Showing papers in "Forensic Toxicology in 2011"

Identification and quantitation of two cannabimimetic phenylacetylindoles JWH-251 and JWH-250, and four cannabimimetic naphthoylindoles JWH-081, JWH-015, JWH-200, and JWH-073 as designer drugs in illegal products

Abstract: Six cannabimimetic indoles have been identified as adulterants in herbal or chemical products being sold illegally in Japan, with four of the compounds being new as adulterants to our knowledge The identifications were based on analyses using gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy The first two compounds were identified as phenylacetyl indoles JWH-251 (2-(2-methylphenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone; 1) and its demethyl-methoxylated analog JWH-250 (2-(2-methoxyphenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone; 2) Compound 2 was identical to that found as an adulterant in the UK and in Germany in 2009 The third compound was naphthoylindole JWH-081 (1-(4-methoxynaphthalenyl)-(1-pentyl-1H-indol-3-yl)methanone; 3), and the fourth was JWH-073 (1-naphthalenyl(1-butyl-1H-indol-3-yl)methanone; 4), which had been identified as an adulterant in our previous study Two additional compounds were JWH-015 (1-naphthalenyl(2-methyl-1-propyl-1H-indol-3-yl)methanone; 5) and JWH-200 (1-naphthalenyl(1-(2-(4-morpholinyl)ethyl)-1H-indol-3-yl)methanone; 6) Compounds 1–4 and 6 were reported to be synthetic cannabinoids with selective affinity for cannabinoid CB1 receptors, while compound 5 was reported to be a selective CB2 receptor agonist causing immunosuppressive effects without psychotropic affects One product contained both CB1 and CB2 receptor agonists in our collection Quantitative analyses of the six cannabimimetic compounds in 20 products revealed that there was large variation in concentrations of the detected compounds among products; for herbal cutting products, the total amounts of these cannabinoids ranged from 26 to 100 mg

Identification and quantitation of a benzoylindole (2-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone and a naphthoylindole 1-(5-fluoropentyl-1H-indol-3-yl)-(naphthalene-1-yl)methanone (AM-2201) found in illegal products obtained via the Internet and their cannabimimetic effects evaluated by in vitro [35S]GTPγS binding assays

Abstract: During our careful surveillance of unregulated drugs in January to February 2011, we found two new compounds used as adulterants in herbal products obtained via the Internet. These compounds were identified by liquid chromatography–mass spectrometry, gas chromatography-mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a benzoylindole (2-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (1), which is a positional isomer of (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (RCS-4, 4). The second compound was 1-(5-fluoropentyl-1H-indol-3-yl)-(naphthalene-1-yl)methanone (AM-2201, 2). The compound 2 has been reported to be a cannabinoid receptor agonist. Because the cannabimimetic effects of compounds 1 and 4 have not been reported to date, their biological activities were evaluated by measuring the activation of [35S] guanosine-5′-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins, together with those of other synthetic cannabimimetic compounds. For quantitation of the above two compounds (1 and 2) and previously identified compounds (AM-694, 3; JWH-122, 5; RCS-4, 4), each product was extracted with methanol under ultrasonication to prepare a sample solution for analysis by liquid chromatography with ultraviolet detection. Each of four commercial products contained some of cannabimimetic indoles 1–5; their contents ranged from 14.8 to 185 mg per pack.

Identification and quantitation of two benzoylindoles AM-694 and (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone, and three cannabimimetic naphthoylindoles JWH-210, JWH-122, and JWH-019 as adulterants in illegal products obtained via the Internet

Abstract: During our careful surveillance of unregulated drugs, we found five new compounds used as adulterants in herbal and drug-like products obtained via the Internet. These compounds were identified by liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a benzoylindole AM-694, which is 1-[(5-fluoropentyl)-1H-indol-3-yl]-(2-iodophenyl)methanone (1). The second compound was (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (2), which was also classified as a benzoylindole. The three other compounds were identified as naphthoylindoles JWH-210 (4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 3), JWH-122 (4-methylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 4), and JWH-019 (1-hexyl-3-(naphthalen-1-oyl)indole; 5). All compounds except compound 2 had been reported to be cannabinoid receptor agonists. For quantitation of the five compounds and previously reported compounds, each product was extracted with methanol under ultrasonication to prepare a test solution for analysis by liquid chromatography with ultraviolet detection. Each compound detected in 43 commercial products showed large variation in content ranging from 4.0 to 359 mg per pack.

