May 8, 2017 - study of 5âfluoroâADB,1 except that the interval of gradient elution ..... tion of 5âfluoroâADBâPINACA and MABâCHMINACA in dubious.
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Journal Code D T A 1 2 3
2
Received: 17 February 2017
Article ID Dispatch: 19.06.17 CE: 2 2 0 No. of Pages: 7 ME: Revised: 22 May 2017
58
Accepted: 25 May 2017
59
DOI: 10.1002/dta.2220
60 61
4 5
SHORT COMMUNICATION
62
6
63
7
64
8 9 10 11 12
Identification and quantification of predominant metabolites of synthetic cannabinoid MAB‐CHMINACA in an authentic human urine specimen
65 66 67 68 69 70
13 14 Q1 Q2 15 16
Koutaro Hasegawa Itaru Yamagishi
|
|
Kayoko Minakata
Kanako Watanabe
|
|
Kunio Gonmori
|
Hideki Nozawa
71
|
72
Osamu Suzuki
73 74
17 18 19 20 21 22 23 24
Department of Legal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan Correspondence Koutaro Hasegawa, Department of Legal Medicine, Hamamatsu University School of Medicine, 1‐20‐1 Handayama, Higashi‐ku, Hamamatsu 431‐3192, Japan. Email: 07484771@hama‐med.ac.jp
25
Q3
An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB‐CHMINACA, was investigated. Although unchanged MAB‐
78
compound could not be detected from a urine specimen. We obtained six kinds of reference
79
standards of MAB‐CHMINACA metabolites from a commercial source. The MAB‐CHMINACA
80
metabolites from the urine specimen of the abuser were extracted using a QuEChERS method
81
including dispersive solid‐phase extraction, and analyzed by liquid chromatography–tandem
82 83
CHMINACA metabolites tested, two predominant metabolites could be identified and
27
84
quantified in the urine specimen of the deceased. After hydrolysis with β‐glucuronidase,
28
85
an increase of the two metabolites was not observed. The metabolites detected were a
29
4‐monohydroxycyclohexylmethyl
30
metabolite
M1
86
(N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐
2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide)
31
and
a
dihydroxyl
87
(4‐
88
hydroxycyclohexylmethyl and tert‐butylhydroxyl) metabolite M11 (N‐(1‐amino‐4‐hydroxy‐3,
32
89
3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide). Their
33
90
concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively.
34
91
Although there is one previous in vitro study showing the estimation of metabolism of MAB‐
35
92
CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and
36
93
quantification of MAB‐CHMINACA metabolites in an authentic human urine specimen.
37 38
94 95
KEY W ORDS
96
39 4‐hydroxycyclohexylmethyl MAB‐CHMINACA, in vivo human metabolism, LC–MS/MS, MAB‐
40
97
CHMINACA metabolites in human urine, tert‐butylhydroxyl MAB‐CHMINACA
41
98 99
42
100
43 44
76 77
CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the
mass spectrometry with or without hydrolysis with β‐glucuronidase. Among the six MAB‐
26
75
1
|
I N T RO D U CT I O N
45
To enforce the laws controlling NPS, it is, of course, essential to
101
identify a controlled drug from seizures, commercial products, and/or
102
46
In recent years, synthetic cannabinoid receptor agonists (SCRAs) have
human specimens by scientific methods. The most preferred specimen
103
47
become one of the largest groups of new psychoactive substances
is urine, because of the noninvasiveness and sufficient amounts to be
104
48
(NPS) in most countries of the world.1-7 They are strictly controlled
collected. However, unfortunately, the levels of unchanged SCRAs in
105
49
under regulation by Pharmaceutical Affairs Law, due to the epidemic
human urine specimens are generally very low at subnanograms per
106
50
abuse in 2013–2015 in Japan. Nowadays, over 2000 NPS including
milliliter,9 and frequently not detectable by usual liquid chromato-
107
51
SCRAs and cathinone derivatives are extensively controlled in Japan.8
graphy–tandem mass spectrometry (LC–MS/MS).10-12 Therefore, as
108
52
The number of such controlled compounds is still increasing, making
substitutes for unchanged SCRAs, various metabolites in human urine
109
53
Japan one of the most stringent countries in the world for the control
specimens have been proposed to prove the intake of illegal
110
54
of NPS.
parent SCRA(s).13
111 112
55
113
56 57
Drug Test Anal. 2017;1–7.
wileyonlinelibrary.com/journal/dta
Copyright © 2017 John Wiley & Sons, Ltd.
