Forensic Toxicology https://doi.org/10.1007/s11419-018-0431-z ORIGINAL ARTICLE Postmortem distribution of mepirapim and acetyl fentanyl in biological fuid and solid tissue specimens measured by the standard addition method Akira Mochizuki1 · Hiroko Nakazawa1 · Noboru Adachi2 · Kenichi Takekawa1 · Hideki Shojo2 Received: 6 May 2018 / Accepted: 26 June 2018 © The Author(s) 2018 Abstract Purpose Mepirapim is a new synthetic cannabinoid. We previously reported that the concentrations of unchanged mepirapim in whole blood and urine were much higher than those of other synthetic cannabinoids. To determine the postmortem distri- bution of mepirapim and acetyl fentanyl in the deceased individual, we established a standard addition method for detailed analysis by liquid chromatography–mass spectrometry (LC–MS) for quantifcation of these drugs. Methods The LC–MS method was fully validated for linearity, extraction recovery, matrix efect and repeatability. Results Good linearities, extraction recoveries, matrix efects and repeatabilities were shown for both target compounds in all specimens. The concentrations of mepirapim and acetyl fentanyl in three body fuid specimens and 12 solid tissue speci- mens were measured. For mepirapim, the highest concentrations were found in the liver and kidney, and the concentrations in the blood and urine specimens were one order of magnitude lower than the high concentrations in the solid tissues except the psoas major muscle. For acetyl fentanyl, the highest concentrations were found in the myocardium, spleen and kidney, and the concentrations in the body fuid specimens were also one order of magnitude lower than the highest concentrations in the solid tissues. There were concentration diferences of mepirapim and acetyl fentanyl among the regions of the brain. Conclusions The concentration of unchanged mepirapim in urine was much higher than those of other synthetic cannabi- noids; the higher dosage, urinary excretion, metabolisms and/or pharmacokinetics of mepirapim may be quite diferent from those of other synthetic cannabinoids. Keywords Mepirapim · Synthetic cannabinoid · Acetyl fentanyl · Postmortem distribution · LC–MS · Standard addition method Introduction by inhalation [4]. Mepirapim is a new and unique synthetic cannabinoid that was frst identifed in herbal blends in Japan Illicit psychoactive substances (e.g., synthetic cannabinoids, [5]. Very recently, the afnities of mepirapim toward ­CB1 cathinone derivatives and synthetic opioids) have become a and ­CB2 in term of Ki values have been reported. The Ki val- serious threat worldwide as designer drugs of abuse [1–3]. ues were 2650 and 1850 nM for ­CB1 and ­CB2, respectively We previously encountered a curious case in which two male [6], but it actually functions as a CB receptor agonist. This subjects self-dosed mepirapim plus acetyl fentanyl by dif- compound difers from JWH-018, because it has a 4-methyl- ferent routes of administration, that is, intravenously and piperazine group in place of the naphthyl group [4]. We thus reported a detailed gas chromatography–tandem mass spec- * Hideki Shojo trometry with the internal standard (IS) method for quanti- [email protected] fcation of mepirapim and acetyl fentanyl in whole blood 1 and urine [4]; the concentrations of unchanged mepirapim Forensic Science Laboratory, Yamanashi Prefectural in whole blood and urine were much higher than those of Police Headquarters, 312‑4 Kubonakajima, Isawa, Fuefuki, Yamanashi 406‑0036, Japan other common synthetic cannabinoids. On the other hand, acetyl fentanyl is a synthetic fentanyl 2 Department of Legal Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, 1110 analog in which the propionyl group of fentanyl is replaced Shimokato, Chuo, Yamanashi 409‑3898, Japan by an acetyl group [7]. Recently, acetyl fentanyl has been Vol.:(0123456789)1 3 Forensic Toxicology encountered in clinical and forensic case studies [8–15]. The (Ann Arbor, MI, USA), and Isolute SLE + (1 mL capacity) number of available reports dealing with determination of columns from Biotage (Uppsala, Sweden). Other common acetyl fentanyl from multiple specimens in authentic cases chemicals used in this study were of the highest purity com- has been limited [3, 10, 11, 15]. In addition, the number of mercially available. specimens was few; one report included fve specimens [11], and another one included four specimens [15]. The most Extraction procedure detailed report was provided by Poklis et al. [10], including seven specimens using 14 fatal cases. In this study, we have To 50 µL of each body fuid or 100 µL solid tissue homoge- quantifed both mepirapim and acetyl fentanyl in as many as nate (2 g tissue in 8 mL 0.01 N HCl solution thoroughly 15 specimens. To our