Recently abused β-keto derivatives of 3,4-methylenedioxyphenylalkylamines: a review of their metabolisms and toxicological analysis

Abstract: β-Keto derivatives of 3,4-methylenedioxyphenylalkylamines (bk-MDPAs), especially 2-methylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (methylone), 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (bk-MBDB), and 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (bk-MDEA), are abused as substitutes for 3,4-methylenedioxyphenylalkylamines in some countries, causing increased social problems. With the widespread abuse of bk-MDPAs, the analysis of bk-MDPAs and their metabolites in human specimens is quite important in forensic and clinical toxicology. In this review, the metabolisms of bk-MDPAs and simultaneous analytical methods for bk-MDPAs and their metabolites by gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, and liquid chromatography–tandem mass spectrometry are presented.

Identification and quantitation of cannabimimetic compound JWH-250 as an adulterant in products obtained via the Internet

Abstract: During our careful survey of unregulated drugs in Tokyo, a new compound was disclosed as an adulterant in herbal and powder products. This compound was found to have a molecular weight of 335 by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, and the accurate mass measurement suggested an elementary composition of C22H26NO2. Using these mass data together with those obtained by nuclear magnetic resonance analysis, the compound was identified as 1-pentyl-3-(2-methoxyphenylacetyl)indole (JWH-250), which had been reported by Huffman and coworkers in 2005. This compound was classified as a phenylacetylindole and a cannabinoid receptor agonist. For quantitation of the compound in herbal and powder products, each product was extracted with methanol under ultrasonication to prepare the solution for analysis by liquid chromatography with ultraviolet detection. The contents of JWH-250 in five products ranged from 77.4 to 165 mg per pack.

Colorimetric detection and chromatographic analyses of designer drugs in biological materials: a comprehensive review

Abstract: A number of analogues of phenethylamine and tryptamine, which are prepared by modification of the chemical structures, are being developed for circulation on the black market. Often called “designer drugs,” they are abused in many countries, and cause serious social problems in many parts of the world. Acute deaths have been reported after overdoses of designer drugs. Various methods are required for screening and routine analysis of designer drugs in biological materials for forensic and clinical purposes. Many sample preparation and chromatographic methods for analysis of these drugs in biological materials and seized items have been published. This review presents various colorimetric detections, gas chromatographic (GC)–mass spectrometric, and liquid chromatographic (LC)–mass spectrometric methods proposed for designer drug analyses. Basic information on extractions, derivatizations, GC columns, LC columns, detection limits, and linear ranges is also summarized.

Optimization of simultaneous analysis of tetrodotoxin, 4-epitetrodotoxin, 4,9-anhydrotetrodotoxin, and 5,6,11-trideoxytetrodotoxin by hydrophilic interaction liquid chromatography–tandem mass spectrometry

Abstract: Recently, liquid chromatography–mass spectrometry (LC–MS) has been recognized as a useful method to analyze tetrodotoxin (TTX), the primary toxin for puffer fish poisoning. TTX usually exists with its chemically interchangeable analogs, such as 4-epiTTX and 4, 9-anhydroTTX. TTX and 4-epiTTX have the same molecular weight and the same fragmentation pattern by tandem mass spectrometry (MS–MS). In this study, we optimized conditions for hydrophilic interaction liquid chromatography (HILC)–MS–MS to quantitate TTX, 4-epiTTX, 4,9-anhydroTTX, and 5,6,11-trideoxyTTX with good separation. The relationships between the applied amounts of each TTX analog to the HILC–MS–MS instrument and the peak areas showed good linearity in the ranges examined. The lower limits of detection (signal-to-noise ratio = 3) were 64 pg on-column for TTX, 128 pg for both 4-epiTTX and 4,9-anhydroTTX, and 180 pg for 5,6,11-trideoxyTTX using an API 2000 mass spectrometer.