1
114
1
2
HASEGAWA
ET AL.
58 59
2 3
In the work reported in this communication, two metabolites of
extraction (SPE) centrifuge tubes with caps (2 mL capacity), one of
60
4
MAB‐CHMINACA detected in a human urine specimen collected from
which contained 25 mg of N‐propylethylenediamine (primary secondary
61
5
a deceased, who abused SCRAs, were identified and determined
amine (PSA)), 25 mg of end‐capped octadecylsilane (C18EC) and 150 mg
62
6
quantitatively.
of magnesium sulfate, and the other of which contained 25 mg of
63
C18EC and 150 mg of magnesium sulfate without PSA, and Captiva
64
ND Lipids cartridges (3 mL capacity) were from Agilent (Santa Clara,
65
CA, USA). Other common chemicals used were of the highest purity
66
commercially available.
67
7 8 9
2
CASE HI STO RY
|
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
The human urine specimen was collected at autopsy performed in October 2014 from the same deceased as described in the reports for demonstration of 5‐fluoro‐ADB11 and MAB‐CHMINACA,12 and stored in the frozen state at −80°C. The deceased was a man in his thirties, and found in a room of a house. Three opened silver‐colored packages with brand names of ‘AL 37’, ‘AP 31’, and ‘GM sapphire’ were also found in his vicinity.14 Upon autopsy, there were neither serious
68
which had been collected at autopsy and stored at −80°C
69
until analysis. For this urine specimen, the metabolites of MAB‐
70
CHMINACA could not be analyzed, because of the unavailability of
71
their reference standards at the times of our previous studies.11,12 A
72
blank urine specimen containing no drug metabolites to be used for
73
method validation experiments was self‐sampled by one of the authors.
74
10,11
studies,
injuries nor disorders related to his death macroscopically. As internal
75
findings, the trachea was filled with a large amount of stomach
3.2
occluding the airway. Such a massive aspiration of stomach contents
A 100 μL volume of urine was added to 900 μL of acetonitrile,
78
into the trachea is usually not observed for subjects with average
followed by mixing with 1 ng of IS dissolved in 10 μL of acetonitrile,
79
physical strength and in a state of clear consciousness. This phenome-
and shaken gently in a test tube. The whole mixture was decanted into
80
non was likely due to lowered consciousness together with vomiting
a QuEChERS SPE centrifuge tube with or without PSA, vortexed for
frequently provoked by the inhalation of SCRA(s).15,16 Other findings
81
30 s, and centrifuged at 10 000 rpm for 2 min. The 600 μL volume
82
were consistent with asphyxia as the direct cause of death; the indirect
of upper acetonitrile layer was passed through a Captiva ND Lipids
83
cause was considered to be SCRA poisoning. Other details of the case
cartridge. The eluate was evaporated to dryness under nitrogen
history were described in our previous reports.11,12,14
84
stream, and reconstituted in 60 μL of mobile phase. A 3.5 μL aliquot
85
of the final extract solution was then subjected to LC–MS/MS for
86
identification and quantification.
87
Routine analysis of blood alcohol using gas chromatography showed a low level of alcohol (0.3 mg/mL) in the blood. Immunochem-
|
Extraction procedure for urine specimen
76
contents, which reached the tracheal bifurcation, thus completely
29 30
The urine specimen was the same as that dealt with in our previous
77
ical drug screening using a Triage Drugs of Abuse panel (Alere,
When the enzymatic hydrolysis of a urine specimen was used, the
88
Waltham, MA, USA) for urine specimens showed a positive result for
following procedure was conducted before the dispersive SPE. A
89
barbiturate drug(s). NAGINATA screening for conventional drugs and
1.0 mL volume of urine specimen was mixed with 200 μL of 2 M sodium
toxic compounds using gas chromatography–mass spectrometry17
90
acetate buffer (pH 4.5), 200 μL of aqueous solution of β‐glucuronidase
91
containing 10 000 units of activity, and 10 ng of IS dissolved in 100 μL
92
of acetonitrile. The mixture solution was incubated at 60°C for 1 h.
