Identification of 5-Fluoro ADB in Human Whole Blood in Four Death Cases

Identification of 5-Fluoro ADB in Human Whole Blood in Four Death Cases Abstract Four deaths that seemed to have been caused by a designer drug occurred within a 3-week period in Sendai, Japan. In each case, the decedent possessed the same sachet, labeled “Heart Shot BLACK”, which contained a dried plant material with an aromatic scent. It was revealed in our analysis that the product contained a synthetic cannabinoid receptor agonist, 5-fluoro ADB (methyl 2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamide]-3,3-dimethylbutanoate, also known as 5-fluoro MDMB-PINACA), which is now classified as a restricted designer drug in Japan after it caused several casualties. For standard samples, the detection of 5-fluoro ADB in whole blood in the calibration range (0.04–4 ng/mL) was successful with recoveries of 94.6–98.1%, limits of detection of 6 pg/mL, and limits of quantification of 40 pg/mL. The intraday and interday precisions were 0.9–4.8% and 1.1–6.6%, respectively. The bias was –1.1 to 2.9%. We were able to confirm that 5-fluoro ADB was present in the blood of all four decedents at a concentration of 0.11–1.92 ng/mL. From the autopsy, toxicological findings, and circumstances surrounding the cases, it was considered that inhalation of 5-fluoro ADB could have contributed to the deaths. However, the extent to which 5-fluoro ADB contributed to the deaths remains unclear due to the current lack of toxicological information on the compound. In future research, the toxicity of 5-fluoro ADB in humans and the mechanism underlying this effect need to be elucidated. Introduction There has been a rapid increase in the use of designer drugs globally. In addition, many accidental intoxications, deaths and driving accidents resulting from the abuse of designer drugs have been reported worldwide. Although several designer drugs have been noted and their use is being regulated, new uncontrolled designer drugs are becoming increasingly available. Furthermore, the observed effects of such drugs are getting more powerful and dangerous, and the number of fatal cases resulting from their use increases yearly (1–4). In Japan, many fatal cases were reported over a short period during the fall of 2014. Hasegawa et al. (5) reported that more than 10 people in Japan died after using products containing 5-fluoro ADB (Figure 1), which is an indazole-3-carboxamide-type synthetic cannabinoid (SC) that has been categorized as a designer drug by the Ministry of Health, Labor and Welfare in Japan; however, there are several, related fatalities that have not been reported. Therefore, it was concluded that 5-fluoro ADB could be harmful to humans. 5-Fluoro ADB has also been linked to four deaths that occurred within 3 weeks in Sendai (Japan) in 2014. In each case, the decedents possessed the same designed sachet, labeled “Heart Shot BLACK”, which contained a dried plant with an aromatic scent (Figure 1). In the present study, we developed a liquid chromatography–tandem mass spectrometry (LC–MS-MS) method for the assay of 5-fluoro ADB in the blood of four victims who had used the drug. The analysis was conducted on whole blood samples taken from the external iliac vein and the heart. Figure 1. View largeDownload slide (A) The structure of 5-fluoro ADB. (B) The appearance of the “Heart Shot BLACK” packet. (C) The dried plant material in the packet. Figure 1. View largeDownload slide (A) The structure of 5-fluoro ADB. (B) The appearance of the “Heart Shot BLACK” packet. (C) The dried plant material in the packet. Experimental 5-Fluoro ADB (≥98%, 10 mg/mL in acetonitrile) and 5-fluoro ADBICA (≥98%, 10 mg/mL in acetonitrile) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Acetonitrile and methanol of liquid chromatography–mass spectrometry grade were purchased from Wako Pure Chemical Industries, Limited (Osaka, Japan). Ammonium formate was purchased from Kanto Chemical Company Inc. (Tokyo, Japan). All other chemicals used were analytical grade. Frozen human whole blood was purchased from Biopredic International (Rennes, France). A QuEChERS pre-packed extraction kit containing 6 g of magnesium sulfate and 1.5 g of sodium acetate, and a dispersive solid-phase extraction kit (for fatty samples) were purchased from Agilent Technologies (Santa Clara, CA, USA). Analysis of the Dried Plant Material in “Heart Shot BLACK” A fraction of the dried plant material in “Heart Shot BLACK” was extracted with methanol, and the resulting liquid was diluted appropriately. One microliter of this solution was then subjected to gas chromatography–mass spectrometry (GC–MS) analysis. A GC–MS instrument (5975C, Agilent Technologies, Santa Clara, CA, USA) equipped with a DB-5ms column (30 m × 0.25 mm i.d.; 0.25 μm film thickness; Agilent Technologies, Santa Clara, CA, USA) was used for detection of the compound. