A case of death caused by abuse of a synthetic cannabinoid N-1-naphthalenyl-1-pentyl-1H-indole-3-carboxamide
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- Sasaki, C., Saito, T., Shinozuka, T. et al. Forensic Toxicol (2015) 33: 165. doi:10.1007/s11419-014-0246-5
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A man aged in his twenties was found dead on the floor in his room. A package containing dried herbal blend labeled “Fairy evolution” and smoking devices were found in the room. The postmortem interval of the deceased was estimated to be 3 days. The autopsy disclosed no marked findings explaining the cause of death. Toxicological analyses by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry revealed the presence of N-1-naphthalenyl-1-pentyl-1H-indole-3-carboxamide (NNEI) in the herbal blend and in specimens taken from the victim. The concentrations in the blood and adipose tissue specimens were 0.64–0.99 and 42.9 ng/ml, respectively. To our knowledge, this is the first report to describe NNEI concentrations in human specimens in the fatal poisoning case.
KeywordsN-1-Naphthalenyl-1-pentyl-1H-indole-3-carboxamide NNEI Synthetic cannabinoid Herbal blend LC–MS–MS GC–MS
A man aged in his twenties was found dead on the floor in a supine position in his room. A package containing dried herbal blend labeled “Fairy evolution” and smoking devices were found in the room. A relative reported that he had a poor appetite and that his body weight had decreased by about 15 kg during the past 10 months. The deceased had no medical history. An autopsy was performed about 3 days after the estimated time of death.
The deceased was 174 cm tall, weighed 69 kg, and was of average physical build. No remarkable injuries were observed by macroscopic observation. The lungs showed marked congestion, the left weighing 818 g and the right weighing 932 g. No obvious changes were observed in other organs. By microscopic observation, the organs showed congestion. In the heart, arteriolar wall hypertrophy, slight interstitial fibrosis, and contraction bands were found. The lungs showed marked congestion and edema, and alveolar macrophage infiltrations were also observed. In the liver, slight lymphocytic infiltrations were found in the Glisson’s sheath. Arteriolar hyalinizations and severe congestion were found in the spleen. Corpora amylacea were observed in part of the corpus callosum in the brain. From the above-described microscopic observation, it was difficult to explain the cause of death.
Samples of whole blood (right atrium, left atrium, right femoral vein, left femoral vein), urine, brain, heart, lung, liver, kidney, and abdominal subcutaneous adipose tissue were collected at autopsy and kept frozen at −30 °C. To prepare plasma, the whole blood samples (right atrium, left atrium) were centrifuged at 3,000 rpm for 10 min and the supernatant was transferred to a new tube; the plasma samples were stored at −30 °C until use. Black hair, 40 cm long, was collected from the parietal region at autopsy and kept at 4 °C.
NNEI and [1-(5-fluoropentyl)-1H-indol-3-yl-2,4,5,6,7-d5]-(4-methyl-1-naphthalenyl)-methanone (MAM-2201-d5) (500 μg/500 μl methanolic solution) were purchased from Cayman Chemical (Ann Arbor, MI, USA); the MonoSpin C18 columns from GL Sciences (Tokyo, Japan); methanol (HPLC grade) and acetonitrile (HPLC grade) from Sigma Aldrich (St. Louis, MO, USA). All other chemicals were analytical grade and purchased from Wako Pure Chemical (Osaka, Japan).
Analysis of the herbal blend by gas chromatography–mass spectrometry
The herbal blend was analyzed by slight modification of a previous method  using gas chromatography–mass spectrometry (GC–MS). About 5 mg of the herbal blend was immersed in 1 ml of methanol. The mixture was vortexed and left at room temperature for 10 min. After centrifugation, the supernatant was analyzed by GC–MS using an Agilent 6890 N GC system with a 5975B mass-selective detector (Agilent, Santa Clara, CA, USA) with an HP-5MS column (30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent) under the conditions described previously .