93
After incubation, 8.5 mL of acetonitrile was added to the mixture, and
94
38
shaken gently in a test tube. A 1.0 mL aliquot of the mixture was placed
95
39
in a QuEChERS dispersive SPE centrifuge tube. The subsequent
96
procedure was exactly the same as described above.
97
31 32 33 34 35 36 37
revealed the presence of low levels of a nicotine metabolite (0.1 μg/mL) in whole blood and quetiapine (0.3 μg/mL) and phenobarbital (0.3 μg/mL) in the urine.
3
MATERIALS AND METHODS
|
40 41 42 43
3.1
|
98
Materials
3.3
|
99
LC–MS/MS conditions
100
N‐(1‐Amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)
44
methyl)‐1H–indazole‐3‐carboxamide (M1), (S)‐2‐(1‐(cyclohexylmethyl)‐
45
1H–indazole‐3‐carboxamido)‐3,3‐dimethylbutanoic acid (M2), (S)‐2‐
46
(1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamido)‐3,
47
3‐dimethylbutanoic
acid
(M3),
3‐(1‐(cyclohexylmethyl)‐1H–
48
indazole‐3‐carboxamido)‐2,2‐dimethylsuccinic
49
(cyclohexylmethyl)‐N‐(4,4‐dimethyl‐2‐oxotetrahydrofuran‐3‐yl)‐1
50
H–indazole‐3‐carboxamide (M10), N‐(1‐amino‐4‐hydroxy‐3,3‐dimethyl‐
51
1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐
acid
(M7),
1‐
The LC conditions were almost the same as described in our previous study of 5‐fluoro‐ADB,1 except that the interval of gradient elution was changed from 10 to 15 min in the present experiments. For selected reaction monitoring (SRM) by tandem MS, the ion transitions were m/z 387 ! 257 for metabolite M1, m/z 403 ! 257 for metabolite M11, and m/z 373 ! 257 for IS; fragmentor voltage and collision energy were 120 and 21 V, respectively, for all target compounds.
101 102 103 104 105 106 107 108 109
52
carboxamide (M11), and (S)‐N‐(1‐amino‐3‐methyl‐1‐oxobutan‐2‐yl)
53
‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide
54
CHMINACA metabolite M1A, internal standard (IS)) were purchased
55
from Cayman Chemical (Ann Arbor, MI, USA). β‐Glucuronidase from
56
Helix pomatia type H‐1 (3 015 000 units/g) was from Sigma‐Aldrich
The product ion spectra of the reference standards of six MAB‐
113
57
(St Louis, MO, USA). Two kinds of QuEChERS dispersive solid‐phase
CHMINACA
114
4
RESULTS AND DISCUSSION
|
110
(AB‐
4.1
|
111
Preliminary results metabolites
were
112 obtained
by
collision‐induced
1
HASEGAWA
3
ET AL.
58 59
2 3
dissociation of the protonated molecules, which gave each suitable
increase in peak areas was not observed for M11 and M1; peak area
60
4
qualifier ion to be used for testing the presence of metabolite(s) in the
ratios of the extract after hydrolysis to that without hydrolysis were
61
5
authentic urine specimen obtained from the abuser (data not shown).
about 0.9 and 0.93 for M11 and M1, respectively.
62
6
Without hydrolysis with β‐glucuronidase, two intense peaks at
For optimization of extraction for M1, M11, and IS from urine
63
7
retention times of 2.4 and 5.3 min appeared by SRM, suggesting the
specimens, we compared the peak areas of the three compounds using
64
8
presence of dihydroxyl (hydroxylated at 4‐cyclohexylmethyl and tert‐
the dispersive SPE centrifuge tubes in the presence of PSA with those
65
9
butyl) metabolite M11 and 4‐hydroxycyclohexylmethyl metabolite
in the absence of PSA. First, we could obtain all three peaks using
66
10 F1 M1 in the authentic urine (panels 2 and 4 of Figure 1). Peaks (signal‐
dispersive SPE centrifuge tubes even without PSA. By using dispersive
67
11
to‐noise ratio > 3) were not detected by SRM for the remaining four
SPE centrifuge tubes with PSA, all peak areas of M1, M11, and IS
68
12
metabolites (data not shown).
increased about 1.5‐fold as compared with those without PSA, because
69
13
After hydrolysis with β‐glucuronidase, the results were almost the
M1, M11, and IS are slightly basic compounds. Therefore, the disper-
70
14
same as those without hydrolysis; no peaks appeared for the four
sive SPE centrifuge tubes in the presence of PSA were used for
71
15
metabolites other than M11 and M1 by SRM. Furthermore, any
extraction in this study.
