In order to prove synthetic cannabinoid abuse, it is necessary to detect intact synthetic cannabinoids or their metabolites from such biological samples as urine or blood. Generally, blood is used as the biological sample because it is usually difficult to detect intact synthetic cannabinoids in urine. Furthermore, a rapid and accurate method for the detection of synthetic cannabinoids in the biological sample is required. Therefore, we examined the applicability of solid-phase dispersive extraction (SPDE)-GC/MS in the rapid detection of intact synthetic cannabinoids in blood. We chose seven synthetic cannabinoids designated as narcotics. To determine the optimum operating conditions for SPDE, we selected Oasis^® HLB as the solid-phase material for pre-treatment and filled it with 10 mg into the equipment, and acetone as the eluent. The pre-treatment resulted in 80-100% recovery. Furthermore, the pre-treatment time was significantly reduced in SPDE compared to solid-phase extraction (SPE). In addition, the pre-treatment protected operators from unnecessary exposure, reduced cross-contamination of chemicals, and decreased operation complexity. The limit of detection (S/N>3) of JWH-018, JWH-122, cannabicyclohexanol (CCH), XLR-11, and was 2.5 ng/mL, and that of JWH-073 and MAM-2201 was 5 ng/mL. The limit of quantification (S/N>10) of JWH-018, JWH-122, CCH, XLR-11, and was 5 ng/mL, and that of JWH-073 and MAM-2201 was 10 ng/mL. The average recoveries of the seven synthetic cannabinoids from pooled serum samples spiked at 25 and 450 ng/mL were 76.9-107.4% (SD: 6.4-10.7%) and 63.1-89.6% (SD: 3.9-8.2%), respectively. (SPDE)-GC/MS was proven to be a useful method for detecting intact synthetic cannabinoids in blood.

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Synthetic cannabinoids and synthetic cathinones are classified as "designated substances", and comprise a new class of illegal drugs. In order to offer rapid and expert testimony on these drugs, a screening method that uses direct analysis in real time (DART ^TM) time-of-flight (TOF) mass spectrometry (MS) was developed. DART enabled the direct introduction of a sample without any pretreatment, such as extraction and cleanup. The positive-ion mode was suitable for ionization in TOF-MS. It was also possible to accurately measure the mass of each drug. The mass spectra clearly showed all peaks representing the protonated molecules of synthetic cannabinoids (14 types) and synthetic cathinones (three types). The analysis of samples of herbal products, plant leaves, and tablets was possible without any pre-processing of the samples. As a result, stimulants, cannabis components, and several synthetic cannabinoids (such as JWH-210, , JWH-203, JWH-081, 4-methylehcathinone, and naphyrone) were identified in each sample. With DART-TOF-MS, the mass spectrum was obtained in a few seconds. The analysis with DART-TOF-MS was proven to be useful for screening controlled substances.

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Cannabis is an important industrial plant, in addition to its illicit drug use. Compound Δ⁹-THC (Δ⁹-tetrahydrocannabinol) is mainly responsible for the hallucinogenic effect on humans. The aminoalkylindole group cannabimimetics targets at the same physiological receptors to mimic the analgesic effects of Δ⁹-THC. Since there is no reliable colorimetric test to detect these synthetic cannabimimetics on site, a simple colorimetric assay for (aminoalkyl)indole group-containing drugs was developed, based on the silica/sulfuric acid-catalyzed Ehrlich reaction of (aminoalkyl)indoles with p-dimethylaminobenzaldehyde. The electrophilic substitution reaction of indoles with carbonyl compounds resulting in the formation of bis(indolyl)alkanes in an acid-catalyzed reaction has been used for the first time for their spectrophotometric determination by color change from yellow to purple/blue. The method was statistically validated against liquid chromatography tandem mass spectrometry, and applied to certain (aminoalkyl)indole derivatives, with 0.5 – 2.5 μg mL⁻¹ detection limits for

-, JWH-081, MAM-2201, JWH-018, JWH-210, JWH-122, 5F-PB-22 and XLR-11.