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The GC oven temperature was maintained at 100°C for 1 min, and then was ramped at 10°C/min to a final temperature of 325°C (hold time, 5 min). The temperature of the injection port was set at 280°C. The mass spectrometer was operated in an electron ionization mode with the source temperature set at 280°C. The mass range used was m/z 50–550. Analytical Conditions for Assaying 5-Fluoro ADB in Whole Blood Preparation of whole blood was carried out according to the modified QuEChERS method reported by Usui et al. (6), with minor modifications. The purified sample was transferred into a clean vial and 5 μL was injected onto an LC–MS/MS. LC analysis was performed using a Nexera LC system (Shimadzu, Kyoto, Japan). An L-column2 ODS column (150 mm × 1.5 mm i.d.; 5 μm particle size; CERI, Tokyo, Japan), equipped with a guard column (OPTI-GUARD 1 mm C18; Optimize Technologies, Inc., Oregon City, OR, USA), was used for chromatographic separation. The mobile phase consisted of 10 mmol/L ammonium formate (95%) and methanol (5%) (solvent A), and 10 mmol/L ammonium formate (5%) and methanol (95%) (solvent B). The solvent gradient was increased linearly from 0 to 100% solvent B in 15 min and was maintained at this composition for 5 min. Subsequently, the gradient was changed to 0% solvent B and maintained for 10 min to re-equilibrate the column. The flow rate of the mobile phase was set at 0.1 mL/min and the column temperature was maintained at 40°C. MS/MS detection was performed using a QTRAP 5500 system (SCIEX, Framingham, MA, USA). Confirmation of 5-fluoro ADB was performed in multiple reaction monitoring (MRM)-enhanced product ion (EPI) scan mode. The EPI scan range used was m/z 50–500, and the collision energies were set at 20, 35 and 50 eV. The dwell time was set at 20 ms for each transition. Quantitation was performed in MRM mode. The MRM transition 378 > 233 was used for quantitation, and the other three transitions (378 > 318, 378 > 145 and 378 > 90) were used as qualifier ions. The MRM transition 362 > 232 was used for 5-fluoro ADBICA (internal standard). All the experiments were conducted in positive ion mode. Method Validation Method validation was carried out based on the report by Peters et al. (7). Selectivity tests were performed using different whole blood samples (n = 6). Seven-point (0.04, 0.1, 0.2, 0.8, 1, 2 and 4 ng/mL) calibration curves were constructed by plotting peak area ratios (5-fluoro ADB/5-fluoro ADBICA) against nominal concentrations of calibration standards. The curves were fitted using weighted least squares linear regression with a weighting factor of 1/x2. Accuracy (bias) and precision of the devised method were determined using quality control samples (QCs) at low (0.08 ng/mL), medium (0.4 ng/mL) and high (3 ng/mL) concentrations relative to the calibration range. Each QC sample was analyzed in duplicate on eight consecutive days. Accuracy (bias) was calculated as the percentage deviation from the mean using calculated and nominal concentrations. Intraday and interday precision values were calculated using one-way analysis of variance and expressed as relative standard deviation (RSD %). The limit of quantification (LOQ) was defined as the lowest concentration of the analyte, which was measured from the calibration curve, to give a signal-to-noise (S/N) ratio >10. The limit of detection (LOD) was determined based on an S/N ratio greater than 3. The recovery (RE), matrix effect (ME) and process efficiency (PE) were determined using six different samples at three concentrations: QC low (0.08 ng/mL), QC medium (0.4 ng/mL) and QC high (3 ng/mL), according to the method reported by Matuszewski et al. (8). Post-preparative stability was determined by analyzing processed samples (QC high and low concentrations) at time intervals of 60 min over 15 h at 4°C. Case Histories Case 1 A male, in his 20s, was found dead in a sitting position, leaning forward against the television stand in his room. The police found an opened sachet labeled “Heart Shot BLACK” on a table. The deceased had been holding a cigarette lighter in his hand tightly. An empty aluminum can, which seemed to have been used for smoking, was also found near his body. There was no medication history and no specific findings were obtained at autopsy. Case 2 A male, in his 50s, was found dead in his room. He was lying on the floor in a supine posture. An opened sachet labeled “Heart Shot BLACK”, a cigarette lighter, and pipes were found on a table. No medical history was reported. At autopsy, it was found that the decedent had ischemic cardiac disease. Case 3 A male, in his 20s, was found dead in a prone position in a hallway. He had been vomiting and bleeding from his nose. An ashtray, cigarette lighter, handmade aluminum foil pipe and opened “Heart Shot BLACK” sachet were found in the kitchen. No medical history was reported. There was no medication history and no specific findings were obtained at autopsy. Case 4 A male with schizophrenia, in his 50s, was found dead in his car in a parking lot. He was lying in the front passenger seat, out of the driver’s seat, and was holding a plastic pipe and an unsealed “Heart Shot BLACK” package tightly in his hands. A cigarette lighter was found on the passenger seat. His medical history indicated that he was taking prescription drugs (risperidone, biperiden and olanzapine) for the treatment of schizophrenia. There were no specific findings at autopsy and the prescription drugs were not detected in his blood. In all cases, ethanol, n-butanol and acetone testing of blood samples by headspace GC were negative. The forensic pathologist assumed that the cause of death in all cases was acute circulatory failure after drug inhalation, and all were classified as undetermined manner of death. Results and Discussion Regarding the analytical method, we confirmed that there were no interfering peaks at the retention times of the analyte and internal standard by analyzing non-spiked whole blood samples that were obtained from six