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Forensic Toxicol (2015) 33:112–121 Identification and quantitation of 5-fluoro-ADB, one of the mostdangerous synthetic cannabinoids, in the stomach contentsand solid tissues of a human cadaver and in some herbal products Koutaro Hasegawa • Amin Wurita • Kayoko Minakata •Kunio Gonmori • Itaru Yamagishi • Hideki Nozawa •Kanako Watanabe • Osamu Suzuki Received: 25 November 2014 / Accepted: 26 November 2014 / Published online: 12 December 2014Ó Japanese Association of Forensic Toxicology and Springer Japan 2014 Since late September 2014, there have been addition method for quantitation to overcome the matrix approximately 10 cases in Japan where people have died effects. The levels of 5-fluoro-ADB in the cadaver from inhaling smoke from herbal blends containing a specimens were generally low; it could not be detected newly emerged synthetic cannabinoid. Although the drug from blood or urine specimens. The levels of 5-fluoro- ADB in solid tissues were 1.17–7.95 ng/g. Because the institutions as 5-fluoro-ADB, to our knowledge, its data highest levels were found for the adipose tissue and heart have not been published in any scientific context. We muscle, the final extracts of the adipose tissue and/or recently encountered an autopsy case in which 5-fluoro- heart muscle were concentrated 10- and 200-fold to ADB was involved. The deceased was a 34-year-old man obtain product ion mass spectra of 5-fluoro-ADB using who was found dead in his room. The postmortem LC–MS–MS and its mass spectrum by gas chromatog- interval was estimated at 35–40 h. The direct cause of the death was asphyxia due to aspiration of stomach contents completely coincided with those obtained from the ref- into the trachea, which likely took place during vomiting erence standard 5-fluoro-ADB, confirming that the target under low-consciousness conditions provoked by inhala- compound was 5-fluoro-ADB. The quantitative results tion of the 5-fluoro-ADB smoke. The cadaver was sub- obtained by selected reaction monitoring of LC–MS–MS jected to autopsy at our department. Femoral vein blood, showed the highest level, at 7.95 ng/g, in the adipose right heart blood, left heart blood, urine, stomach con- tissue, followed by stomach contents, brain, heart muscle, tents, and nine solid tissues including the adipose tissue pancreas, and spleen. For the lung, liver, kidney, and were collected and frozen until analysis. The extraction skeletal muscle, levels were below the quantitation limit of 5-fluoro-ADB and internal standard 5-fluoro-AMB was (about 0.5 ng/g), although very small peaks above the performed using a modified QuEChERS method plus detection limit (about 0.1 ng/g) could be observed for all filtration through Captiva ND Lipids cartridges, followed of the above solid tissues. The low levels of 5-fluoro- by liquid chromatography–tandem mass spectrometry ADB in the solid tissues were likely as a result of only a (LC–MS–MS) analysis. Because this study dealt with small amount of 5-fluoro-ADB incorporated into the body various kinds of human matrices, we used the standard via the lungs due to the short period from the beginningof smoking the herb to the fatal asphyxia resulting fromaspiration of a massive amount of stomach contents intothe trachea under low-consciousness conditions. In addi-tion, we measured the content of 5-fluoro-ADB in three K. Hasegawa and A. Wurita contributed equally to this work.
packages, all of which were opened, that were found K. Hasegawa (&)
A. Wurita
K. Minakata
K. Gonmori
under a pillow near the deceased; their levels of 5-fluoro- I. Yamagishi
H. Nozawa
K. Watanabe
O. Suzuki ADB were 49.2 mg/g, 12.2 lg/g, and 0.77 lg/g. To our Department of Legal Medicine, Hamamatsu University School knowledge, this is the first reported identification and of Medicine, 1-20-1 Handayama, Higashi-ku, quantitation of 5-fluoro-ADB in human specimens and Hamamatsu 431-3192, Japane-mail: [email protected] herbal products.