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  Recently, illegal herbal or liquid products containing psychoactive compounds have been a serious problem damaging human health and causing numerous traffic accidents. Reports indicate that most of those herbal products contain various types of synthetic cannabinoids. There are many on-site drug-testing devices; however, synthetic cannabinoids are not targeted compounds for such devices. In this study, we evaluated the on-site drug-testing device “K2/Spice Test” for the detection of 12 different types of 38 synthetic cannabinoids (including 13 naphthoylindole-type synthetic cannabinoids) and a natural cannabinoid (Δ⁹-tetrahydrocannabinol). Although this device is primarily used for the detection of metabolites of naphthoylindole-type synthetic cannabinoids in urine samples, we applied it to detect synthetic cannabinoids in illegal herbal products for rapid screening analyses. As a result of the on-site examination of synthetic cannabinoids, 10 naphthoylindole-type synthetic cannabinoids [five narcotics (JWH-018, JWH-073, -, MAM-2201, and JWH-122); five designated substances (JWH-015, JWH-200, AM-1220, JWH-019, and JWH-020)], and two other types of synthetic cannabinoid [designated substances (a benzoylindole AM-694 and a naphthoylnaphthalene CB-13)] showed positive results (the limit of detection ranged from 50 to 250 μg/mL). Furthermore, MeOH extracts of illegal herbal products containing naphthoylindole-type synthetic cannabinoids also showed positive results (the limit of detection ranged from 2.5 to 10 mg herbal products/mL). Therefore, we found that this device may be useful for the on-site examination of some naphthoylindole-type synthetic cannabinoids not only in urine samples but also in illegal herbal products.

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 Several synthetic cannabinoids such as - contain the 3-carbonyl-N-fluoropentylindole structure. This structure has fluorine positional isomers on the alkyl chain. In most cases, legal controls are placed only on the 5-fluoro analogue. Thus, differentiation of isomers is a significant issue in forensic science. In this study, we developed a method for the differentiation of positional isomers of 3-carbonyl-N-fluoropentylindole derivatives utilizing multiple-stage mass spectrometry using an ion trap tandem mass spectrometer. In addition, the analogues whose fluorine atom was replaced with a chlorine atom or hydroxyl group were also examined.
 With respect to each positional isomer of fluorine and chlorine, the ion at m/z 232 or m/z 248, obtained by MS² analysis of [M＋H]⁺, were selected as the precursor ions for MS³ analysis. The ion at m/z 232 and m/z 248 corresponded to the 3-carbonyl-N-fluoropentylindole and 3-carbonyl-N-chloropentylindole structures. Furthermore, the ion at m/z 212, corresponding to the de-halogenated fragments of the 3-carbonyl-N-fluoropentylindole- and 3-carbonyl-N-chloropentylindole-structures, was selected as the precursor ion for MS⁴ analysis. Consequently, combination of these MSⁿ analysis achieved differentiation of all the positional isomers.
 With respect to positional isomers with the hydroxyl group, however, the fragment ion at m/z 212 was not observed from the MS³ analysis of m/z 230, which corresponds to the 3-carbonyl-N-fluoropentylindole structure. Therefore, differentiation of each positional isomer was not achieved by MSⁿ analysis.
 This method is useful for the differentiation of positional isomers of 3-carbonyl-N-fluoropentylindole and 3-carbonyl-N-chloropentylindole derivatives.

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  In recent years, many analogs of narcotics have been widely distributed as easily available psychotropic substances and have become a serious problem in Japan. To counter the spread of these non-controlled substances, the Pharmaceutical Affairs Law in Japan was amended in 2006 to establish a new category; Designated Substances in order to more strictly control these substances. In April 2007, 31 compounds and 1 plant were first controlled as Designated Substances. Before 2007, the major compounds distributed in the Japanese illegal drug market were tryptamines, phenethylamines and piperazines. Alkyl nitrites, such as isobutyl nitrite and isopentyl nitrite, were also widely distributed. After they were listed as Narcotics or Designated Substances in 2007, these compounds, especially the tryptamines, quickly disappeared from the market. In their place, cathinone derivatives have been widely distributed, as well as different phenethylamines and piperazines. Additionally, in recent years, new herbal products containing synthetic cannabinoids have appeared globally. As at July 2012, 78 substances (including 1 plant; Salvia divinorum) were listed in the category of Designated Substances. They were 13 tryptamines, 17 phenethylamines, 11 cathinones, 4 piperazines, 23 synthetic cannabinoids, 6 alkyl nitrites, 3 other compounds and 1 plant. In this review, we show our survey of the spread of new designer drugs in Japan, focusing especially on synthetic cannabinoids and cathinone derivatives. Also, the prevalence and legal status of these substances in other countries will be presented.