different sources. The validation parameters of the developed procedure for whole blood analysis are summarized in Table I. Concerning the post-preparative stability test, 5-fluoro ADB and 5-fluoro ADBICA were both stable for at least 15 h at 4°C. All other validation parameters were within their respective accepted ranges. Table I. Validation parameters of the developed method Calibration range (ng/mL)  0.04–4 (0.04, 0.1, 0.2, 0.8, 1, 2 and 4)  r  0.999  LOD (ng/mL)  0.006  LOQ (ng/mL)  0.04  Calibration range (ng/mL)  0.04–4 (0.04, 0.1, 0.2, 0.8, 1, 2 and 4)  r  0.999  LOD (ng/mL)  0.006  LOQ (ng/mL)  0.04  QCs (ng/mL)  Precision (RSD%)  Bias (%)  Intraday run  Interday run  LOQ (0.04)  1.1  1.1  0.1  Low (0.08)  0.9  1.3  −1.1  Medium (0.4)  4.8  6.6  1.1  High (3)  1.4  1.4  2.9  QCs (ng/mL)  Precision (RSD%)  Bias (%)  Intraday run  Interday run  LOQ (0.04)  1.1  1.1  0.1  Low (0.08)  0.9  1.3  −1.1  Medium (0.4)  4.8  6.6  1.1  High (3)  1.4  1.4  2.9  QCs (ng/mL)  Low (0.08)  Medium (0.4)  High(3)  RE % (RSD %)  98.1 (1.9)  94.6 (3.8)  96.5 (3.8)  ME % (RSD %)  94.7 (0.9)  96.1 (3.7)  93.7 (3.0)  PE % (RSD %)  95.2 (2.9)  90.2 (3.0)  89.8 (1.6)  QCs (ng/mL)  Low (0.08)  Medium (0.4)  High(3)  RE % (RSD %)  98.1 (1.9)  94.6 (3.8)  96.5 (3.8)  ME % (RSD %)  94.7 (0.9)  96.1 (3.7)  93.7 (3.0)  PE % (RSD %)  95.2 (2.9)  90.2 (3.0)  89.8 (1.6)  From the GC–MS analysis, a peak with retention time at 20.1 min was observed. The electron ionization–mass spectrum of this compound was found to be identical to that of standard 5-fluoro ADB. There were no other drug peaks seen in the chromatogram. All of the samples from the “Heart Shot BLACK” sachets in the four cases gave the same results. In each case, 5-fluoro ADB was identified in whole blood. Figure 2 shows merged MRM chromatograms and MS/MS spectra at three different collision energies using the [M + H]+ of 5-fluoro ADB. These results were identical to those for the standard 5-fluoro ADB. An LC-quadrupole time-of-flight (SCIEX, Triple TOF 5600) method, which can detect over 1,500 compounds, such as benzodiazepines, antidepressants, pesticides, natural toxins, controlled drugs and designer drugs, was used for a general, unknown screening of the blood samples. However, apart from the study compound, no other drugs were detected in any of the blood samples from the four decedents. Figure 2. View largeDownload slide Merged multiple reaction monitoring (MRM) chromatograms of standard 5-fluoro ADB and 5-fluoro ADB detected in the whole blood samples of the deceased. (B) Product ion spectra obtained by analyzing whole blood samples of the deceased at three different collision energies (20, 35 and 50 eV). Figure 2. View largeDownload slide Merged multiple reaction monitoring (MRM) chromatograms of standard 5-fluoro ADB and 5-fluoro ADB detected in the whole blood samples of the deceased. (B) Product ion spectra obtained by analyzing whole blood samples of the deceased at three different collision energies (20, 35 and 50 eV). 5-Fluoro ADB is an indazole-type SC with a tert-leucine methyl ester moiety in its structure. This designer drug is similar to indole/indazole-3-carboxamide-type SCs, such as 5-fluoro AMB, AMB-FUBINACA, MDMB-FUBINACA, 5-fluoro MDMB-PICA and MDMB-CHMICA, which contain an N-1-methoxycarbonylalkyl group. Although it is clear that many SCs are potent agonists of human cannabinoid receptor type-1 (CB1) and type-2 (CB2), there are still many unanswered questions about the mechanism of action of these compounds. Banister et al. (9) have reported on the pharmacological properties of these SCs. In their studies, many common SCs were tested in fluorometric assays and found to be highly potent agonists of CB1 (concentration that gives half-maximal response, EC50 = 0.45–36 nM) and CB2 (EC50 = 4.6–128 nM). In addition, the authors indicated that, 5-fluoro ADB, which had the second-highest potency (0.59 nM) among the agonists studied, showed more than ~290 times the potency of Δ9−ΤΗC (171 nM). Furthermore, they demonstrated that 5-fluoro MDMB-PICA, which has a similar indole-type structure as 5-fluoro ADB does, was the most potent among the compounds tested (i.e., 380 times more potent than Δ9−THC). Table II shows the concentrations of 5-fluoro ADB in the blood samples that we analyzed. Currently, there is limited data on 5-fluoro ADB intoxication, although several incidents have occurred in Japan. Hasegawa et al. (5) reported on a fatal case of 5-fluoro ADB intoxication and detected 5-fluoro ADB in various tissues of the victim; however, the compound was not detected in the blood. Barcelo et al. (10) have also reported on acute cases of intoxication with 5-fluoro ADB. The authors detected two possible metabolites of 5-fluoro ADB (the 5-OH-pentyl and ester hydrolysis forms) in the urine samples of the patients; however, there were no data on the concentrations of the metabolites We also obtained the spectra of these two potential metabolites from blood samples, but could not positively identify them by comparing to the spectra of authentic compounds. Currently, there are no available data on the concentrations of 5-fluoro ADB in blood. Table II. Concentration of 5-fluoro ADB in the four cases Case  Iliac venous blood (ng/mL)  Right heart blood (ng/mL)  Left heart blood (ng/mL)  1  0.12  0.24  0.45  2  0.23  1.35  NAa  3  0.16  0.14  0.11  4  1.38  1.92  NA  Case  Iliac venous blood (ng/mL)  Right heart blood (ng/mL)  Left heart blood (ng/mL)  1  0.12  0.24  