Forensic Toxicol (2015) 33:112–121 5-Fluoro-ADB
Methyl (R)-2-[1-(5- encountered an autopsy case in which 5-fluoro-ADB was involved. Three silver-colored packages containing herbal dimethylbutanoate
Synthetic cannabinoid
Adipose blend mixtures were also found in the room of the deceased tissue
Postmortem distribution
Herbal blend products by law enforcement, and were delivered to our laboratoriesfor analysis. In this study, we identify and quantitate5-fluoro-ADB in various specimens collected at autopsy as well as in the illegal herbal products. To our knowledge,this is the first report to identify and quantitate the new In recent years, synthetic cannabinoids [–] and cathi- designer drug 5-fluoro-ADB from biological and herbal nones have become widely distributed, and are now blend specimens.
causing social problems throughout many parts of theworld. Although the traditional marijuana and hashishcontaining D9-tetrahydrocannabinol as the psychoactive compound have been associated with almost no fatalitiesthus far, two reports have described fatalities caused by the The deceased was a 34-year-old man, who was found dead in synthetic cannabinoids MAM-2201 ] and NNEI his room in a house owned by his mother. He was lying in a We also reported a fatal case due to the combined use of prone position on his futon. There were remarkable post- two synthetic cannabinoids, AB-CHMINACA and 5-flu- mortem vibices on the front chest, abdomen, and the front oro-AMB, and the N-methyl-D-aspartate receptor channel parts of both thighs. He was grasping a small handmade blocker diphenidine ].
aluminum foil pipe in his right hand. Around his nostrils and Very recently, a new and very dangerous synthetic mouth, there were vomit debris attached. Three opened sil- cannabinoid has emerged; since late September 2014, there ver-colored packages of different brands of herbal blends have been approximately 10 deaths in Japan caused by were found under his pillow. Medical examination at the smoking this compound (Kikura-Hanajiri, personal com- scene found no evidence of no criminality except for the munication). Although it was tentatively identified by some suspected drug abuse by the deceased; the cadaver was rel- drug-monitoring institutions as 5-fluoro-ADB (see its atively fresh, and no injuries were found on the body surface.
structure in Fig. ), to our knowledge, data have not The cadaver was stored in a refrigerated morgue at 2 °C for been published in any scientific context. We recently 1 day. The autopsy was performed at our department.
Fig. 1 Structures of 5-fluoro-ADB and 5-fluoro-AMB(internal standard, IS) Forensic Toxicol (2015) 33:112–121 At the beginning of the autopsy, the postmortem interval Chemical (Ann Arbor, MI, USA). Other common chemi- was estimated to be 35–40 h. External macroscopic cals used were of the highest purity commercially avail- observations noted that livor mortis was remarkably asso- able. Plastic centrifuge tubes with caps (5-ml capacity, ciated with vibices over wide areas of the front body sur- 6 9 1.5 cm external diameter) and stainless beads (5 mm face. There was remarkable congestion of the face, and external diameter) for crushing solid tissues were pur- petechiae were noted on both palpebral and bulbar chased from TAITEC, Saitama, Japan. The QuEChERS dispersive SPE centrifuge tubes with caps (2-ml capacity), There were no serious injuries related to death. Inter- each of which contained 25 mg of primary-secondary nally, the trachea was filled with a large amount of amine (PSA), 25 mg of end-capped octadecylsilane stomach contents, which reached the tracheal bifurcation, (C18EC), and 150 mg of magnesium sulfate, and Captiva thus completely occluding the airway. Such a massive ND Lipids cartridges (3-ml capacity) were purchased from aspiration of stomach contents into the trachea is usually Agilent (Santa Clara, CA, USA).
not observed for persons with average physical strength Whole blood specimens from the right and left atria and and in a state of clear consciousness. This phenomenon is femoral vein, urine, stomach contents, and solid tissue likely due to lowered consciousness together with vom- specimens (brain, lung, heart muscle, liver, spleen, kidney, iting provoked by the inhalation of synthetic cannabi- pancreas, skeletal muscle, and adipose tissue) were col- noid(s). There were petechiae on the inside surface of the lected from the cadaver at autopsy and kept frozen at scalp and on the surface of both lungs and the heart.