Differential inhibition of human cytochrome P450 2A6 and 2B6 by major phytocannabinoids

Abstract: Inhibitory effects of Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), and cannabinol (CBN) on the catalytic activities of human recombinant cytochrome P450 (CYP) 2A6 and CYP2B6 were investigated. Δ9-THC, CBD, and CBN noncompetitively inhibited coumarin 7-hydroxylase activity of recombinant CYP2A6 with the apparent K i values of 28.9, 55.0, and 39.8 μM, respectively. On the other hand, Δ9-THC, CBD, and CBN inhibited 7-benzoxyresorufin O-debenzylase activity of recombinant CYP2B6 in a mixed fashion with the K i values of 2.81, 0.694, and 2.55 μM, respectively. Because the inhibition of CYP2B6 by CBD was the most potent, investigation was conducted to determine which moiety of the CBD structure was responsible for the inhibition. Olivetol and d-limonene, the partial structure of CBD, inhibited the CYP2B6 activity to some extent. Inhibitory effects of CBD-2′-monomethyl ether and CBD-2′,6′-dimethyl ether attenuated with the number of methylations on the phenolic hydroxyl groups in the resorcinol moiety of CBD. Cannabidivarin, a CBD analogue having a propyl side chain, inhibited the CYP2B6 activity less potently than CBD possessing a pentyl side chain. Therefore, both structures of pentylresorcinol and terpene moieties of CBD were suggested to play important roles in the CYP2B6 inhibition. Δ9-THC, CBD, and CBN showed metabolism-dependent inhibition for CYP2A6 but not for CYP2B6. Furthermore, Δ9-THC and CBN were characterized as mechanism-based inhibitors for CYP2A6. The k inact and K I values of Δ9-THC were 0.0169 min−1 and 0.862 μM, respectively; the k inact and K I values of CBN were 0.00909 min−1 and 1.01 μM, respectively. These results indicated that Δ9-THC, CBD, and CBN showed differential inhibition against CYP2A6 and CYP2B6.

Imaging of methamphetamine incorporated into hair by MALDI-TOF mass spectrometry

Abstract: Matrix-assisted laser desorption ionization (MALDI)–time-of-flight (TOF) mass spectrometry (MS) was used for visual demonstration of methamphetamine (MA) incorporation into human hair. Longitudinal sections of human scalp hair shafts from chronic MA users were directly subjected to imaging MS. Numerous MA-positive spots with various intensities were observed in the specimens, which probably reflect habitual MA abuse and the different MA blood levels upon each administration. This imaging MS method for drugs in hair seems to give much more accurate chronological information on drug use, and clearer discrimination between deliberate drug use and passive exposure, using only a single hair shaft. This is the first report of imaging MS applied to forensic toxicology. This method is expected open a new field in analyses of drugs in hair.

Mushroom toxins: a forensic toxicological review

Abstract: Mushrooms are ubiquitous in the world. Amateur hunters harvest mushrooms growing in forests to enjoy eating them as seasonal delicacies, and occasionally they cause poisonings and even deaths. In this review, mushroom toxins are tabulated according to mushroom species, symptoms, toxicities and analytical methods on the basis of references. Second, because we constructed a method for analysis of amatoxins, the most virulent mushroom toxins, by liquid chromatography-time-of-flight-mass spectrometry, we introduce it for use in forensic toxicology. Third, an extensive poisoning incident after consumption of the usually edible mushroom Pleurocybella porrigens took place in nine prefectures in Japan from September to December 2004, resulting in 59 poisoned people including 19 deaths; this incident is briefly described and discussed in relation to its causative toxin(s). Finally, we present the chemical structures of new toxins purified from the highly toxic mushrooms Podostroma cornu-damae and Russula subnigricans; their structures were very unique, and the toxicities were comparable to those of amatoxins. From the forensic toxicological point of view, reports on sophisticated methodology for analyses of mushroom toxins seem to be too scant even for the well-known toxins. Hereafter, a number of toxic mushrooms and their new toxins are expected to be disclosed, especially because of environmental changes such as the global warming phenomenon.

