood, plasma, urine and faeces was measured using liquid scintillation counting (LSC) to obtain the mass balance of niraparib. Moreover, metabolite profiling was performed on selected plasma, urine and faeces samples using liquid chromatography - tandem mass spectrometry (LC-MS/MS) coupled to off-line LSC. Mean TRA recovered over 504 h was 47.5% in urine and 38.8% in faeces, indicating that both renal and hepatic pathways are comparably involved in excretion of niraparib and its metabolites. The elimination of 14C-radioactivity was slow, with t1/2 in plasma on average 92.5 h. Oral absorption of 14C-niraparib was rapid, with niraparib concentrations peaking at 2.49 h, and reaching a mean maximum concentration of 540 ng/mL. Two major metabolites were found: the known metabolite M1 (amide hydrolysed niraparib) and the glucuronide of M1. Based on this study it was shown that niraparib undergoes hydrolytic, and conjugative metabolic conversions, with the oxidative pathway being minimal.

Micelle-enhanced direct spectrofluorimetric method for the determination of linifanib: Application to stability studies.

A new simple stability-indicating spectrofluorimetric method has been developed and validated for the determination of the tyrosine kinase inhibitor, linifanib (LNF). The proposed method makes use of the native fluorescence characteristics of LNF in a micellar system. Compared with aqueous solutions, the fluorescence intensity of LNF was greatly enhanced upon the addition of Tween-80. The relative fluorescence intensity of LNF was measured in a diluting solvent composed of 2% Tween-80: phosphate buffer pH 8.0 (20: 80, v/v) using excitation and emission wavelengths of 290 and 450 nm, respectively. The proposed method was fully validated as per the ICH guidelines. The recorded fluorescence intensity of LNF was rectilinear over a concentration range of 0.3-2 µg/ml with a high correlation coefficient (r = 0.9990) and low limits of detection (0.091 µg/ml) and quantitation (0.275 µg/ml). The applicability of the method was extended to study the inherent stability of LNF under different stress degradation conditions including, alkaline, acidic, oxidative, photolytic and thermal degradation. Moreover, the method was utilized to study the kinetics of the alkaline and oxidative degradation of LNF. The pseudo-first order rate constants and half-lives were calculated.

Identification of three novel new psychoactive substances 4F-AB-BUTINACA, AB-PHETINACA, and 2F-NENDCK.

The identification of new psychoactive substances (NPS) is an active and cutting-edge topic in forensic science. With the emergence of a large number of NPS, their timely identification to prevent spread can pose a challenge to clinical and forensic toxicology laboratories. Three emerging NPS had been identified in recently seized materials, including two synthetic cannabinoids [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobutyl)-1H-indazole-3-carboxamide (4F-AB-BUTINACA) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-phenethyl-1H-indazole-3-carboxamide (AB-PHETINACA)] and a ketamine-like substance [2-(2-fluorophenyl)-2-(ethylamino) cyclohexan-1-one(2F-NENDCK)]. The three compounds were first identified by Fourier transform infrared spectrometry (FT-IR), gas chromatography-mass spectrometry (GC-MS), ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UHPLC-QTOF-MS), and nuclear magnetic resonance (NMR). These data may assist forensic analysts in analyzing the same substances or their homologous compounds.

Distribution of primary and specialized metabolites in Nigella sativa seeds, a spice with vast traditional and historical uses.

Black cumin (Nigella sativa L., Ranunculaceae) is an annual herb commonly used in the Middle East, India and nowadays gaining worldwide acceptance. Historical and traditional uses are extensively documented in ancient texts and historical documents. Black cumin seeds and oil are commonly used as a traditional tonic and remedy for many ailments as well as in confectionery and bakery. Little is known however about the mechanisms that allow the accumulation and localization of its active components in the seed. Chemical and anatomical evidence indicates the presence of active compounds in seed coats. Seed volatiles consist largely of olefinic and oxygenated monoterpenes, mainly p-cymene, thymohydroquinone, thymoquinone, γ-terpinene and α-thujene, with lower levels of sesquiterpenes, mainly longifolene. Monoterpene composition changes during seed maturation. γ-Terpinene and α-thujene are the major monoterpenes accumulated in immature seeds, and the former is gradually replaced by p-cymene, carvacrol, thymo-hydroquinone and thymoquinone upon seed development. These compounds, as well as the indazole alkaloids nigellidine and nigellicine, are almost exclusively accumulated in the seed coat. In contrast, organic and amino acids are primarily accumulated in the inner seed tissues. Sugars and sugar alcohols, as well as the amino alkaloid dopamine and the saponin α-hederin accumulate both in the seed coats and the inner seed tissues at different ratios. Chemical analyses shed light to the ample traditional and historical uses of this plant.

Detection of ADB-BUTINACA Metabolites in Human Urine, Blood, Kidney and Liver.

