Identification trial for 5-fluoro-ADB-PINACA and MAB-CHMINACA

The extracts from all specimens were subjected to detection of peaks of 5-fluoro-ADB-PINACA by LC–MS–MS in various detection modes. The protonated molecular ion at m/z 363 could not be detected in any specimens of the deceased. However, in the selected reaction monitoring (SRM) mode, a very small peak at m/z 318 [above the limit of detection (LOD) and the below the limit of quantitation (LOQ)] could be detected only for the stomach contents; for all other specimens, any peak at m/z 318 was below the LOD in the SRM mode.

In contrast to 5-fluoro-ADB-PINACA in the specimens, the concentrations of MAB-CHMINACA were found at much higher concentrations than those of 5-fluoro-ADB-PINACA. It was possible to obtain product ion mass spectra from femoral vein whole blood and liver tissue specimens. Figure 1 shows an example of product ion mass spectra obtained from the extracts of femoral vein whole blood and liver tissue in comparison with that of the reference standard MAB-CHMINACA. The spectra obtained from the two extracts coincided with that of the reference standard MAB-CHMINACA, with no impurity peaks, confirming the detected compound was MAB-CHMINACA.

Fig. 1 Product ion mass spectra of the extracts of femoral vein whole blood and liver tissue specimens collected from the deceased in comparison with that of the reference standard MAB-CHMINACA, recorded by liquid chromatography–tandem mass spectrometry (LC–MS–MS) together with the probable fragmentation mode Full size image

Validation of the method

Figure 2 shows an example of the SRM chromatograms for the target compound and the IS extracted from femoral vein whole blood and liver tissue. The target compound MAB-CHMINACA and IS AB-CHMINACA appeared at retention times of 8.72 and 7.93 min, respectively. The bottom panel of Fig. 2 shows the absence of AB-CHMINACA in the liver tissue extract, though a very small peak was detected at the same retention time as that of AB-CHMINACA with 100-fold magnification of the vertical axis. It seems reasonable to regard the small peak as a carryover of the AB-CHMINACA spiked into the liver tissue just before the run without the spiking of the IS. It should be noted that the backgrounds were generally very low, and there were no impurity peaks interfering with the target or IS peaks.

Fig. 2 Selected reaction monitoring chromatograms using LC–MS–MS for the reference standard MAB-CHMINACA, the extracts of the femoral vein whole blood and liver tissue specimens, the internal standard (IS) AB-CHMINACA spiked into the liver tissue, and the extract of the liver tissue without addition of the IS Full size image

Table 1 shows the standard addition calibration equations for AMB-CHMINACA in 14 specimens, except for urine, in which the target compound was below the LOD. The correlation coefficients for all specimens were greater than 0.999. The LOD (signal-to-noise ratio ≥3) for the compound using the present method was around 0.1 ng/ml or g. The LOQ (signal-to-noise ratio ≥10) was around 0.5 ng/ml or g.

Table 1 Standard addition calibration equations for MAB-CHMINACA in body fluids and solid tissues of the deceased Full size table

Because we employed the standard addition method for quantitation, without the use of blank specimens, it was impossible to present the usual accuracy and precision data. Instead, as shown in Table 2, we repeated intraday and interday determinations of MAB-CHMINACA in the femoral vein whole blood and liver tissue specimens as an example. The repeatability, expressed as relative standard deviations, was not greater than 14.8 %.

Table 2 Examples of intraday and interday repeatability for determination of MAB-CHMINACA in postmortem femoral vein blood and in the liver of the deceased Full size table

Although the standard addition method can overcome matrix effects and low recovery rates, we checked them under the present extraction conditions. In this study, we used acetonitrile deproteinization plus QuEChERS dispersive solid-phase extraction plus filtration through a Captiva ND Lipids cartridge coupled to an LC–MS–MS system. The matrix effects for MAB-CHMINACA were 76.9 ± 1.70 and 69.4 ± 2.17 % (n = 3 in each) for the femoral vein whole blood and liver tissue specimens, respectively. The recovery rates of the test compound calculated (for calculation method, see Ref. [13]]) were excellent at 88.8 ± 11.2 and 109 ± 5.82 % (n = 3 in each) for the femoral vein whole blood and liver tissue specimens, respectively.