Detection of saxitoxin in counterterrorism using a commercial lateral flow immunoassay kit

Abstract: The neurotoxin saxitoxin (STX) is registered in the list of the Chemical Weapons Convention. In preparation against potential terrorism by STX use, we investigated the performance of a commercially available rapid test kit for paralytic shellfish poisoning (PSP), which is essentially a lateral flow immunoassay kit. Pink lines in the test and control zones appeared after 35 min and were observed by the naked eye and were recorded by a digital scanner. The competitive displacement of gold-labeled antitoxin analog antibody by STX in the test zone was quantitatively shown using the ratio of the intensity of the test zone line to that of control zone line. As the STX concentration increased, the intensity of the pink line in the test zone on the strip decreased. The limit of detection was defined as the STX concentration that gave a ratio half that of the STX blank test, and was calculated to be about 12 ng/ml. Various matrix components, such as wheat flour and the decontamination chemical hypochlorite, were examined for their effects on false positive and false negative results in the determination of STX by the PSP Rapid Test system.

Oripavine as a new marker of opiate product use

Abstract: During our extensive surveillance of opiates in urine specimens of opium users, we noticed the appearance of an unknown peak (compound X) in total ion chromatograms obtained by gas chromatography-mass spectrometry (GC–MS) after enzymatic hydrolysis and trimethylsilyl (TMS) derivatization. We identified the compound X as oripavine. Oripavine was found to be a new and useful putative marker of opium/poppy seed use in differentiation from heroin, pharmaceutical codeine, and pharmaceutical morphine use. The presence of oripavine in the urine of opium users is probably the result of O-demethylation of the opium alkaloid thebaine. Analytical method optimization for GC–MS detection of oripavine in urine was also undertaken. Underivatized oripavine could not be detected by GC–MS, and trials for derivatization of oripavine with acetic anhydride and propionic anhydride were unsuccessful. Trials were successful with bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane. It was also disclosed that almost all amounts of oripavine in human urine existed in the unconjugated form; it was absolutely necessary to hydrolyze the conjugate before TMS derivatization of oripavine for its GC–MS analysis.

8-Hydroxy-2′-deoxyguanosine and arsenic compounds in urine and serum of a 4-year-old child suffering from acute promyelocytic leukemia during treatment with arsenic trioxide

Abstract: Arsenic trioxide (ATO) is an effective agent for acute promyelocytic leukemia (APL). In this study, the concentrations of dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), As (V), As (III), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine and/or serum of a 4-year-old APL patient were followed during ATO treatment. In comparison with a similar analysis previously published for an 85-year-old APL patient, the levels of arsenic compounds and the percentages of MMA and DMA in the present child patient were lower than those of the old patient. Significant positive correlation of 8-OHdG was observed only with DMA, and not with other arsenic compounds. These results are quite different from those of our previous study on an old APL patient during ATO treatment. When arsenic poisoning is diagnosed, it seems important to take into account the differences in the arsenic metabolism according to the ages of subjects. This study is the first to show the relationship between the levels of 8-OHdG and arsenic compounds in urine of a child APL patient treated with ATO.

A study on mechanisms of toxic actions of ciguatoxins: existence of functional relationship between CTX3C and charged residues of voltage sensors in Nav1.4 sodium channel

Abstract: Ciguatoxins, a group of virulent marine toxins, are bound to "site 5" inside voltage-dependent sodium channels and alter their kinetics dramatically; this is probably responsible for clinical symptoms of ciguatoxin poisoning or ciguatera. Although ciguatoxins are known to affect the voltage dependency of both activation and inactivation kinetics, site 5 has not been functionally clarified. This study, therefore, targeted putative voltage sensors as a receptor for ciguatoxins. We constructed mutants, in which 1 of 23 basic residues was substituted with glutamine in the S4 segment for each of four domains. We then examined the effects of a synthetic ciguatoxin congener CTX3C on these mutants. Notably, the suppressive effect of CTX3C on the sodium current (INa) amplitude of domain 2 mutants, which carried a mutation in the basic S4 residue of domain 2, was either reduced or eliminated. Kinetic analyses of domain 2 mutants in comparison with those of the wild type and other mutants revealed that the negative shift of the steady-state inactivation curve (V1/2inactΔ) was significantly decreased. The resistance of domain 2 mutants to CTX3C in terms of changes in V1/2inact is suggested to be closely related to resistance to the suppressive effect of CTX3C on the INa amplitude. This is the first report to demonstrate the existence of a functional relationship between a ciguatoxin congener and voltage sensor segments in domain 2 of the α-subunit of voltage-dependent sodium channels, although further studies are needed to locate a ciguatoxin receptor.