-80 °C until analysis; the adipose tissue specimen was Congestion of both lungs was remarkable. These findings obtained from the abdominal subcutaneous area.
are consistent with asphyxia as the direct cause of death;the indirect cause appeared to be synthetic cannabinoid Extraction procedure for human specimens except adipose tissue Routine analysis of blood alcohol using gas chroma- tography showed a low level of alcohol (0.3 mg/ml) in One gram or 1 ml of solid tissue or fluid specimens, the blood. Immunochemical drug screening using the respectively, was placed in a 5-ml plastic tube with a cap Triage Drugs of Abuse panel (Alere, Waltham, MA, containing 4-ml acetonitrile and 1,000 ng of 5-fluoro- USA) for urine specimens showed a positive result for AMB (IS) dissolved in 10 ll acetonitrile. Tissue speci- barbiturate drugs. NAGINATA screening for conventional mens were minced with clean surgical scissors; this step drugs and toxic compounds in whole blood using gas was skipped for fluid or stomach content specimens. Five stainless beads were added to the mixture, and the tube revealed the presence of a low level of quetiapine and a was capped, held to a bead beater-type homogenizer nicotine metabolite. The deceased had a history of (Beads Crusher lT-12; TAITEC), and vigorously shaken admission to a mental health hospital because of heavy at 3,200 rpm for 5 min. All of the suspension solution dependence on the designated drugs for about 3 months in except the beads was transferred to a large test tube, and 2013 and 2014. Upon discharge in 2014, which had 5 ml of acetonitrile was added, and the solution was occurred just several days earlier, he had been prescribed gently shaken. Six 1-ml portions were taken from the quetiapine, olanzapine, levomepromazine, biperiden, and 10-ml homogenate suspension, and were prepared, with troxipide by the psychiatrist. However, the NAGINATA and without the addition of different amounts of 5-fluoro- screening test identified only quetiapine, and the reason ADB dissolved in 10 ll of acetonitrile, in 1.5-ml plastic for the failure to detect the other psychotropic drugs was centrifuge tubes with caps to construct a standard addition calibration curve, vortexed for 30 s, and centrifuged at10,000 rpm for 2 min. The clear supernatant was dec-anted into the QuEChERS dispersive SPE centrifuge tube Materials and methods containing PSA, C18EC, and magnesium sulfate, vortexedfor 30 s, and centrifuged at 10,000 rpm for 2 min. The upper acetonitrile layer was passed through a Captiva NDLipids cartridge. A 3.5-ll aliquot of the eluate was then by Dr. R. Kikura-Hanajiri of the National Institute of When the product ion mass spectrum via LC–MS–MS Health Sciences, Tokyo, Japan. 5-Fluoro-AMB, used as an or mass spectrum via GC–MS was recorded, the final internal standard (IS) (see its structure in Fig. ) for ana- acetonitrile eluate was condensed approximately tenfold or lysis of 5-fluoro-ADB, was purchased from Cayman 200-fold, respectively, by evaporation.
Forensic Toxicol (2015) 33:112–121 Extraction procedure for the adipose tissue specimen Eclipse Plus C18 column (100 9 2.1 mm internal diame-ter, particle size 1.8 lm; Agilent) was used. The LC con- One gram of solid adipose tissue was placed in a 5-ml ditions were as follows: injection volume, 3.5 ll; flow rate, plastic tube with a cap containing 4 ml of acetonitrile and 0.25 ml/min; elution mode, gradient with 10 mM ammo- 5,000 ng of IS dissolved in 10 ll of acetonitrile. The nium formate/0.1 % formic acid in distilled water (A) and specimen was minced with clean surgical scissors. The acetonitrile (B) from 60 % A/40 % B to 100 % B over 5-ml plastic tube containing the mixture was heated at 15 min, followed by isocratic elution with the final solvent 80 °C for 10 min, and the five stainless steel beads were composition for 10 min. The column and autosampler were added to the mixture. The tube was capped, held to the operated at room temperature.