72
16
73
17
74
18
75
19
76
20
77
21
78
22
79
23
80
24
81
25
82
26
83
27
84
28
85
29
86
30
87
31
88
32
89
33
90
34
91
35
92
36
93
37
94
38
95
39
96
40
97
41
98
42
99
43
100
44
101
45
102
46
103
47
104
48
105
49
106
50
107
51
108
52
109
53
110
54
111
55
112
56 57
FIGURE 1 Product ion spectra of reference standards M1 and M11 of MAB‐CHMINACA together with spectra obtained from the extract of an authentic human urine of an abuser, using LC–MS/MS. in the bottom panel, a product ion spectrum of IS metabolite M1A is also presented
113 114
4
1
HASEGAWA
ET AL.
4.2
3 4
imen of the deceased. We concentrated the final eluate extracted from
7
the authentic urine specimen about ten‐fold for obtaining product ion
8
spectra of M1 and M11, because good enough mass spectra of these
9
metabolites for profiling could not be obtained without concentration.
10
The precursor ions were the protonated molecules at m/z 387, 403,
11
and 373 for M1, M11, and IS, respectively.
12
In the product ion spectra of M1 and M11 in extracts from the
13
authentic human urine, the parent ion peaks also appeared at m/z
14
387 and 403, respectively, though these peaks could not be observed
15
markedly in the product ion spectra of reference standards M1 and
16
M11. Although each product ion profile of the human urine extract
17
agreed well with that of the corresponding reference standard, the
18
21 22
Product ion spectra and SRM chromatograms
standards M1 and M11, and those of the extracts from the urine spec-
6
20
|
Figure 1 shows an example of product ion spectra of the reference
5
19
58 59
2 Intra‐day (n = 5) and inter‐day (n = 5) accuracy and precision data
60
were evaluated by using quality control samples at 1.0, 5.0, and
61
10.0 ng/mL for M1 and M11 prepared with blank human urine
62
(Table 3). The accuracy values ranged from 93.4 to 119% and the T3 63 precision values from 3.8 to 6.7% for all compounds examined,
64
showing generally good reproducibility for this method.
65
Stability of the target compounds in urine samples was also
66
assessed under various conditions. We prepared urine samples spiked
67
with M1 and M11 at 1.0, 5.0, and 10.0 ng/mL, and stored each sample
68
at room temperature, 4° and −30°C. Concentrations of M1 and M11 in
69
each sample were determined after 7, 14, 21, and 28 days by
70
LC–MS/MS. Metabolites M1 and M11 in urine specimens were found
71
to be relatively stable under all conditions examined (ranged from
72
70.9 to 124%), as was also reported in another article showing excellent
73
stabilities of synthetic cannabinoid metabolites in human urine.19
74 75
fragmentation of each parent compound in the urine extracts seems
T1 somewhat suppressed, probably by the matrix effect (Table 1). resulting in the appearance of the parent ion peaks of M1 and M11 (Figure 1).
F2
23 24 25 26 27 28
Figure 2 shows an example of SRM chromatograms for the reference standards M1 and M11, and M1, M11, and IS in the extract of the authentic urine specimen. The retention times of M1, M11, and IS were 5.3, 2.4, and 3.4 min, respectively. It was confirmed that the urine specimen in this case contained no M1A which enabled us to use M1A as IS. All three peaks appeared sufficiently sharp, and background levels were low without marked impurity peaks.
4.4 | Concentrations of metabolites M1 and M11 in authentic human urine sample
76 77 78
The means ± standard deviation of the concentrations for M1 and M11
79
in the authentic urine of the abuser were found to be 2.17 ± 0.15 and
80
10.2 ± 0.31 ng/mL (n = 3 each), respectively.
81
M1 is monohydroxylated in the 4‐position of the cyclohexyl
82
moiety; M11 is dihydroxylated in the 4‐position of the cyclohexyl
83
moiety and also at the tert‐butyl moiety in the amide linker (their
84
structures are shown in Figure 1).