0.45  2  0.23  1.35  NAa  3  0.16  0.14  0.11  4  1.38  1.92  NA  aNA, not available. A fatal case following the use of 5-fluoro AMB, which is a structural analog of 5-fluoro ADB, was reported by Shanks et al. (11). In that case, 5-fluoro AMB was detected in the subclavian vein blood at a concentration of 0.3 ng/mL. Furthermore, Hess et al. (12) have reported that 5-fluoro AMB (0.19 ng/mL) and other SCs were detected in the femoral venous blood of a victim of diabetic ketoacidosis. These quantitative results and the findings of other studies on the blood concentrations of SCs following SC intoxication support our current findings (13, 14). Incidentally, it has been reported that tert-leucinate-type cannabinoids, such as 5-fluoro ADB, are more potent agonists of CB1 and CB2 than valinate-type cannabinoids, such as 5-fluoro AMB. Furthermore, 5-fluoro ADB, which has a 5-fluoropentyl substituent in its structure, is considered to be a more powerful SC than other structurally similar SCs (such as MDMB-FUBINACA (4-fluorobenzyl substituent), MDMB-CHMINACA (cyclohexyl substituent), and MDMB-PINACA (pentyl substituent). These findings suggest that 5-fluoro ADB is more toxic to humans than other SCs are. However, since there are no pharmacokinetic, pharmacodynamic or toxicological data on 5-fluoro ADB currently, it is difficult to explain the causal relationship between blood concentrations of 5-fluoro ADB and the occurrence of death in the present cases. Nevertheless, from the autopsies, toxicological findings, and circumstances surrounding the cases, we believe that inhalation of 5-fluoro ADB had an impact on the deaths of the four males in this study. However, unfortunately, we are unable to determine to what extent 5-fluoro ADB contributed to the deaths. Therefore, further studies are required to better understand the toxicity, metabolism and mechanism of action of 5-fluoro ADB. References 1 Adams, A.J., Banister, S.D., Irizarry, L., Trecki, J., Schwartz, M., Gerona, R. ( 2017) “Zombie” outbreak caused by the synthetic cannabinoid AMB-FUBINACA in New York. The New England Journal of Medicine , 376, 235– 242. Google Scholar CrossRef Search ADS PubMed  2 European Drug Report 2016 Trends and Developments. ( 2016) European Monitoring Centre for Drugs and Drug Addiction. http://www.emcdda.europa.eu/edr2016 (accessed March 21, 2017). 3 Langer, N., Lindigkeit, R., Schiebel, H.M., Papke, U., Ernst, L., Beuerle, T. ( 2016) Identification and quantification of synthetic cannabinoids in “spice-like” herbal mixtures: update of the German situation for the spring of 2016. Forensic Science International , 269, 31– 41. Google Scholar CrossRef Search ADS PubMed  4 Lee, J., Yang, S., Kang, Y., Han, E., Feng, L.Y., Li, J.H., et al.  . ( 2017) Prevalence of new psychoactive substances in Northeast Asia from 2007 to 2015. Forensic Science International , 272, 1– 9. Google Scholar CrossRef Search ADS PubMed  5 Hasegawa, K., Wurita, A., Minakata, K., Gonmori, K., Yamagishi, I., Nozawa, H., et al.  . ( 2015) Identification and quantitation of 5-fluoro-ADB, one of the most dangerous synthetic cannabinoids, in the stomach contents and solid tissues of a human cadaver and in some herbal products. Forensic Toxicology , 33, 112– 121. Google Scholar CrossRef Search ADS   6 Usui, K., Hayashizaki, Y., Hashiyada, M., Funayama, M. ( 2012) Rapid drug extraction from human whole blood using a modified QuEChERS extraction method. Legal Medicine , 14, 286– 296. Google Scholar CrossRef Search ADS PubMed  7 Peters, F.T., Drummer, O.H., Musshoff, F. ( 2007) Validation of new methods. Forensic Science International , 165, 216– 224. Google Scholar CrossRef Search ADS PubMed  8 Matuszewski, B.K., Constanzer, M.L., Chavez-Eng, C.M. ( 2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Analytical Chemistry , 75, 3019– 3030. Google Scholar CrossRef Search ADS PubMed  9 Banister, S.D., Longworth, M., Kevin, R., Sachdev, S., Santiago, M., Stuart, J., et al.  . ( 2016) Pharmacology of valinate and tert-leucinate synthetic cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues. ACS Chemical Neuroscience , 7, 1241– 1254. Google Scholar CrossRef Search ADS PubMed  10 Barcelo, B., Pichini, S., Lopez-Corominas, V., Gomila, I., Yates, C., Busardo, F.P., et al.  . ( 2017) Acute intoxication caused by synthetic cannabinoids 5F-ADB and MMB-2201: a case series. Forensic Science International , 273, e10– e14. Google Scholar CrossRef Search ADS PubMed  11 Shanks, K.G., Behonick, G.S. ( 2016) Death after use of the synthetic cannabinoid 5F-AMB. Forensic Science International , 262, e21– e24. Google Scholar CrossRef Search ADS PubMed  12 Hess, C., Stockhausen, S., Kernbach-Wighton, G., Madea, B. ( 2015) Death due to diabetic ketoacidosis: induction by the consumption of synthetic cannabinoids? Forensic Science International , 257, e6– e11. Google Scholar CrossRef Search ADS PubMed  13 Kronstrand, R., Roman, M., Andersson, M., Eklund, A. ( 2013) Toxicological findings of synthetic cannabinoids in recreational users. Journal of Analytical Toxicology , 37, 534– 541. Google Scholar CrossRef Search ADS PubMed  14 Kneisel, S., Auwarter, V. ( 2012) Analysis of 30 synthetic cannabinoids in serum by liquid chromatography-electrospray ionization tandem mass spectrometry after liquid-liquid extraction. Journal of Mass Spectrometry , 47, 825– 835. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Analytical Toxicology Oxford University Press

Identification of 5-Fluoro ADB in Human Whole Blood in Four Death Cases