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Drug abusers most often smoke ‘herbal incense’ as a cigarette or inhale it using a smoking tool. Smoking may cause pyrolysis of the drug and produce decomposed products of which biological effect has never been investigated. The synthetic cannabinoid UR-144 is known to undergo thermal degradation, giving a ring-opened isomer, so-called UR-144 degradant. The present study demonstrates by using UR-144 as a model drug that the smoke of burned UR-144 contains the UR-144 degradant. The UR-144 degradant showed approximately four fold higher agonist activity to human CB₁ receptor and augmented hypothermic and akinetic actions in mice compared to UR-144. These results indicate that smoking behavior may increase psychological actions of the certain synthetic cannabinoids.

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2011年頃から，危険ドラッグが関与したと考えられる健康被害や自動車事故等の他害事件の報告が急増し，大きな社会問題となった．2016年4月時点で，医薬品医療機器等法下，指定薬物として規制されている物質数は2,343物質であるが，その中でカンナビノイド受容体に対し作用を有する合成物質群（合成カンナビノイド）の数は最も多く，全体の約40％を占める（包括指定を除く）．本稿では，合成カンナビノイドの流通実態およびその法規制について解説する．

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  Prior to the designation of illegal drugs (psychoactive drugs) by prefectural regulations, the Tokyo Metropolitan Government conducts surveys on the risk of drugs, reports the results to the governor through the Tokyo Metropolitan Government Advisory Committee on Illegal Drugs, an affiliated organization, and provides the central government with information. The Tokyo Metropolitan Institute of Public Health conducts identification of the constituents of drugs and biological effect tests to help the committee analyze and assess information on the risks of drugs. Narcotics and stimulants increase the concentrations of dopamine, serotonin, and norepinephrine, i.e., neurotransmitters, in the presynaptic clefts, exerting an excitatory effect. In the postsynaptic region, these neurotransmitters are considered to directly combine with the receptors and activate guanine nucleotide-binding proteins, causing activation. We have developed nine types (categorized into three groups) of simple, high-throughput measurement systems and examined their measurement methods. The systems are designed to assess the following properties of drugs: effects of: 1) inhibiting reuptake; 2) stimulating the release of neurotransmitters in the presynaptic region; and 3) activating G proteins in the postsynaptic region. The systems provide useful information in that they allow searches for the effects of a variety of psychoactive substances that are expected to become widespread, e.g., designer drugs, hallucinogenic plants and synthetic cannabinoids; they also allow you to conduct a test using micrograms of a drug, facilitating testing even when it is not available in a large quantity.

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  Cases of people experiencing disturbed consciousness or dyspnea, causing traffic accidents, or requiring ambulance transport to hospital due to abuse of law-evading chemical substances have become a serious social problem in Japan. Most law-evading herbal products are marketed as incense or herbs and consist of finely chopped, dry vegetative matter mixed with chemical substances (drugs). Analysis of the chemical substances in these herbal products has demonstrated that they contain synthetic cannabinoids. Because there are many cannabinoid compounds, even if a particular drug is regulated, similar compounds that differ only slightly in structure may be added in their place. Therefore a cat-and-mouse game exists between regulations on chemical substances and their propagation. This paper summarizes the pharmacological actions and dangers of chemical substances contained in law-evading herbal products by focusing on synthetic cannabinoids, as a group of chemical substances contained in these products. Furthermore, comprehensive designations of synthetic cannabinoids have been introduced as a new method of regulation that emphasizes the similarity of chemical structures; this paper also outlines the comprehensive designations. We established a psychic-dependence liability and cytotoxicity screening system for synthetic cannabinoids using animals (behavioral analysis in vivo) and cell cultures (cytotoxicity analysis in vitro). With our drug-screening system, we were able rapidly to evaluate and quantify psychic-dependence liabilities and cytotoxicity of synthetic cannabinoids contained in law-evading herbal products. These scientific data using our screening system contributed to the establishment of legislation for comprehensive designations of synthetic cannabinoids.