85
29
Very recently, Carlier et al.20 reported in vitro metabolism of
86
30
MAB‐CHMINACA, detecting 10 metabolites after 3 h incubation of
87
MAB‐CHMINACA with human hepatocytes. Although they did not
88
31
4.3
|
Validation of the method
32
The method validation was performed essentially according to
make quantitative analyses, the ranking of the metabolites was roughly
89
33
Matuszewski et al.18 The recovery rates of M1, M11, and IS from blank
tried according to the peak areas of extracted ion chromatograms; the
90
34
human urine specimen determined at 1.0 and 10.0 ng/mL for each
most predominant metabolites were those monohydroxlated at the
91
35
compound are shown in Table 1. Recovery rates for all compounds
cyclohexylmethyl moiety including M1 (rank = 2), and the most
92
36
examined were not lower than 96.3% and were satisfactory. The matrix
minor metabolites were those monohydroxylated (the location of
93
37
effects of M1, M11, and IS in blank human urine specimen at 1.0 and
monohydroxylation not known; rank = 9) and dihydroxylated
94
38
10.0 ng/mL for each compound were somewhat suppressive with
exclusively at the cyclohexylmethyl moiety (the exact location of two
95
39
biases ranging from −16.0 to −36.6%, showing that relatively moderate
hydroxylations not known; rank = 10) together with M11 (rank = 8).
96
19
40
suppressive effects took place for ionization of analytes in extracts
The peak area of M1 was 18‐fold larger than that of M11.
Although
97
41
even after the dispersive SPE and Captiva ND Lipids cartridge filtration.
our study dealt with only one human authentic case, the concentrations
98
Table 2 shows regression equations for M1 and M11 in human
of M1 and M11 in the present study were 2.17 and 10.2 ng/mL,
99
urine specimens. Good linearity was obtained with correlation coeffi-
respectively. Our result is quite different from that of the previous
100
42 T2 43
20
44
cients not lower than 0.999 in the range 0.5–20.0 ng/mL (five plots)
in vitro study.
There seems to be distinct difference between the
101
45
for each compound. The detection limit (signal‐to‐noise ratio = 3) of
metabolite profiles obtained by in vitro experiments and by the present
102
46
M1 and M11 by this method for urine specimens was about 0.1 ng/mL.
in vivo analysis of urinary metabolites. The main causative factor may be
103 104
47 48 49 50 51 52 53
TABLE 1
Matrix effects and recovery rates for determination of MAB‐CHMINACA metabolites M1 and M11 and AB‐CHMINACA metabolite M1A (IS) spiked into human urine at different concentrations Compound
Concentration added (ng/mL)
Matrix effect (%bias)
Recovery rate (%)
MAB‐CHMINACA metabolite M1
1.0 10.0
−19.2 ± 3.1 −22.0 ± 2.4
97.6 ± 4.3 98.7 ± 2.7
MAB‐CHMINACA metabolite M11
1.0 10.0
−16.0 ± 3.0 −19.8 ± 1.8
96.3 ± 2.4 96.9 ± 1.7
1.0 10.0
−35.6 ± 2.1 −36.6 ± 1.1
97.7 ± 1.8 97.4 ± 1.4
54 55 56 57
AB‐CHMINACA metabolite M1A (IS)
Data given as mean ± standard deviation from five determinations.
105 106 107 108 109 110 111 112 113 114
1
HASEGAWA
5
ET AL.
58
2
59
3
60
4
61
5
62
6
63
7
64
8
65
9
66
10
67
11
68
12
69
13
70
14
71
15
72
16
73
17
74
18
75
19
76
20
77
21
78
22
79
23
80
24
81
25
82
26
83
27
84
28
85
29
86
30
87
31
88
32
89
33
90
34
91
35
92
36
93
37
94
38
95
39
96
40
97 98
41 42 43
FIGURE 2
Selected reaction monitoring chromatograms using LC–MS/MS for reference standards of M1 and M11 and those from the extract of an authentic human urine of an abuser. In the bottom panel, the chromatogram for reference standard of IS (metabolite M1A) is also presented
46
100 101
44 45
99
TABLE 2
102
Calibration equations for MAB‐CHMINACA metabolites M1 and M11 in human urine Equation
Correlation coefficient (r)
48
MAB‐CHMINACA metabolite M1
0.5–20.0
y = 19.38× − 0.1415
0.999
105
49
MAB‐CHMINACA metabolite M11
0.5–20.0
y = 6.80× + 0.1404
0.999
106
50 51
Compound
103
Range (ng/mL)
47
Each equation was constructed with five different concentrations (0.5, 1.0, 5.0, 10, and 20 ng/mL) by plotting the triplicated mean peak area ratios of test compound to IS.