The N-butyl indazole derivative, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3-carboxamide (ADB-BUTINACA or ADB-BINACA), currently a drug of abuse in Russia, is reported to have a cannabinoid receptor potency and efficacy almost three times higher than JWH-018. ADB-BUTINACA was detected in blood from patients with suspected drug intoxications, as well as in blood, kidney and liver samples collected during postmortem investigations. Using liquid chromatography-time-of-flight-mass spectrometry, a number of ADB-BUTINACA metabolites were tentatively identified in urine samples. These include products of mono- and dihydroxylation, hydroxylation of the N-butyl side chain and dehydrogenation, formation of a dihydrodiol, hydrolysis of the terminal amide group, N-dealkylation of the indazole and a combination of these reactions. The dihydrodiol was found to be the predominant metabolite, with its chromatographic peak area exceeding those of other metabolites by almost an order of magnitude. For the routine analysis of blood, liver and kidney samples, the dihydrodiol and monohydroxylated metabolites along with the parent compound are recommended as target analytes. The same metabolites in free and glucuronidated forms are also recommended for analytical confirmation in urine samples.

Identification of a novel bromoindazole synthetic cannabinoid analogue in seized e-cigarette liquid: ADB-BRINACA.

A wide variety of new synthetic cannabinoids have emerged around the world in recent years, and because of this rapid emergence, the detection and monitoring of this class of abused drugs remain a challenge. In this study, a new cannabimimetic indazole-3-carboxamide derivative, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)- 5-bromo-1 H-indazole-3-carboxamide, was identified from seized e-cigarette liquid samples and newly named as ADB-BRINACA by referring to the names of other known indazole-class synthetic cannabinoids. Structure identification was accomplished based on gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-high resolution quadrupole mass spectrometry (HPLC-QTOF-MS/MS), and nuclear magnetic resonance spectrometry (NMR). The concentration range of ADB-BRINACA in six e-cigarette liquid samples was found to be 2228-4203 mg/L using ERETIC 2, a quantitative NMR method, which is advantageous in the absence of a reference material. As there have been no chemical or pharmaceutical reports on ADB-BRINACA until now, this is the first report presenting a comprehensive analytical characterization of ADB-BRINACA.

Phenanthrene-fused azo-ene-ynes: synthesis of dibenzo[f,h]cinnoline and dibenzo[e,g]isoindazole derivatives.

The cyclization reactions of a phenanthreno-fused azo-ene-yne compound have been studied both experimentally and computationally. Experimental results show that this system is prone to dimerization, more so than previously studied naphthalene- and benzene-based analogues. Calculations reveal that pyrazoles and arene-fused pyrazoles strongly stabilize carbenes in the 5-position through "coarctate conjugation", suggesting a stationary concentration of the carbenes/carbenoids during cyclization that is high enough for dimerization.

Identification and quantification of 10 indole/indazole carboxamide synthetic cannabinoids in 36 herbal blends by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy.

Herbal blends containing synthetic cannabinoids have become popular alternatives to marijuana. The number of synthetic cannabinoids and speed of their emergence enable this group of compounds particularly challenging in terms of detection, monitoring, and responding. In this work, both gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR) methods were developed for the identification and quantification of synthetic cannabinoids in herbal blends. Ten types of indole/indazole carboxamide synthetic cannabinoids, which showed different types of substitutions connected to nitrogen of the indole/indazole carboxamide, were detected in 36 herbal blends. The GC-MS fragmentation routes of indole/indazole carboxamide synthetic cannabinoids were discussed in detail for structure identification purpose. The concentration range of synthetic cannabinoid in 36 herbal blends was 1.9-50.6 mg/g using GC-MS method, while 1.5-49.0 mg/g by NMR method. Nicotine in herbal blends was quantified by NMR method without using reference material, and showed a variation of 5.3-44.7 mg/g. For quantitative analysis, NMR method showed great advantage in the absence of reference material, while GC-MS method showed great merit for multiple-compound analysis when reference material was available. Therefore, for the quantitative analysis of new emerged synthetic cannabinoid in herbal blends, different methods could be chosen by considering whether reference material is available, as well as the number and types of synthetic cannabinoids detected in a single sample.

Profiling of synthesis-related impurities of the synthetic cannabinoid Cumyl-5F-PINACA in seized samples of e-liquids via multivariate analysis of UHPLC-MSn data.

Vaping of synthetic cannabinoids via e-cigarettes is growing in popularity. In the present study, we tentatively identified 12 by-products found in a pure sample of the synthetic cannabinoid Cumyl-5F-PINACA (1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide), a prevalent new psychoactive substance (NPS) in e-liquids, via high-resolution mass spectrometry fragmentation experiments (HRMS/MS). Furthermore, we developed a procedure to reproducibly extract this synthetic cannabinoid and related by-products from an e-liquid matrix via chloroform and water. The extracts were submitted to flash chromatography (F-LC) to isolate the by-products from the main component. The chromatographic impurity signature was subsequently assessed by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) and evaluated by automated integration. The complete sample preparation sequence (F-LC + UHPLC-MS) was validated by comparing the semi-quantitative signal integrals of the chromatographic impurity signatures of five self-made e-liquids with varying concentrations of Cumyl-5F-PINACA [0.1, 0.2, 0.5, 0.7 and 1.0% (w/w)], giving an average relative standard deviation of 6.2% for triplicate measurements of preparations of the same concentration and 10.5% between the measurements of the five preparations with different concentrations. Lastly, the chromatographic signatures of 14 e-liquid samples containing Cumyl-5F-PINACA from police seizures and Internet test purchases were evaluated via hierarchical cluster analysis for potential links. For the e-liquid samples originating from test purchases, it was found that the date of purchase, the identity of the online shop, and the brand name are the critical factors for clustering of samples.