bead beater-type homogenizer, and vigorously shaken at The tandem MS conditions were as follows: interface, 3,200 rpm for 5 min. Despite vigorous shaking, the lique- ESI mode; polarity, positive ion mode; ion source tem- fied fat layer and acetonitrile layer did not mix well. All of perature, 320 °C; ion source voltage, 500 V; quantitation, the mixture except the beads was transferred to a scaled selected reaction monitoring (SRM) mode, using peak area; 50-ml conical flask, and total volume of up to 50 ml was ion transitions, m/z 378 ? 233 for 5-fluoro-ADB and m/z made by the addition of acetonitrile, followed by gentle 364 ? 233 for 5-fluoro-AMB (IS); fragmentor voltage and shaking. At this stage, the liquid fat and acetonitrile were collision energy, 120 and 21 V for 5-fluoro-ADB, and 120 completely mixed. Six 1-ml portions taken from the 50-ml and 17 V for 5-fluoro-AMB, respectively.
homogenate in acetonitrile were prepared with and without Data acquisition, peak integration, and calculations were the addition of different amounts of 5-fluoro-ADB dis- performed on an Agilent MassHunter computer worksta- solved in 10 ll of acetonitrile in 1.5-ml plastic centrifuge tion (Revision: Acquisition, B02.01; Qualification, B03.01; tubes with caps, vortexed for 30 s, and centrifuged at SP2 and Quantification, B04.00).
10,000 rpm for min. Each supernatant fraction was sub-jected to QuEChERS dispersive solid-phase extraction and GC–MS conditions filtered through the Captiva ND Lipids cartridge, asdescribed above; a 3.5-ll aliquot of the eluate was ana- The GC–MS instrument used was an Agilent 6850 gas lyzed using LC–MS–MS.
chromatograph connected to a 5975 Series mass spec-trometer (Agilent). GC conditions were as follows: sepa- Extraction procedure for herbal blend specimens ration column, Agilent HP-5 ms fused-silica capillary(30 m 9 0.25 mm internal diameter, 0.25 lm film thick- For analysis of 5-fluoro-ADB in the three herbal blend ness); injector temperature, 250 °C; interface temperature, products, 10 mg of each herbal debris was treated as 280 °C; injection mode, splitless; injection volume, 1 ll; described in a previous report [Briefly, 10 mg of the carrier gas (He) pressure, 151 kPa; oven temperature pro- plant debris was sonicated in 1.0 ml of acetonitrile for gram, initial temperature at 60 °C (2-min hold) followed 10 min, and centrifuged at 10,000 rpm for 2 min. The by ramp at 20 °C/min up to 300 °C. MS conditions were as supernatant layer was decanted into a test tube, followed by follows: ion source temperature, 230 °C; ionization mode, appropriate dilution with acetonitrile (from no dilution to electron ionization (EI) at 70 eV; emission current, 35 lA; 1,000 dilution; overall 100–100,000 dilution). To 1.0 ml of detection gain, 1,118 V; identification, scan mode; scan each diluted acetonitrile extract solution, 10 ll of aceto- range, m/z 50–400; scan speed, 2.86 scans/s.
nitrile solution, with or without an appropriate amount ofreference standard 5-fluoro-ADB, was added and the Standard addition method mixture shaken gently to construct a standard additioncalibration curve. For quantitation of 5-fluoro-ADB in Although the standard addition method is frequently used herbal mixtures, IS was not used; instead, only units of for analysis with atomic absorption spectroscopy in order to peak area were used. A 3.5-ll aliquot of the final solution overcome matrix effects it is not popular in the field of was injected into the LC–MS–MS instrument.
forensic toxicology. We began using the standard additionmethod in our laboratories in a study analyzing ethylene LC–MS–MS conditions glycol (EG) and propylene glycol (PG) in whole bloodspecimens collected from non-occupational and healthy LC–MS–MS with electrospray ionization (ESI) was con- subjects ], where non-negligible concentrations of EG ducted on an Agilent 1200 LC-SL system containing a and PG were found in whole blood of healthy subjects. In microdegasser and high-performance autosampler, which order to measure the preexisting compounds, the standard was connected to a 6460 Triple Quad LC/MS tandem addition method had to be employed. Since then, we have MS instrument (Agilent). For LC separation, a ZORBAX realized that the standard addition method is very useful for Forensic Toxicol (2015) 33:112–121 investigating the distribution of xenobiotics in human body obtain a peak area designated as B. Finally, a 3.5-ll fluid and solid tissue specimens, as the matrices collected aliquot of the reference standard 5-fluoro-ADB acetoni- from human cadavers have quite different properties. In an trile solution at either 10- or 50-fold lower concentration investigation involving measurement of postmortem dis- was injected into the LC–MS–MS system to obtain a tribution of a-pyrrolidinovalerophenone using LC–MS–MS peak area designated as C. The matrix effect and in a case of fatal poisoning, we were embarrassed by the recovery rate was calculated for both matrices, as fol- remarkably different matrix effects among different human lows: matrix effect (%) = [(A - B)/C] 9 100; recovery body fluids and organs [], and the standard addition rate (%) = [B/(A - B)] 9 100.