Postmortem distribution of MAB-CHMINACA in body fluids and solid tissues of the deceased

Table 3 shows the postmortem distribution of MAB-CHMINACA in body fluids, stomach contents, and nine solid tissues. The concentration of the compound in urine was below the LOD (about 0.1 ng/ml), despite detection of the compound in whole blood specimens. This suggests that the interval between inhalation of MAB-CHMINACA and cardiac arrest was so short that the compound could not reach the urinary bladder via the kidney and ureter. In the previous studies, synthetic cannabinoids were reported to accumulate in the adipose tissue at higher concentrations [9, 13, 22]. However, for MAB-CHMINACA in the present case, its concentration in the adipose tissue was not high, but was second from the lowest among the nine solid tissue specimens. This phenomenon may be also due to the shortness of interval between inhalation of the compound and cardiac arrest; it seems likely that it takes some time for MAB-CHMINACA to accumulate up to relatively high levels in the fat of the adipose tissue. The concentration of MAB-CHMINACA was outstandingly highest in the liver, followed by the kidney, pancreas and heart muscle.

Table 3 Concentrations of MAB-CHMINACA in body fluids and solid tissues of the deceased Full size table

According to our previous study [13], it is evident that this victim smoked the herbal blend of “GM sapphire.” In another study [15], we also disclosed a high concentration of MAB-CHMINACA from the same herbal blend item “GM sapphire,” and here we detected MAB-CHMINACA from body fluid and solid tissue specimens collected from the same cadaver as used in the previous study [13]. These results show that 5-fluoro-ADB and MAB-CHMINACA coexisted in the “GM sapphire” herbal blend that was smoked by the victim. It should be concluded, therefore, that 5-fluoro-ADB and MAB-CHMINACA synergically exerted their toxicities, leading to death after a short interval.

It seems useful to discuss the difference in distribution among the specimens collected from the same deceased for 5-fluoro-ADB and MAB-CHMINACA. In addition, the contents of both compounds in “GM sapphire” are known; they were 49.2 ± 2.46 [13] and 133 ±4.5 mg/g [15], respectively. The first point to be mentioned is the much higher concentrations of MAB-CHMINACA than those of 5-fluoro-ADB in the specimens; the concentrations of 5-fluoro-ADB were only 3.18, 1.90, 1.82, 1.17, 1.61 and 7.95 ng/g in stomach contents, the brain, heart muscle, spleen, pancreas and adipose tissue, respectively, and were below the LOD for body fluid specimens; very small peaks appeared for the lung, liver, kidney, and skeletal muscle, but they were below the LOQ [13]. Even if we take into consideration the fact that the concentration of MAB-CHMINACA is 2.7-fold higher than that of 5-fluoro-ADB in the herbal blend product “GM sapphire," the concentrations of MAB-CHMINACA in various human specimens are much higher than the values expected (Table 3) when compared with those of 5-fluoro-ADB [13]. In addition, the concentrations of MAB-CHMINACA were 6.05–10.6 ng/ml in whole blood specimens, while those of 5-fluoro-ADB in whole blood were below the LOD. These differences in concentrations between the two compounds may be largely due to the stability of the compounds; MAB-CHMINACA has a primary amino group at the terminal part and a cyclohexylmethyl moiety attached to the nitrogen, both of which are much more stable and resistant to chemical decomposition and/or metabolism than the methoxy group and the 5-fluoropentyl moiety present in the 5-fluoro-ADB structure in an alive/dead human body.

Although the outstandingly high concentration of MAB-CHMINACA in the liver is difficult to explain, the second-highest concentration of the compound in the kidney (Table 3) may be due to the ability of this compound to excrete into urine via the kidney, while the concentration of 5-fluoro-ADB in the kidney was below the LOQ [13]. This contrast seems to be due to the hydrophilic nature of the compounds; MAB-CHMINACA is more hydrophilic due to the presence of a primary amino group than is 5-fluoro-ADB with the methoxy group.