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Metabolomics is an essential tool for not only understanding the pathophysiology of various diseases but also for searching for initial clues to unknown toxic effects of drugs. Mass spectrometry-based metabolomics has achieved highly sensitive and selective analysis of metabolites, and gas chromatography mass spectrometry remains a gold standard because of its robustness and usability. However, it is tedious to annotate metabolites with electron ionization (EI)-based mass spectra; thus, gas chromatography tandem mass spectrometry (GC/MS/MS)-based metabolome analysis has played an important role in metabolomics. In particular, the selected reaction monitoring (SRM) mode achieves higher selectivity and an improved signal-to-noise ratio, which leads to easier metabolite identification. In this mini review, we concisely outline the pros and cons of GC/MS/MS-based metabolome analysis and provide its applications to pathophysiological analysis of disease and drug-induced toxicity in animal models based on our previous studies. Finally, future perspectives for newly developed high-throughput metabolome analysis are briefly described.

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Legally regulated synthetic cannabinoids (SCs) are continuously being created by making minor positional modifications to pre-existing analogs; thus, compounds with minor structural differences must be isolated and identified accurately. For iodo-benzoylindole derivatives of SCs, only specific isomers are currently the target of legal control, and it is necessary to establish an analytical method for accurately identifying positional isomers. In this study, we synthesized a series of 57 designer drugs and developed a screening method for identifying halogen positional isomers on the phenyl ring of benzoylindole derivative SCs in serum. Analytical methods using the Discovery F5 pentafluorophenyl column gave the best selectivity and retention of the positional isomer analytes. Some of the meta and para iodo-substituted SCs were eluted at similar retention times and were difficult to separate by liquid chromatography (LC). However, they were identified via the relative abundance of the two product ions in the collision-induced dissociation reaction using LC-hybrid quadrupole/orbitrap high-resolution mass spectrometry. Our synthesized halogen-substituted positional isomer SC library and method for differentiating positional isomers of halogenated benzoylindole SC derivatives could provide an indispensable analysis tool for identifying illegal drugs in serum of drug users.

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  Abuses of new psychoactive substances have recently become serious social problems. When synthetic cannabinoids are consumed, most of the drugs are not detected in unchanged form from the urine. In that cases, it is difficult to determine what drug was consumed in drug testing. If blood and saliva of suspects are obtained immediately after drug consumption, unchanged drugs could be detected from the specimens. Therefore, on-site sampling of these specimens are effective to determine the consumed drugs. We examined how police officers easily obtain blood and saliva of suspects on site and what drug concentrations are needed to detect drugs in blood and saliva obtained by the sampling methods. First, blood and saliva sampling methods were examined using various collecting tools. For blood sampling method, it was effective to bleed from a fingertip with a lancet and then to absorb the blood to paper pulp. Blood of approximately 5 μL was obtained by this safe and simple method. For saliva sampling method, dropping saliva directly into a 25 mL centrifuge tube (direct sampling method) was convenient for drug analysts. However, because some subjects felt it unpleasant that the sampling situation was watched by the observer, the alternative sampling method, absorbing saliva in a mouth with a cotton swab was also used. Saliva of at least 50 μL was obtained by the two methods. Next, five drugs (JWH 018, 5F-APINACA, 4-MeO α-PVP, 4-Cl AMP and MeBP) in blood and saliva were analyzed using a liquid chromatograph-ion trap mass spectrometer to estimate the concentrations required for drug detection. The limits of detection of five drugs were in the range of 0.1-10 ng/mL for blood (5 μL) and 0.01-1 ng/mL for saliva (50 μL) obtained by the direct sampling method. On the other hand, absorbing saliva by a swab made drug detection difficult because synthetic cannabinoids, JWH 018 and 5F-APINACA, were strongly adsorbed in the swab. It is considered that saliva obtained by the direct sampling method is effective for drug testing because the sampling is rapid and simple, a large volume of saliva is obtained, and the drug concentrations in abusers' saliva are generally high as compared with those in blood.