method was very useful for overcoming the matrix effectsand different recovery rates. The standard addition methodseems to be the best choice in particular for comparison of Results and discussion concentrations in matrices with fairly different properties,and this method was employed in the current study. The Identification of 5-fluoro-ADB using LC–MS–MS details of the procedure and calculation of the results have been detailed in our previous reports ].
Because the preliminary results showed that the adipose Matrix effects and recovery rates tissue and heart muscle contained the highest concentra-tions of the target compound, we took the final acetonitrile In order to obtain the values of matrix effects and extract solutions from the two solid tissues, concentrated recovery rates for 5-fluoro-ADB in human specimens, 10- and 200-fold for LC–MS–MS and GC–MS analysis, the starting point is to measure all concentrations of a respectively. Figure shows the product ion mass spectra target compound in all specimens. In this experiment, of LC–MS–MS obtained from the reference standard the testing of the matrix effects and recovery rates of 5-fluoro-ADB, heart muscle extract, and adipose tissue 5-fluoro-ADB in the heart muscle and adipose tissue are extract. The spectra obtained from both tissues coincided simply provided as examples. After measuring 5-fluoro- well with that of the reference standard 5-fluoro-ADB, with ADB concentrations in both matrices according to the no impurity peaks. The base peak appeared at m/z 233, methods previously described, we prepared two con- which was used for quantitative analysis.
centrations of reference standard 5-fluoro-ADB dissolved Figure shows the mass spectra of GC–MS obtained in pure acetonitrile for each matrix: one with concen- from the reference standard 5-fluoro-ADB and from the trations 10 times higher and 10 times lower than those in adipose tissue extract. The spectrum obtained from the the matrix for the heart muscle specimen; and one adipose tissue also coincided with that of the reference concentrations two times and 50 times lower than those standard 5-fluoro-ADB. Therefore, we were able to con- in the matrix for the adipose tissue specimen. Then, 1 g clude that 5-fluoro-ADB was identified in the adipose tis- of the heart muscle or adipose tissue was crushed in sue and heart muscle of the cadaver.
acetonitrile with addition of IS, diluted, and gentlyshaken according to each method as previously descri- Validation of the method bed. Although we usually take six 1-ml portions from 10or 50 ml of tissue homogenate in acetonitrile for con- Figure shows an example of the SRM chromatograms for structing a standard addition calibration curve, in this the target compound and IS extracted from the heart case we took four 1-ml portions from each matrix muscle and adipose tissue. The target compound 5-fluoro- homogenate, which were subjected to the centrifugation, ADB and IS 5-fluoro-AMB appeared at retention times of the QuEChERS dispersive solid-phase extraction, and 9.58 and 8.78 min, respectively, under our analytical con- the filtration through Captiva ND Lipids cartridges, also ditions. The bottom panel of Fig. shows the absence of as previously described, with no additions. The final 5-fluoro-AMB in the adipose tissue extract, indicating that three eluates from the cartridges were combined, and 5-fluoro-AMB was able to be used as IS in this study. It 10 ll of the reference standard 5-fluoro-ADB acetonitrile should be noted that the backgrounds were generally very solution at either tenfold or twofold higher concentration low, and there were no impurity peak interfering with the was added to 1.0 ml of the combined extract acetonitrile target or IS peaks.