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Abstract

Abstract Four deaths that seemed to have been caused by a designer drug occurred within a 3-week period in Sendai, Japan. In each case, the decedent possessed the same sachet, labeled “Heart Shot BLACK”, which contained a dried plant material with an aromatic scent. It was revealed in our analysis that the product contained a synthetic cannabinoid receptor agonist, 5-fluoro ADB (methyl 2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamide]-3,3-dimethylbutanoate, also known as 5-fluoro MDMB-PINACA), which is now classified as a restricted designer drug in Japan after it caused several casualties. For standard samples, the detection of 5-fluoro ADB in whole blood in the calibration range (0.04–4 ng/mL) was successful with recoveries of 94.6–98.1%, limits of detection of 6 pg/mL, and limits of quantification of 40 pg/mL. The intraday and interday precisions were 0.9–4.8% and 1.1–6.6%, respectively. The bias was –1.1 to 2.9%. We were able to confirm that 5-fluoro ADB was present in the blood of all four decedents at a concentration of 0.11–1.92 ng/mL. From the autopsy, toxicological findings, and circumstances surrounding the cases, it was considered that inhalation of 5-fluoro ADB could have contributed to the deaths. However, the extent to which 5-fluoro ADB contributed to the deaths remains unclear due to the current lack of toxicological information on the compound. In future research, the toxicity of 5-fluoro ADB in humans and the mechanism underlying this effect need to be elucidated. Introduction There has been a rapid increase in the use of designer drugs globally. In addition, many accidental intoxications, deaths and driving accidents resulting from the abuse of designer drugs have been reported worldwide. Although several designer drugs have been noted and their use is being regulated, new uncontrolled designer drugs are becoming increasingly available. Furthermore, the observed effects of such drugs are getting more powerful and dangerous, and the number of fatal cases resulting from their use increases yearly (1–4). In Japan, many fatal cases were reported over a short period during the fall of 2014. Hasegawa et al. (5) reported that more than 10 people in Japan died after using products containing 5-fluoro ADB (Figure 1), which is an indazole-3-carboxamide-type synthetic cannabinoid (SC) that has been categorized as a designer drug by the Ministry of Health, Labor and Welfare in Japan; however, there are several, related fatalities that have not been reported. Therefore, it was concluded that 5-fluoro ADB could be harmful to humans. 5-Fluoro ADB has also been linked to four deaths that occurred within 3 weeks in Sendai (Japan) in 2014. In each case, the decedents possessed the same designed sachet, labeled “Heart Shot BLACK”, which contained a dried plant with an aromatic scent (Figure 1). In the present study, we developed a liquid chromatography–tandem mass spectrometry (LC–MS-MS) method for the assay of 5-fluoro ADB in the blood of four victims who had used the drug. The analysis was conducted on whole blood samples taken from the external iliac vein and the heart. Figure 1. View largeDownload slide (A) The structure of 5-fluoro ADB. (B) The appearance of the “Heart Shot BLACK” packet. (C) The dried plant material in the packet. Figure 1. View largeDownload slide (A) The structure of 5-fluoro ADB. (B) The appearance of the “Heart Shot BLACK” packet. (C) The dried plant material in the packet. Experimental 5-Fluoro ADB (≥98%, 10 mg/mL in acetonitrile) and 5-fluoro ADBICA (≥98%, 10 mg/mL in acetonitrile) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Acetonitrile and methanol of liquid chromatography–mass spectrometry grade were purchased from Wako Pure Chemical Industries, Limited (Osaka, Japan). Ammonium formate was purchased from Kanto Chemical Company Inc. (Tokyo, Japan). All other chemicals used were analytical grade. Frozen human whole blood was purchased from Biopredic International (Rennes, France). A QuEChERS pre-packed extraction kit containing 6 g of magnesium sulfate and 1.5 g of sodium acetate, and a dispersive solid-phase extraction kit (for fatty samples) were purchased from Agilent Technologies (Santa Clara, CA, USA). Analysis of the Dried Plant Material in “Heart Shot BLACK” A fraction of the dried plant material in “Heart Shot BLACK” was extracted with methanol, and the resulting liquid was diluted appropriately. One microliter of this solution was then subjected to gas chromatography–mass spectrometry (GC–MS) analysis. A GC–MS instrument (5975C, Agilent Technologies, Santa Clara, CA, USA) equipped with a DB-5ms column (30 m × 0.25 mm i.d.; 0.25 μm film thickness; Agilent Technologies, Santa Clara, CA, USA) was used for detection of the compound. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The GC oven temperature was maintained at 100°C for 1 min, and then was ramped at 10°C/min to a final temperature of 325°C (hold time, 5 min). The temperature of the injection port was set at 280°C. The mass spectrometer was operated in an electron ionization mode with the source temperature set at 280°C. The mass range used was m/z 50–550. Analytical Conditions for Assaying 5-Fluoro ADB in Whole Blood Preparation of whole blood was carried out according to the modified QuEChERS method reported by Usui et al. (6), with minor modifications. The purified sample was transferred into a clean vial and 5 μL was injected onto an LC–MS/MS. LC analysis was performed using a Nexera LC system (Shimadzu, Kyoto, Japan). An L-column2 ODS column (150 mm × 1.5 mm i.d.; 5 μm particle size; CERI, Tokyo, Japan), equipped with a guard column (OPTI-GUARD 1 mm C18; Optimize Technologies, Inc., Oregon City, OR, USA), was used for chromatographic separation. The mobile phase consisted of 10 mmol/L ammonium formate (95%) and methanol (5%) (solvent A), and 10 mmol/L ammonium formate (5%) and methanol (95%) (solvent B). The solvent gradient was increased linearly from 0 to 100% solvent B in 15 min and was maintained at this composition for 5 min. Subsequently, the gradient was changed to 0% solvent B and maintained for 10 min to re-equilibrate the column. The flow rate of the mobile phase was set at 0.1 mL/min and the column temperature was maintained at 40°C. MS/MS detection was performed using a QTRAP 5500 system (SCIEX, Framingham, MA, USA). Confirmation of 5-fluoro ADB was performed in multiple reaction monitoring (MRM)-enhanced product ion (EPI) scan mode. The EPI scan range used was m/z 50–500, and the collision energies were set at 20, 35 and 50 eV. The dwell time was set at 20 ms for each transition. Quantitation was performed in MRM mode. The MRM transition 378 > 233 was used for quantitation, and the other three transitions (378 > 318, 378 > 145 and 378 > 90) were used as qualifier ions. The MRM transition 362 > 232 was used for 5-fluoro ADBICA (internal standard). All the experiments were conducted in positive ion mode. Method Validation Method validation was carried out based on the report by Peters et al. (7). Selectivity tests were performed using different whole blood samples (n = 6). Seven-point (0.04, 0.1, 0.2, 0.8, 1, 2 and 4 ng/mL) calibration curves were constructed by plotting peak area ratios (5-fluoro ADB/5-fluoro ADBICA) against nominal concentrations of calibration standards. The curves were fitted using weighted least squares linear regression with a weighting factor of 1/x2. Accuracy (bias) and precision of the devised method were determined using quality control samples (QCs) at low (0.08 ng/mL), medium (0.4 ng/mL) and high (3 ng/mL) concentrations relative to the calibration range. Each QC sample was analyzed in duplicate on eight consecutive days. Accuracy (bias) was calculated as the percentage deviation from the mean using calculated and nominal concentrations. Intraday and interday precision values were calculated using one-way analysis of variance and expressed as relative standard deviation (RSD %). The limit of quantification (LOQ) was defined as the lowest concentration of the analyte, which was measured from the calibration curve, to give a signal-to-noise (S/N) ratio >10. The limit of detection (LOD) was determined based on an S/N ratio greater than 3. The recovery (RE), matrix effect (ME) and process efficiency (PE) were determined using six different samples at three concentrations: QC low (0.08 ng/mL), QC medium (0.4 ng/mL) and QC high (3 ng/mL), according to the method reported by Matuszewski et al. (8). Post-preparative stability was determined by analyzing processed samples (QC high and low concentrations) at time intervals of 60 min over 15 h at 4°C. Case Histories Case 1 A male, in his 20s, was found dead in a sitting position, leaning forward against the television stand in his room. The police found an opened sachet labeled “Heart Shot BLACK” on a table. The deceased had been holding a cigarette lighter in his hand tightly. An empty aluminum can, which seemed to have been used for smoking, was also found near his body. There was no medication history and no specific findings were obtained at autopsy. Case 2 A male, in his 50s, was found dead in his room. He was lying on the floor in a supine posture. An opened sachet labeled “Heart Shot BLACK”, a cigarette lighter, and pipes were found on a table. No medical history was reported. At autopsy, it was found that the decedent had ischemic cardiac disease. Case 3 A male, in his 20s, was found dead in a prone position in a hallway. He had been vomiting and bleeding from his nose. An ashtray, cigarette lighter, handmade aluminum foil pipe and opened “Heart Shot BLACK” sachet were found in the kitchen. No medical history was reported. There was no medication history and no specific findings were obtained at autopsy. Case 4 A male with schizophrenia, in his 50s, was found dead in his car in a parking lot. He was lying in the front passenger seat, out of the driver’s seat, and was holding a plastic pipe and an unsealed “Heart Shot BLACK” package tightly in his hands. A cigarette lighter was found on the passenger seat. His medical history indicated that he was taking prescription drugs (risperidone, biperiden and olanzapine) for the treatment of schizophrenia. There were no specific findings at autopsy and the prescription drugs were not detected in his blood. In all cases, ethanol, n-butanol and acetone testing of blood samples by headspace GC were negative. The forensic pathologist assumed that the cause of death in all cases was acute circulatory failure after drug inhalation, and all were classified as undetermined manner of death. Results and Discussion Regarding the analytical method, we confirmed that there were no interfering peaks at the retention times of the analyte and internal standard by analyzing non-spiked whole blood samples that were obtained from six different sources. The validation parameters of the