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 From 2012 to 2014 in Japan, a total of 214 cases of motor vehicle collisions were attributed to the use of illegal drugs by drivers. In 93 out of 96 investigated cases, the causative agents were 22 kinds of synthetic cannabinoids (SCs). Those SCs can be grouped into three groups according to the timeline of use and their chemical structures. The first generation SC naphtoyl indole derivatives, such as MAM-2201, were used in 2012 and disappeared by governmental overall regulations in Spring, 2013. Instead, quinolinyl ester indoles (second generation SC, such as 5F-PB-22) and indazole carboxamides (third generation SC, such as 5F-AB-PINACA) appeared thereafter with much stronger potencies. An outbreak of SC occurred in Summer, 2014 with one of the strongest SCs, 5F-ADB. The common signs observed in the SC-abused drivers are impaired consciousness, anterograde amnesia, catalepsy with muscle rigidity, tachycardia, and vomiting or drooling. Since the third generation SCs are extremely potent CB1 agonists (only a small amount is required) and instable in blood, it is very difficult to detect SCs in biological samples. Actually, only in one third of the cases, SC could be detected in blood or urine.

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  We previously developed a rapid method for morphological examination of cannabis and its related herbal products. In this study, general methods for chemical examination by thin layer chromatography (TLC) and gas chromatography/mass spectrometry (GC/MS) were improved. In TLC, a development using toluene-hexane-diethylamine (25:10:3, by vol.) at 40°C allowed sufficient separation of cannabinoids and rapid analysis. It was also confirmed that the TLC conditions were applicable to the discrimination of cannabinoids and the other components in herbs. In the pretreatment for GC/MS, using a 96-well plate and a multi-channel pipette allowed to prepare many analytical samples at the same time. In GC/MS, using a short and narrow bore capillary column allowed rapid analysis (ca. 10 min/sample). Based on the chemical examination method in this study and the morphological examination method that we previously developed, a rapid procedure to examine cannabis and its related herbal products was proposed. In the morphological and chemical examination methods that we developed, the total time required for morphological examination by microscopy, color test by Fast Blue B reagent, and chemical examination by TLC was approximately 3 h for 96 samples.

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危険ドラッグの蔓延が社会問題となっている．出現する危険ドラッグは，主に中枢神経作用薬であり，これまでに流通が確認されていない「新規の化学物質」である場合が多い．代表的な薬物としては，合成カンナビノイド，カチノン系化合物，フェンシクリジン類似の催幻覚薬等である．こうした危険ドラッグに適切な規制を施すためには，薬物依存性，毒性などの有害作用に関する科学的根拠を収集することが必須であり，迅速な作用評価システムの構築が重要である．行動薬理学的手法による中枢神経に関する評価法：自発運動量の変化を指標にすることで，薬物の中枢興奮作用もしくは中枢抑制作用の有無を推測できる．また，条件付け場所嗜好性試験による報酬効果の解析から，薬物の精神依存性を予測できる．一方，多くの類縁化合物が存在する場合や催幻覚薬の評価においては，既存の規制薬物を訓練薬物として，その自覚効果の類似性を評価する薬物弁別試験は効率的な手法である．細胞を利用する評価法：薬物の毒性評価に有用である．危険ドラッグの作用点が明確なもの，例えば，合成カンナビノイドであればカンナビノイド受容体であるが，カンナビノイド受容体を強制発現させた細胞による合成カンナビノイドの毒性評価は，迅速かつ高感度での解析が可能である．危険ドラッグの作用点に着目した検出法は，薬物自体の化学構造に依存しない検出法として，化学構造が変化しても有害作用の発現が危惧される「危険性の検出」が可能となり，現場での迅速かつ効果的な流通規制が可能となる．薬物規制のために科学的根拠を収集することは，まさに薬物の作用機序を解明するための基礎研究の一部であると捉えることができる．登場する危険ドラッグは刻一刻と変化することから，新規薬物の検出システムの開発を進めるとともに，薬物乱用防止の啓発および薬物依存症対策の強化が急務である．

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