Postmortem distribution of mepirapim and acetyl fentanyl in biological fluid and solid tissue specimens measured by the standard addition method

Mepirapim hydrochloride, acetyl fentanyl, JWH-200 and acetyl fentanyl-d₅ were purchased from Cayman Chemical (Ann Arbor, MI, USA), and Isolute SLE + (1 mL capacity) columns from Biotage (Uppsala, Sweden). Other common chemicals used in this study were of the highest purity commercially available.

To 50 µL of each body fluid or 100 µL solid tissue homogenate (2 g tissue in 8 mL 0.01 N HCl solution thoroughly homogenized with a blender) were added 20 µL of methanol solution containing a known amount of mepirapim and acetyl fentanyl as standard additions, 20 µL of methanol solution containing 50 ng of JWH-200 and 10 ng of acetyl fentanyl-d₅ as ISs for mepirapim and acetyl fentanyl, respectively, and 0.75 mL of 1% Na₂CO₃. The mixture was mixed on a vortex mixer and transferred onto an Isolute SLE + (1 mL capacity) column. The column was eluted by using 5 mL of methyl tert-butyl ether. The eluent was evaporated, and the residue was reconstituted in 200 µL methanol. A 2-µL aliquot was used for analysis by LC–MS.

Quantitative analysis was performed by selected-ion monitoring (SIM) analysis of LC–MS. Briefly, LC–MS was performed on an ACQUITY Arc LC system connected to an ACQUITY QDa mass spectrometer (Waters, Milford, MA, USA). The LC conditions were as follows: XBridge BEH C18 column (75 × 3.0 mm i.d., particle size, 2.5 µm; Waters); injection volume, 2 µL; flow rate, 0.5 mL/min; elution mode, isocratic with 10 mM ammonium acetate in water/methanol (45:55, v/v); column temperature, 40 °C. The MS conditions were as follows: interface, electrospray ionization mode; polarity, positive; probe temperature, 600 °C; capillary voltage, 1.2 kV; scan mode, SIM; monitoring ion and cone voltage, m/z 314 and 10 V for mepirapim, m/z 323 and 20 V for acetyl fentanyl, m/z 385 and 20 V for JWH-200 and m/z 328 and 20 V for acetyl fentanyl-d₅, respectively.

The standard addition method can completely overcome matrix effects. The method does not require blank human body fluid or solid tissue specimens that are negative for target compounds as described before. Note that when investigating the distribution of a xenobiotic compound with different types of matrices, the standard addition method is required to construct a calibration curve to obtain a single concentration value [16, 17]. To obtain each value, six different concentrations of the target compounds and fixed concentrations of ISs were added to homogenate portions of the same matrix, respectively, for construction of a calibration curve. The slope of the calibration curve crosses the y-intercept (intensity) and intersects the x-axis (target compound concentration) at the negative side; the absolute value of which indicates the preexisting concentration of the target compounds in the matrix.

To determine the matrix effects and recovery rates for mepirapim and acetyl fentanyl in body fluid and solid tissue specimens, we used the method reported previously [16, 17]. Briefly, we first measured all concentrations of the two target compounds in matrices by using the standard addition method. Then, the same extraction procedure without standard addition was again performed twice for each matrix. At the final step of the extraction, in which 200 µL of methanol was added to reconstitute the extract residue after evaporation of the eluent, we used 200 µL of pure methanol (unspiked) in one sample and also used 200 µL of a methanol solution (spiked) containing the same amounts of mepirapim and acetyl fentanyl contained in the initial 50 µL body fluid or 100 µL solid tissue homogenate as those already measured by the standard addition method for each cadaver specimen in another sample. After reconstitution with the methanol solutions, 2 µL aliquots were injected into the LC–MS system. The peak area obtained by reconstitution with pure methanol was designated as B; the peak area obtained by reconstitution with methanol containing the same amount of the target compound in a specimen of the deceased was designated as A. A 2-µL volume of the above methanol solution (neat sample) containing the same amount as that contained at the initial step without any reconstitution was injected into the LC–MS system; the resulting peak area was designated as C.

Each matrix effect and recovery rate was calculated according to the following equations:

To confirm the linearity of the calibration curve for mepirapim and acetyl fentanyl, the standard addition calibration curves were constructed in suitable concentration ranges as shown in Table 1 for the body fluid and solid tissue specimens by using six plot points at different concentrations each (n = 5 each). Good linearity was obtained for both compounds and for all specimens, with correlation coefficients not smaller than 0.9941 (Table 1).

Figure 1 shows the SIM chromatograms of mepirapim and acetyl fentanyl extracted from femoral vein whole blood, and JWH-200 and fentanyl-d₅ spiked into the whole blood as ISs. The chromatograms showed very symmetrical peaks for the target compounds and low backgrounds in all specimens.

Given that the standard addition method was used in this study, it was impossible to present the usual accuracy and precision data using blank human specimens spiked with different concentrations of mepirapim and acetyl fentanyl (quality controls), because we did not use blank human specimens obtained from another suitable autopsy for ethical reasons. Instead, as shown in Table 2, we tested the intraday and interday repeatability for the two target compounds in femoral vein whole blood and liver specimens as examples. For mepirapim, the repeatability expressed as relative standard deviations was ≤ 10.7%. For acetyl fentanyl, the repeatability was ≤ 10.8%.

We used a specially devised method for calculation of matrix effects and recovery rates, as summarized in Table 3. The matrix effects on mepirapim and acetyl fentanyl for all specimens were small, with biases of ≤ 29.6 and ≤ 13.7%, respectively (n = 5 each). The recovery rates of mepirapim and acetyl fentanyl were ≥ 45.6 and ≥ 62.4%, respectively (n = 5 each).

By dilution of each specimen, the detection limits of mepirapim and acetyl fentanyl by this method were estimated to be approximately 20 and 2 ng/mL or g, respectively.

As shown in Table 4, the concentrations of mepirapim in heart whole blood, femoral vein whole blood and urine were as high as 587, 554 and 309 ng/mL, respectively, as compared with those of other common synthetic cannabinoids. The concentrations in the solid tissue specimens except psoas major muscle were one order of magnitude higher than those of the blood and urine specimens. The highest concentrations of mepirapim among these specimens were found in the liver and kidney at 5410–6300 ng/g.

For acetyl fentanyl, the concentrations in heart whole blood, femoral vein whole blood and urine were 212, 170 and 169 ng/mL, respectively. The highest concentrations of acetyl fentanyl among these specimens were found in the myocardium, followed by the spleen and kidney at 1140–1180 ng/g.

Within the brain, both mepirapim and acetyl fentanyl distributed unevenly. The highest concentrations of both compounds were found in the pons, followed by the cerebrum or cerebellum. The lowest concentrations were found in the medulla oblongata for both compounds.