solution and shaken gently. A 3.5-ll aliquot was injected Table shows standard addition calibration equations into the LC–MS–MS system to obtain a peak area des- for 5-fluoro-ADB in the specimens in which the test ignated as A. A 3.5-ll aliquot of the final eluate from the compound could be quantitated. The correlation coefficient fourth 1.0-ml portion of the homogenate, with no addi- values obtained for all six specimens were greater than tion, was also injected into the LC–MS–MS system to 0.999. The detection limit (signal-to-noise ratio C3) for the Forensic Toxicol (2015) 33:112–121 Fig. 2 Product ion mass spectra of the extracts of the heart muscle recorded by using liquid chromatography–tandem mass spectrometry and adipose tissue specimens collected from the deceased cadaver in (LC–MS–MS) together with the probable fragmentation mode comparison with that of the reference standard 5-fluoro-ADB, compound using this method was around 0.1 ng/ml or g.
Because we employed the standard addition method for The lower quantitation limit (signal-to-noise ratio C10) quantitation, without the use of blank specimens, it was was around 0.5 ng/ml or g.
impossible to present the usual accuracy and precision data.
Forensic Toxicol (2015) 33:112–121 Standard 5-fluoro-ADB 150 200 250 300 350 400 (m/z) 150 200 250 300 350 400 (m/z) Fig. 3 Mass spectrum of the extract of the cadaver adipose tissue chromatography–electron ionization–mass spectrometry together with specimen extract of the deceased in comparison with that of the the probable fragmentation mode reference standard of 5-fluoro-ADB, recorded by using gas Instead, as shown in Table , we repeated intraday and heart muscle and adipose tissue specimens, respectively.
interday determinations of 5-fluoro-ADB in the heart The recovery rates of the test compound were also excel- muscle and adipose tissue specimens as examples. The lent, at 91.8 ± 1.43 and 101 ± 1.12 % (n = 3 in each) for repeatability, expressed as relative standard deviations, was the heart muscle and adipose tissue specimens, respec- not greater than 7.26 %.
tively. The final treatment with the Captiva ND Lipids Although the standard addition method can overcome cartridge appears to be very useful in preventing the matrix effects and low recovery rates, the matrix effects for depressive matrix effects likely caused by ionized 5-fluoro-ADB in various matrices under the present extraction conditions are of interest. In this study, we usedacetonitrile deproteinization plus QuEChERS dispersive Postmortem distribution of 5-fluoro-ADB in the various solid-phase extraction plus filtration through a Captiva ND cadaver specimens Lipids cartridge coupled to an LC–MS–MS analysis sys-tem.
Table shows the postmortem distribution of 5-fluoro- 87.7 ± 2.98 and 98.8 ± 1.20 % (n = 3 in each) for the ADB in the body fluids, stomach contents, and nine solid Forensic Toxicol (2015) 33:112–121 Forensic Toxicol (2015) 33:112–121 b Fig. 4 Selected reaction monitoring chromatograms by using LC– Table 3 Concentrations of 5-fluoro-ADB in in body fluids and solid MS–MS for the reference standard 5-fluoro-ADB, the extracts of the tissues of the deceased heart muscle and adipose tissue specimens, the internal standard5-fluoro-AMB spiked into the adipose tissue, and the extract of the Concentration (ng/ml or g) adipose tissue without the addition of IS Femoral vein blood Right heart blood Table 1 Standard addition calibration equations for 5-fluoro-ADB in solid tissues of the deceased, in which 5-fluoro-ADB was quantifiable y = 0.001037x ? 0.00189 0.0008867x ?0.001039 y = 0.00092x ? 0.001479 y = 0.001028x ? 0.00327 Data given as mean ± SD obtained by triplicate determinations ND Not detectable (below the detection limit, 0.1 ng/ml) a If y = 0, the preexisting concentration (x) can be calculated as aminus value direct cause of his death was judged as asphyxia due toaspiration of a massive amount of stomach content vomit tissues, including the adipose tissue of the cadaver. No into the trachea. If the reduction in consciousness and concentrations of 5-fluoro-ADB were detected in any blood vomiting are characteristic effects of 5-fluoro-ADB, the or urine specimens. Although 5-fluoro-ADB concentrations low levels of 5-fluoro-ADB in the victim's specimens are in solid tissues were generally much lower than that of AB- not surprising.