developed procedure for whole blood analysis are summarized in Table I. Concerning the post-preparative stability test, 5-fluoro ADB and 5-fluoro ADBICA were both stable for at least 15 h at 4°C. All other validation parameters were within their respective accepted ranges. Table I. Validation parameters of the developed method Calibration range (ng/mL)  0.04–4 (0.04, 0.1, 0.2, 0.8, 1, 2 and 4)  r  0.999  LOD (ng/mL)  0.006  LOQ (ng/mL)  0.04  Calibration range (ng/mL)  0.04–4 (0.04, 0.1, 0.2, 0.8, 1, 2 and 4)  r  0.999  LOD (ng/mL)  0.006  LOQ (ng/mL)  0.04  QCs (ng/mL)  Precision (RSD%)  Bias (%)  Intraday run  Interday run  LOQ (0.04)  1.1  1.1  0.1  Low (0.08)  0.9  1.3  −1.1  Medium (0.4)  4.8  6.6  1.1  High (3)  1.4  1.4  2.9  QCs (ng/mL)  Precision (RSD%)  Bias (%)  Intraday run  Interday run  LOQ (0.04)  1.1  1.1  0.1  Low (0.08)  0.9  1.3  −1.1  Medium (0.4)  4.8  6.6  1.1  High (3)  1.4  1.4  2.9  QCs (ng/mL)  Low (0.08)  Medium (0.4)  High(3)  RE % (RSD %)  98.1 (1.9)  94.6 (3.8)  96.5 (3.8)  ME % (RSD %)  94.7 (0.9)  96.1 (3.7)  93.7 (3.0)  PE % (RSD %)  95.2 (2.9)  90.2 (3.0)  89.8 (1.6)  QCs (ng/mL)  Low (0.08)  Medium (0.4)  High(3)  RE % (RSD %)  98.1 (1.9)  94.6 (3.8)  96.5 (3.8)  ME % (RSD %)  94.7 (0.9)  96.1 (3.7)  93.7 (3.0)  PE % (RSD %)  95.2 (2.9)  90.2 (3.0)  89.8 (1.6)  From the GC–MS analysis, a peak with retention time at 20.1 min was observed. The electron ionization–mass spectrum of this compound was found to be identical to that of standard 5-fluoro ADB. There were no other drug peaks seen in the chromatogram. All of the samples from the “Heart Shot BLACK” sachets in the four cases gave the same results. In each case, 5-fluoro ADB was identified in whole blood. Figure 2 shows merged MRM chromatograms and MS/MS spectra at three different collision energies using the [M + H]+ of 5-fluoro ADB. These results were identical to those for the standard 5-fluoro ADB. An LC-quadrupole time-of-flight (SCIEX, Triple TOF 5600) method, which can detect over 1,500 compounds, such as benzodiazepines, antidepressants, pesticides, natural toxins, controlled drugs and designer drugs, was used for a general, unknown screening of the blood samples. However, apart from the study compound, no other drugs were detected in any of the blood samples from the four decedents. Figure 2. View largeDownload slide Merged multiple reaction monitoring (MRM) chromatograms of standard 5-fluoro ADB and 5-fluoro ADB detected in the whole blood samples of the deceased. (B) Product ion spectra obtained by analyzing whole blood samples of the deceased at three different collision energies (20, 35 and 50 eV). Figure 2. View largeDownload slide Merged multiple reaction monitoring (MRM) chromatograms of standard 5-fluoro ADB and 5-fluoro ADB detected in the whole blood samples of the deceased. (B) Product ion spectra obtained by analyzing whole blood samples of the deceased at three different collision energies (20, 35 and 50 eV). 5-Fluoro ADB is an indazole-type SC with a tert-leucine methyl ester moiety in its structure. This designer drug is similar to indole/indazole-3-carboxamide-type SCs, such as 5-fluoro AMB, AMB-FUBINACA, MDMB-FUBINACA, 5-fluoro MDMB-PICA and MDMB-CHMICA, which contain an N-1-methoxycarbonylalkyl group. Although it is clear that many SCs are potent agonists of human cannabinoid receptor type-1 (CB1) and type-2 (CB2), there are still many unanswered questions about the mechanism of action of these compounds. Banister et al. (9) have reported on the pharmacological properties of these SCs. In their studies, many common SCs were tested in fluorometric assays and found to be highly potent agonists of CB1 (concentration that gives half-maximal response, EC50 = 0.45–36 nM) and CB2 (EC50 = 4.6–128 nM). In addition, the authors indicated that, 5-fluoro ADB, which had the second-highest potency (0.59 nM) among the agonists studied, showed more than ~290 times the potency of Δ9−ΤΗC (171 nM). Furthermore, they demonstrated that 5-fluoro MDMB-PICA, which has a similar indole-type structure as 5-fluoro ADB does, was the most potent among the compounds tested (i.e., 380 times more potent than Δ9−THC). Table II shows the concentrations of 5-fluoro ADB in the blood samples that we analyzed. Currently, there is limited data on 5-fluoro ADB intoxication, although several incidents have occurred in Japan. Hasegawa et al. (5) reported on a fatal case of 5-fluoro ADB intoxication and detected 5-fluoro ADB in various tissues of the victim; however, the compound was not detected in the blood. Barcelo et al. (10) have also reported on acute cases of intoxication with 5-fluoro ADB. The authors detected two possible metabolites of 5-fluoro ADB (the 5-OH-pentyl and ester hydrolysis forms) in the urine samples of the patients; however, there were no data on the concentrations of the metabolites We also obtained the spectra of these two potential metabolites from blood samples, but could not positively identify them by comparing to the spectra of authentic compounds. Currently, there are no available data on the concentrations of 5-fluoro ADB in blood. Table II. Concentration of 5-fluoro ADB in the four cases Case  Iliac venous blood (ng/mL)  Right heart blood (ng/mL)  Left heart blood (ng/mL)  1  0.12  0.24  0.45  2  0.23  1.35  NAa  3  0.16  0.14  0.11  4  1.38  1.92  NA  Case  Iliac venous blood (ng/mL)  Right heart blood (ng/mL)  Left heart blood (ng/mL)  1  0.12  0.24  0.45  2  0.23  1.35  NAa  3  0.16  0.14  0.11  4  