knowledge, this is the most detailed homogenized with a blender) were added 20 µL of metha- study to date for the distribution of acetyl fentanyl, and also nol solution containing a known amount of mepirapim is the frst demonstration of distribution of mepirapim in and acetyl fentanyl as standard additions, 20 µL of metha- an authentic fatal poisoning case. Such detailed investiga- nol solution containing 50 ng of JWH-200 and 10 ng of tion of postmortem distribution is very useful for evalu- acetyl fentanyl-d5 as ISs for mepirapim and acetyl fentanyl, ating the cause(s) of death. In addition, we have used the respectively, and 0.75 mL of 1% Na 2CO3. The mixture was standard addition method for analysis by liquid chromatog- mixed on a vortex mixer and transferred onto an Isolute raphy–mass spectrometry (LC–MS), which can overcome SLE + (1 mL capacity) column. The column was eluted by the diferent matrix efects and recovery rates in diferent using 5 mL of methyl tert-butyl ether. The eluent was evapo- specimens, and also does not need blank human matrices rated, and the residue was reconstituted in 200 µL methanol. for validation experiments. A 2-µL aliquot was used for analysis by LC–MS. LC–MS conditions Case history Quantitative analysis was performed by selected-ion moni- The deceased in the autopsy case used in our analysis was toring (SIM) analysis of LC–MS. Briefy, LC–MS was per- one of two subjects who had abused drugs together in formed on an ACQUITY Arc LC system connected to an December 2013. According to the confession statement of ACQUITY QDa mass spectrometer (Waters, Milford, MA, the surviving individual, the deceased individual (a male USA). The LC conditions were as follows: XBridge BEH in his 60s) self-administered the drug (approximately C18 column (75 × 3.0 mm i.d., particle size, 2.5 µm; Waters); 50–60 mg) via intravenous injection. Approximately 10 h injection volume, 2 µL; fow rate, 0.5 mL/min; elution mode, after dosing, the surviving individual recognized that the isocratic with 10 mM ammonium acetate in water/methanol other individual had died. An autopsy was performed, and (45:55, v/v); column temperature, 40 °C. The MS conditions postmortem biological fuid (heart whole blood, femoral were as follows: interface, electrospray ionization mode; vein whole blood and urine) and solid (cerebrum, cerebel- polarity, positive; probe temperature, 600 °C; capillary lum, pons, medulla oblongata, lung, myocardium, liver, pan- voltage, 1.2 kV; scan mode, SIM; monitoring ion and cone creas, kidney, adrenal gland, spleen and psoas major muscle) voltage, m/z 314 and 10 V for mepirapim, m/z 323 and 20 V tissue specimens were collected. The postmortem interval for acetyl fentanyl, m/z 385 and 20 V for JWH-200 and m/z was estimated to be 64 h, but because the cadaver had been 328 and 20 V for acetyl fentanyl-d5, respectively. stored at 4 °C until autopsy, it was relatively fresh at autopsy. All specimens were stored at − 80 °C until analysis. The Standard addition method ‘Angela’ drug product found at the scene was seized and analyzed; the powder consisted of 73.2 ± 0.4% mepirapim The standard addition method can completely overcome and 18.9 ± 0.2% acetyl fentanyl (w/w) [4]. Drug analyses matrix efects. The method does not require blank human from the deceased were performed at our department by the body fuid or solid tissue specimens that are negative for request of judicial authorities. target compounds as described before. Note that when inves- tigating the distribution of a xenobiotic compound with dif- ferent types of matrices, the standard addition method is Materials and methods required to construct a calibration curve to obtain a single concentration value [16, 17]. To obtain each value, six dif- Materials ferent concentrations of the target compounds and fxed concentrations of ISs were added to homogenate portions Mepirapim hydrochloride, acetyl fentanyl, JWH-200 and of the same matrix, respectively, for construction of a cali- acetyl fentanyl-d5 were purchased from Cayman Chemical bration curve. The slope of the calibration curve crosses the 1 3 Forensic Toxicology y-intercept (intensity) and intersects the x-axis (target com- methanol solution (neat sample) containing the same amount pound concentration) at the negative side; the absolute value as that contained at the initial step without any reconstitution of which indicates the preexisting concentration of the target was injected into the LC–MS system; the resulting peak area compounds in the matrix. was designated as C. Each matrix effect and recovery rate was calculated Determination of matrix efects and recovery rates according to the following equations: Matrix effect (%) = (A − B)∕C × 100 To determine the matrix effects