Possible involvement of Cyp3a enzymes in the metabolism of tetrahydrocannabinols by mouse brain microsomes

Abstract: Δ8-Tetrahydrocannabinol (THC) and Δ9-THC were mainly oxidized on the pentyl side chain at the 4′-position by mouse brain microsomes. 5′-Hydroxy-THCs were also formed as minor metabolites. However, 11-hydroxy metabolites of THCs, which are major metabolites of the cannabinoids produced by mouse hepatic microsomes, were not detectable as the metabolites formed by mouse brain microsomes. Cytochrome P450 (CYP) enzymes involved in the brain microsomal metabolism of Δ8-THC and Δ9-THC were identified by using CYP-selective inhibitors. The 4′- and 5′-hydroxylations of THCs were strongly inhibited by ketoconazole and troleandomycin, the known inhibitors of CYP3A enzymes, but not by other isozyme-selective inhibitors, such as 7,8-benzoflavone (CYP1A), quinidine (CYP2D), and sulfaphenazole (CYP2C). These results indicate that Cyp3a enzymes are responsible at least in part for the metabolism of Δ8-THC and Δ9-THC by mouse brain microsomes. Our present results using mouse brain seem to support the idea that the mode of THC metabolism by CYP enzymes in human brain is different from that in human liver.

Determination of acephate and methamidophos in tissues: appearance of matrix effect in gas chromatography–mass spectrometry

Abstract: A simple and reliable method to determine acephate and methamidophos in mammalian tissues is presented. The method includes solid-phase extraction of tissue extracts with active carbon cartridges followed by gas chromatography–mass spectrometry analysis. During the study, a matrix effect was observed especially at low concentrations of acephate and methamidophos in serum and in brain. To minimize the effect, we prepared calibration curves with relatively short ranges. The validation data, such as the linearity of calibration curves, limits of detection, and coefficients of variation for recovery rates, were generally satisfactory. The present method is useful for determination of acephate and methamidophos in mammalian tissues because of its simplicity and speed, keeping in mind the presence of the matrix effect.

Urinary morphine and codeine concentrations after ingestion of bean-jam buns decorated with poppy seeds