CHMINACA, as published in a previous report [thehighest concentrations were consistently observed in the Identification and quantitation of 5-fluoro-ADB adipose tissue. This high concentration of synthetic can- in herbal blend products nabinoids in adipose tissue is due to the high lipophilicityof the compounds, and also likely due to the very low Three silver-colored packages of herbal mixtures were levels or absence of metabolizing enzymes in the found near the deceased. The brand names, handwritten with a marker pen, were ‘‘GM sapphire'', ‘‘AP 31,'' and The low levels of 5-fluoro-ADB in all solid tissue ‘‘AL 37,'' For each of the herbal blends, 10 mg was specimens could be explained by the fact that only a small extracted as described in a previous report ], and sub- amount of 5-fluoro-ADB in the herbal smoke was inhaled jected to measurements of mass spectra using LC–MS–MS in a very short period of time, as this potent synthetic and GC–MS after appropriate dilution of the extract solu- cannabinoid likely exerted its powerful action on con- tions. Spectra for all of these products were in complete sciousness and provoked vomiting symptom very shortly agreement with those shown in Figs. and , indicating after the victim inhaled the drug smoke. In this case, the that all mixtures contained 5-fluoro-ADB. We then Table 2 Examples of intraday and interday repeatability for determination of 5-fluoro-ADB in postmortem heart muscle and adipose tissue ofthe deceased Concentration found (ng/ml)a Repeatability (%RSD) Concentration found (ng/ml)a Repeatability (%RSD) RSD Relative standard deviationa Data given as mean ± standard deviation (SD) Forensic Toxicol (2015) 33:112–121 quantitated the concentration of the compound in each of designer drugs newly detected in illegal products. Forensic the three products; the results were 49.2 ± 2.46 mg/g, Toxicol 32:266–281 6. Namera A, Urabe S, Saito T, Torikoshi-Hatano A, Shiraishi H, 12.2 ± 0.21 lg/g, and 766 ± 13.7 ng/g for ‘‘GM sap- Arima Y, Nagao M (2013) A fatal case of 3,4-methylenediox- phire'', ‘‘AP 31,'' and ‘‘AL 37,'' respectively.
ypyrovalerone poisoning: coexistence of a-pyrrolidinobutiophe-none and a-pyrrolidinovalerophenone in blood and/or hair.
Forensic Toxicol 31:338–343 7. Namera A, Konuma K, Kawamura M, Saito T, Nakamoto A, Yahata M, Ohta S, Miyazaki S, Shiraishi H, Nagao M (2014)Time-course profile of urinary excretion of intravenously To our knowledge, this is the first scientific description of administered a-pyrrolidinovalerophenone and a-pyrrolidinobuti- the identification and quantitation of 5-fluoro-ADB in ophenone in a human. Forensic Toxicol 32:68–74 8. Hasegawa K, Suzuki O, Wurita A, Minakata K, Yamagishi I, postmortem human specimens and herbal products. This Nozawa H, Gonmori K, Watanabe K (2014) Postmortem distri- compound is one of the most dangerous synthetic can- bution of a-pyrrolidinovalerophenone and its metabolite in body nabinoids ever known. Inhalation of the smoke from this fluids and solid tissues in a fatal poisoning case measured by LC– compound is thought to have been responsible for about 10 MS–MS with the standard addition method. Forensic Toxicol32:225–234 deaths, and the product has very recently become regulated 9. Hasegawa K, Wurita A, Minakata K, Gonmori K, Nozawa H, as a designated substance in Japan. These cases involving Yamagishi I, Suzuki O, Watanabe K (2014) Identification and deaths likely caused by this compound have become quantitation of a new cathinone designer drug PV9 in an ‘‘aroma identified only since late September 2014. Characteristic liquid'' product, antemortem whole blood and urine specimens,and a postmortem whole blood specimen in its fatal poisoning symptoms after inhalation included rapid loss of con- case. Forensic Toxicol 32:243–250 sciousness and cardiopulmonary arrest. Everyone should 10. Saito T, Namera A, Miura N, Ohta S, Miyazaki S, Osawa M, be alert to the life-threatening effects induced by 5-fluoro- Inokuchi S (2013) A fatal case of MAM-2201 poisoning.