1.38  1.92  NA  aNA, not available. A fatal case following the use of 5-fluoro AMB, which is a structural analog of 5-fluoro ADB, was reported by Shanks et al. (11). In that case, 5-fluoro AMB was detected in the subclavian vein blood at a concentration of 0.3 ng/mL. Furthermore, Hess et al. (12) have reported that 5-fluoro AMB (0.19 ng/mL) and other SCs were detected in the femoral venous blood of a victim of diabetic ketoacidosis. These quantitative results and the findings of other studies on the blood concentrations of SCs following SC intoxication support our current findings (13, 14). Incidentally, it has been reported that tert-leucinate-type cannabinoids, such as 5-fluoro ADB, are more potent agonists of CB1 and CB2 than valinate-type cannabinoids, such as 5-fluoro AMB. Furthermore, 5-fluoro ADB, which has a 5-fluoropentyl substituent in its structure, is considered to be a more powerful SC than other structurally similar SCs (such as MDMB-FUBINACA (4-fluorobenzyl substituent), MDMB-CHMINACA (cyclohexyl substituent), and MDMB-PINACA (pentyl substituent). These findings suggest that 5-fluoro ADB is more toxic to humans than other SCs are. However, since there are no pharmacokinetic, pharmacodynamic or toxicological data on 5-fluoro ADB currently, it is difficult to explain the causal relationship between blood concentrations of 5-fluoro ADB and the occurrence of death in the present cases. Nevertheless, from the autopsies, toxicological findings, and circumstances surrounding the cases, we believe that inhalation of 5-fluoro ADB had an impact on the deaths of the four males in this study. However, unfortunately, we are unable to determine to what extent 5-fluoro ADB contributed to the deaths. Therefore, further studies are required to better understand the toxicity, metabolism and mechanism of action of 5-fluoro ADB. References 1 Adams, A.J., Banister, S.D., Irizarry, L., Trecki, J., Schwartz, M., Gerona, R. ( 2017) “Zombie” outbreak caused by the synthetic cannabinoid AMB-FUBINACA in New York. The New England Journal of Medicine , 376, 235– 242. Google Scholar CrossRef Search ADS PubMed  2 European Drug Report 2016 Trends and Developments. ( 2016) European Monitoring Centre for Drugs and Drug Addiction. http://www.emcdda.europa.eu/edr2016 (accessed March 21, 2017). 3 Langer, N., Lindigkeit, R., Schiebel, H.M., Papke, U., Ernst, L., Beuerle, T. ( 2016) Identification and quantification of synthetic cannabinoids in “spice-like” herbal mixtures: update of the German situation for the spring of 2016. Forensic Science International , 269, 31– 41. Google Scholar CrossRef Search ADS PubMed  4 Lee, J., Yang, S., Kang, Y., Han, E., Feng, L.Y., Li, J.H., et al.  . ( 2017) Prevalence of new psychoactive substances in Northeast Asia from 2007 to 2015. Forensic Science International , 272, 1– 9. Google Scholar CrossRef Search ADS PubMed  5 Hasegawa, K., Wurita, A., Minakata, K., Gonmori, K., Yamagishi, I., Nozawa, H., et al.  . ( 2015) Identification and quantitation of 5-fluoro-ADB, one of the most dangerous synthetic cannabinoids, in the stomach contents and solid tissues of a human cadaver and in some herbal products. Forensic Toxicology , 33, 112– 121. Google Scholar CrossRef Search ADS   6 Usui, K., Hayashizaki, Y., Hashiyada, M., Funayama, M. ( 2012) Rapid drug extraction from human whole blood using a modified QuEChERS extraction method. Legal Medicine , 14, 286– 296. Google Scholar CrossRef Search ADS PubMed  7 Peters, F.T., Drummer, O.H., Musshoff, F. ( 2007) Validation of new methods. Forensic Science International , 165, 216– 224. Google Scholar CrossRef Search ADS PubMed  8 Matuszewski, B.K., Constanzer, M.L., Chavez-Eng, C.M. ( 2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Analytical Chemistry , 75, 3019– 3030. Google Scholar CrossRef Search ADS PubMed  9 Banister, S.D., Longworth, M., Kevin, R., Sachdev, S., Santiago, M., Stuart, J., et al.  . ( 2016) Pharmacology of valinate and tert-leucinate synthetic cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues. ACS Chemical Neuroscience , 7, 1241– 1254. Google Scholar CrossRef Search ADS PubMed  10 Barcelo, B., Pichini, S., Lopez-Corominas, V., Gomila, I., Yates, C., Busardo, F.P., et al.  . ( 2017) Acute intoxication caused by synthetic cannabinoids 5F-ADB and MMB-2201: a case series. Forensic Science International , 273, e10– e14. Google Scholar CrossRef Search ADS PubMed  11 Shanks, K.G., Behonick, G.S. ( 2016) Death after use of the synthetic cannabinoid 5F-AMB. Forensic Science International , 262, e21– e24. Google Scholar CrossRef Search ADS PubMed  12 Hess, C., Stockhausen, S., Kernbach-Wighton, G., Madea, B. ( 2015) Death due to diabetic ketoacidosis: induction by the consumption of synthetic cannabinoids? Forensic Science International , 257, e6– e11. Google Scholar CrossRef Search ADS PubMed  13 Kronstrand, R., Roman, M., Andersson, M., Eklund, A. ( 2013) Toxicological findings of synthetic cannabinoids in recreational users. Journal of Analytical Toxicology , 37, 534– 541. Google Scholar CrossRef Search ADS PubMed  14 Kneisel, S., Auwarter, V. ( 2012) Analysis of 30 synthetic cannabinoids in serum by liquid chromatography-electrospray ionization tandem mass spectrometry after liquid-liquid extraction. Journal of Mass Spectrometry , 47, 825– 835. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Journal of Analytical ToxicologyOxford University Press

Published: Mar 1, 2018

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