Abstract: Dear Editor: Poppy seeds are a common food ingredient. Cakes and bread decorated with or containing poppy seeds are quite popular in many countries. In Japan, one of the most popular foods with poppy seeds is bean-jam buns decorated with poppy seeds. However, the so-called poppy seeds defense has been often used in forensic urinalysis for opiates. It is sometimes difficult to determine the source of morphine and codeine in urine, because morphine-positive results can be caused by ingestion of foods containing poppy seeds [1–3]. Following a number of studies on opiates in poppy seeds, it has become clear that the opiate levels inside the seeds are very low; the alkaloid content of the seeds is affected by external contamination during harvest and changes in harvesting technology [2]. The seeds are contaminated with the chyle of unripe poppy capsules during harvesting. Other factors affecting morphine contents of poppy seeds are the poppy variety, geographical origin, time of harvest, and the processing method for foods containing poppy seeds [2]. In some countries, governmental food agencies have identified products with high morphine contents and removed them from the market. This has led to reduced morphine levels in poppy seeds over the past 4 years, especially in the German market [2]. It is actually easy for producers to reduce morphine contents in poppy seeds by washing or soaking them in water. However, with the reported variation of morphine content in poppy seeds, more data on the urinary levels of morphine and its metabolites after intake of foods containing poppy seeds are required. To date, some investigators have reported urinary concentrations of morphine and codeine after ingestion of poppy seeds or foods containing poppy seeds [4–14]. Furthermore, as a recent trend, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry equipped with databases [15, 16] are now being utilized for drug screening in place of conventional immunochemical methods. The instrumental screening methods give much more sensitive and reliable results than those of the immunochemical tests, and show positive results even in very low concentration ranges of drugs in blood and urine, including opiates after ingestion of foods containing poppy seeds. This is the second reason why we need such data at low concentrations of opiates of food origin in human urine for discrimination from opiate abuse. In this brief communication, we report the concentrations of morphine and codeine in urine after ingesting the most commonly eaten amounts of poppy seeds (i.e., two bean-jam buns decorated with poppy seeds). We determined the urinary concentrations of free (i.e., unconjugated form) opiates as well as those of total (i.e., sum of free and conjugated forms) opiates. Bean-jam buns decorated with poppy seeds (Kimuraya Bakery, Tokyo, Japan) were purchased from a local market. Healthy volunteers (5 males and 9 females, aged 14–60 years old) who had no history of drug abuse or medical record of taking opiates, each ingested two beanjam buns. Urine specimens were collected between 6 and 11 h after consumption. Morphine hydrochloride and codeine phosphate were purchased from Takeda Pharmaceutical (Osaka, Japan); N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane from K. Yamaguchi (&) M. Hayashida M. Nihira Y. Ohno Department of Legal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan e-mail: yamakoji@nms.ac.jp

In vivo incorporation of fenfluramine and norfenfluramine into pigmented and nonpigmented hair of rats measured by HPLC-fluorescence detection

Abstract: The incorporation profiles for fenfluramine (Fen) and its metabolite norfenfluramine (Norf) into black hair and white hair of Zucker rats and into white hair of Wistar rats after intraperitoneal (i.p.) administration of Fen or N-nitrosofenfluramine (N-Fen) were studied in great detail. The target compounds were determined by high-performance liquid chromatography with fluorescence detection using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a derivatization reagent. After repeated i.p. administration of Fen (5 mg/kg) for 4 days to Zucker rats, shaft and root samples of black and white hair were obtained 1 week after the first administration. It was surprising that Fen and Norf levels in root samples of white hair were much higher than those in shaft or root samples of black hair, strongly suggesting that unknown mechanisms other than the action of melanin take place in the white hair root. Time course profiles for Fen and Norf after administration of a single i.p. dose of Fen or N-Fen were constructed for Zucker and Wistar rats. The percent level of Fen or Norf in white hair was 15–50% of that in black hair at any interval within 600 min after a single administration of Fen in Zucker rats. Even with Wistar rats having only white hair, we could demonstrate the time courses for incorporation of Fen and Norf into white hair. Finally, time course profiles for Fen and Norf were also followed after a single i.p. administration of N-Fen; this experiment showed that the levels of Norf were much higher than those of Fen for both black and white hair samples of Zucker rats at any interval tested.

A model system for prediction of the in vivo metabolism of designer drugs using three-dimensional culture of rat and human hepatocytes

Abstract: The in vitro metabolisms of 4-bromo-2,5-dimethoxyphenethylamine (2C-B), 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), and 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7) were studied using TESTLIVER™-rat and TESTLIVER™-human, which are new three-dimensional rat and human hepatocyte culture systems, respectively. The metabolites produced in the incubation media were measured by liquid chromatography–tandem mass spectrometry or gas chromatography–mass spectrometry. The data obtained with the in vitro system of rat origin were carefully compared with those obtained by rat in vivo experiments; most of the metabolites found in the in vivo experiments could be reproduced in the present three-dimensional in vitro experiments, although quantitative metabolite distribution patterns obtained with the three-dimensional system was somewhat different from those of in vivo experiments. Because human in vivo experiments are not possible, especially for dubious designer drugs, the in vitro experiments using the three-dimensional human hepatocyte culture system seem very useful for studying human metabolism of new designer drugs as an alternative to human experiments.