Analysis of NNEI in body fluids, solid tissues, and hair of the victim
NNEI in the deceased’s blood, plasma, urine, the brain, heart, lung, liver, kidney, and abdominal subcutaneous adipose tissue were determined by the previously described method  using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS–MS). MAM-2201-d5 was used as internal standard (IS). The body fluids were extracted with a MonoSpin C18 column. The tissues were homogenized and extracted with acetonitrile .
To confirm whether NNEI attached to the hair of the victim during smoking of the herbal blend, the wash solvent used on the hair of the victim was also analyzed. Several hair shafts that were not washed were weighed, and washed with methanol by vortex mixer. The methanol layer was transferred, and dried under a nitrogen stream. The residue was dissolved in acetonitrile and injected into the LC–ESI–MS–MS.
For direct analysis of the hair, the strand of hair was cut into 13 segments. Segments 1–10 were cut into 2-cm sections from the hair root; segments 11 and 12 were 5 cm long, and segment 13 was the strand from the distal end of segment 12 to the hair end. Each sample was washed with methanol and pure water, extracted using the method reported by Kim et al. , and analyzed by LC–ESI–MS–MS.
LC–ESI–MS–MS analysis was conducted with an Agilent 1200 LC system with a 6410 Triple Quad tandem mass spectrometer (Agilent). Chromatographic separation was performed on an Inert-Sustain C18 HP 3-μm (100 × 3 mm) column at 40 °C. Gradient elution was used for chromatographic separation with mobile phase A of 0.1 % acetic acid aqueous solution, and mobile phase B of acetonitrile. Linear gradient elution started from 80 % A/20 % B, changed to 100 % B over 5 min with a 1-min hold, and returned to 80 % A/20 % B over 4 min for the next run. The flow rate was 0.6 ml/min. The injection volume was 3 μl. The ESI–MS–MS system was operated in the positive-ionization mode, with ions monitored in the selected reaction monitoring (SRM) mode. The ESI source parameters were: high-purity drying gas, nitrogen; flow rate, 6 l/min; temperature, 300 °C; capillary voltage, 4,000 V; nebulizer, 103 kPa; fragmentor and collision energies, 140 and 21 V for NNEI and 170 and 25 V for IS, respectively; ion transitions for selected reaction monitoring, m/z 357.2 → 214.1 for NNEI and m/z 379.2 → 169.1 for IS.
Results and discussion
No alcohol was detected in the blood and urine samples using head-space gas chromatography with flame-ionization detection. Triage Drugs of Abuse Panel Plus TCA (Biosite, San Diego, CA, USA) showed negative results for the urine sample. A drug screening test was also performed by GC–MS with NAGINATA software (Nishikawa Keisoku, Tokyo, Japan) for the plasma and urine samples; the result was negative.
NNEI concentrations in the body fluids and tissues of the victim
Concentration (ng/ml or g)
Right femoral vein
Left femoral vein
Abdominal adipose tissue
Segmental analysis of NNEI concentrations in the deceased’s hair after washing
0 (scalp side)–2 cm
On macroscopic examination of the deceased, no marked changes were observed except for congestion. Histological examination showed arteriolar hyalinizations, which tend to be observed in aged and hypertensive patients, in the spleen, and slight interstitial fibrosis was observed in the heart. Because the deceased was still in his twenties, it was considered that his long abuse of herbal blends caused hypertension and hyperactivity of cardiac function. Poisoning cases with synthetic cannabinoids have been reported [2, 3]. In the present case, no obvious changes were observed on either macroscopic or microscopic examination that could explain the cause of death, and only NNEI was detected in the specimens collected from the victim. Thus, we concluded that the acute circulatory disturbance was induced by NNEI poisoning. To our knowledge, this is the first report describing a fatal case of poisoning by NNEI.
Conflict of interest
There are no financial or other relations that could lead to a conflict of interest.