The phenomenon of para-Fluorophenylpiperazine (pFPP) in combination with the synthetic cannabinoid AMB-FUBINACA in seized plant material in New Zealand.

New psychoactive substances have emerged as a vast and diverse group of illicit drugs over the past decade, with synthetic cannabinoids comprising the largest of the categories. Commonly, a single synthetic cannabinoid is applied to plant material, creating a product that is designed to be smoked by the user. The clandestine preparation process can result in an unevenly distributed product, with varying concentration within and between plant materials. This investigation describes the novel co-detection of the synthetic cannabinoid AMB-FUBINACA, with the piperazine para-fluorophenylpiperazine (pFPP), in a number of plant material samples analysed in New Zealand in 2017. Of 157 samples of plant material containing AMB-FUBINACA, pFPP was detected in 55 of them. A range of pFPP concentrations was observed between the plant material samples, as well as intra-batch variation. The presence of both drugs may be designed to enhance, prolong or balance the psychoactive effects caused from smoking the plant material. However the intended purpose has not been verified. This is the first reported combination of a synthetic cannabinoid and a piperazine in plant material.

Fatal intoxication with new synthetic cannabinoids AMB-FUBINACA and EMB-FUBINACA.

Introduction: Synthetic cannabinoids are currently the largest group of new psychoactive substances. Those that have been subjected to legal control are replaced by newer uncontrolled substances, which causes constant and dynamic changes to the drug market. Some of the most recent synthetic cannabinoids that have appeared on the "legal highs" market are AMB-FUBINACA and EMB-FUBINACA. Case history: A 27-year-old man was found dead on a bed in an apartment. At autopsy, congestion of internal organs, pulmonary oedema and left-sided pleural adhesions were found. The medical examiner concluded that the man died due to acute respiratory failure. The autopsy materials (blood, urine, liver, kidney, stomach, intestine, lung and brain) were collected for further toxicological analyses. Methods: The synthetic cannabinoids AMB-FUBINACA and EMB-FUBINACA were isolated from autopsy materials by precipitation with acetonitrile. The quantitative analyses were carried out by LC-MS/MS. Results: AMB-FUBINACA and EMB-FUBINACA were detected and quantified in all post-mortem materials except the blood. The determined concentrations of these compounds in solid tissues were in the range of 0.2-0.9 ng/g and 0.2-3.5 ng/g. The highest concentrations of AMB-FUBINACA and EMB-FUBINACA were revealed in the stomach content (5.8 and 36.2 ng/mL, respectively). Discussion: The presented case demonstrates that even in cases of fatalities, it is possible that the parent substance will not be present in the blood, while being present in other autopsy materials. The determined concentrations of the compounds may indicate oral administration of synthetic cannabinoids. It can also be assumed that AMB-FUBINACA and EMB-FUBINACA probably contributed to death. Conclusion: The presented case shows that synthetic cannabinoids can be undetected in the blood of even seriously or fatally intoxicated people. This situation means that the analysis of only blood samples may not confirm poisoning. The presented case also suggests that AMB-FUBINACA and EMB-FUBINACA use is dangerous to health and may lead to fatal intoxication.

Fatalities related to new psychoactive substances in Singapore-A case series.

The use of New Psychoactive Substances (NPS) has become a serious global issue with increasing number of reports of their toxicities and fatalities. Likewise, in Singapore, the number of exhibits containing NPS detected had increased 80% from 2011 to 2014. This is a case series of the first four autopsy cases of fatalities due to or related to the use of NPS in Singapore. In one case, we present the first reported case of death due directly to ADB-FUBINACA toxicity (post-mortem blood concentration of 56ng/ml). Another case was due to 25B-NBOMe toxicity (post-mortem blood concentration of 10ng/ml) while the last two cases were deaths related to 5-Fluoro ADB, where the metabolites of the drug were detected.

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB-CHMINACA in an authentic human urine specimen.

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB-CHMINACA, was investigated. Although unchanged MAB-CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB-CHMINACA metabolites from a commercial source. The MAB-CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry with or without hydrolysis with ß-glucuronidase. Among the six MAB-CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with ß-glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4-monohydroxycyclohexylmethyl metabolite M1 (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide) and a dihydroxyl (4-hydroxycyclohexylmethyl and tert-butylhydroxyl) metabolite M11 (N-(1-amino-4-hydroxy-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB-CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB-CHMINACA metabolites in an authentic human urine specimen.