Forensic Toxicol 31:333–337 11. Sasaki C, Saito T, Shinozuka T, Irie W, Murakami C, Maeda K, Nakamaru N, Oishi M, Nakamura S, Kurihara K (2014) A case of The authors are very grateful to Dr. R. Kikura- death caused by abuse of a synthetic cannabinoid N-1-naphtha- Hanajiri of the National Institute of Health Science, Tokyo, Japan, for lenyl-1-pentyl-1H-indole-3-carboxamide. Forensic Toxicol. doi providing us with the reference standard of 5-fluoro-ADB.
12. Hasegawa K, Wurita A, Minakata K, Gonmori K, Nozawa H, Conflict of interest There are no financial or other relations that Yamagishi I, Watanabe K, Suzuki O (2014) Postmortem distri- could lead to a conflict of interest.
bution of AB-CHMINACA, 5-fluoro-AMB and diphenidine inbody fluids and solid tissues in a fatal poisoning case: usefulnessof the adipose tissue for detection of the drugs in unchanged forms. Forensic Toxicol. 13. Kudo K, Ishida T, Hikiji W, Hayashida M, Uekusa K, Usumoto 1. Zuba D, Byrska B (2013) Analysis of the prevalence and coex- Y, Tsuji A, Ikeda N (2009) Construction of calibration-locking istence of synthetic cannabinoids in ‘‘herbal high'' products in databases for rapid and reliable drug screening by gas chroma- Poland. Forensic Toxicol 31:21–30 tography-mass spectrometry. Forensic Toxicol 27:21–31 2. Kikura-Hanajiri R, Uchiyama N, Kawamura M, Goda Y (2013) 14. Wurita A, Hasegawa K, Minakata K, Watanabe K, Suzuki O Changes in the prevalence of synthetic cannabinoids and cathi- (2014) A large amount of new designer drug diphenidine coex- none derivatives in Japan until early 2012. Forensic Toxicol isting with a synthetic cannabinoid 5-fluoro-AB-PINACA found in a dubious herbal product. Forensic Toxicol 32:331–337 3. Chung H, Choi H, Heo S, Kim E, Lee J (2014) Synthetic can- 15. Bonilla E (1978) Flameless atomic absorption spectrophotometric nabinoids abused in South Korea: drug identification by the determination of manganese in rat brain and other tissues. Clin National Forensic Service from 2009 to June 2013. Forensic Chem 24:471–474 Toxicol 32:82–88 16. Wurita A, Suzuki O, Hasegawa K, Gonmori K, Minakata K, 4. Uchiyama N, Shimokawa Y, Matsuda S, Kawamura M, Kikura- Yamagishi I, Nozawa H, Watanabe K (2013) Sensitive determi- Hanajiri R, Goda Y (2014) Two new synthetic cannabinoids, nation of ethylene glycol, propylene glycol and diethylene glycol AM-2201 benzimidazole analog (FUBIMINA) and (4-methylpi- in human whole blood by isotope dilution gas chromatography– mass spectrometry, and the presence of appreciable amounts of and three phenethylamine derivatives, 25H-NBOMe 3,4,5-tri- the glycols in blood of healthy subjects. Forensic Toxicol methoxybenzyl analog, 25B-NBOMe, and 2C-N-NBOMe, iden- tified in illegal products. Forensic Toxicol 32:105–115 17. Wurita A, Hasegawa K, Minakata K, Gonmori K, Nozawa H, 5. Uchiyama N, Shimokawa Y, Kawamura M, Kikura-Hanajiri R, Yamagishi I, Suzuki O, Watanabe K (2014) Postmortem distri- Hakamatsuka T (2014) Chemical analysis of a benzofuran bution of a-pyrrolidinobutiophenone in body fluids and solid derivative, 2-(2-ethylaminopropyl)benzofuran (2-EAPB), eight tissues of a human cadaver. Legal Med 16:241–246 synthetic cannabinoids, five cathinone derivatives, and five other

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