104
107 108 109
52 53
the lipophilicity of the metabolites. The time of clearance of a lipophilic
lipophilic than the M1 metabolite with monohydroxylation. There-
110
54
metabolite from a human body may be longer than that of a less
fore, it is reasonable that the urinary concentration of M11 was
111
55
lipophilic one, because the former may be readily distributed to adipose
higher than that of M1 in the present study, despite that the in vitro
112
56
tissues, and also bound to intra‐ and extracellular lipids and
study showed that the production of M1 was much higher than that
113
57
lipoproteins. The M11 metabolite with dihydroxylation is clearly less
of M11.20
114
1
6
HASEGAWA
ET AL.
3 4
TABLE 3 Intra‐day and inter‐day accuracy and precision for MAB‐CHMINACA metabolites M1and M11 in human urine at different concentrations obtained by the present method
5 6
Compound
7
MAB‐CHMINACA metabolite M1
8 9 10
MAB‐CHMINACA metabolite M11
11 12
Intra‐day (n = 5)
Inter‐day (n = 5)
Concentration added (ng/mL)
Accuracy (%)
Precision (RSD, %)
Accuracy (%)
Precision (RSD, %)
1.0 5.0 10.0
97.2 93.8 93.4
6.7 5.5 4.8
97.4 102 102
4.7 5.2 4.9
1.0 5.0 10.0
117 97.2 98.9
3.8 4.1 4.1
119 116 108
3.9 5.6 5.3
In this study, the hydrolysis with β‐glucuronidase neither
15
18 19 20
increased levels of M1 and M11 nor resulted in the appearance of other metabolites, showing the absence of phase II metabolites. Only two phase I metabolites could be found. These results suggest that the time interval between the intake of MAB‐CHMINACA and death was relatively short. In our previous study of identification and quantification of metab-
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
olites of AB‐CHMINACA, an analog of the present MAB‐CHMINACA (the isopropyl moiety in the amide side chain of AB‐CHMINACA is only replaced by the tert‐butyl moiety in MAB‐CHMINACA), in a urine specimen of an abuser, we could detect a metabolite monohydroxylated in the 4‐position of cyclohexyl moiety and another metabolite carboxylated at the terminus of the amide linker.21 Erratico et al.22 also reported in vitro and in vivo metabolism of AB‐CHMINACA; they detected 26 metabolites by in vitro incubation of AB‐CHMINACA with human liver microsomes, but the in vitro results did not include a metabolite dihydroxylated in the 4‐position of the cyclohexyl moiety and also at the isopropyl moiety in the amide linker. The absence of such a dihydroxylated metabolite of AB‐CHMINACA was also true for a urine sample of an abuser.20 It is of great interest that such a minor difference in their structures, i.e. the propyl and tert‐butyl moieties for AB‐CHMINACA and MAB‐CHMINACA, respectively, causes a great difference in metabolic pathways both in vitro and in vivo.
38 39 40
61 62 63 64 65 66 67 68 70
14
17
60
69
RSD, relative standard deviation.
13
16
58 59
2
5
|
C O N CL U S I O N
41 42
In this report, two metabolites of MAB‐CHMINACA, which is one of
43
the most widely abused SCRAs in the world,2,5,12,16 have been
44
identified and quantified in a human urine specimen obtained
45
from an abuser using LC–MS/MS. In terms of results, a 4‐
46
monohydroxycyclohexylmethyl MAB‐CHMINACA metabolite (M1)
47
and an MAB‐CHMINACA metabolite dihydroxylated at the same
48
position as that of M1 and also at the tert‐butyl moiety (M11) could
49
be detected; the level of M11 was much higher than that of M1.
50
By identifying either metabolite in the urine specimen, it can be
51
concluded that the subject really used MAB‐CHMINACA without
52
doubt; both of them can be a useful marker for MAB‐CHMINACA
53
consumption. In addition, the method established by us for analysis
54
of the MAB‐CHMINACA metabolites in human urine seems useful in
55
toxicological analysis and clinical drug testing. This report is the first
56
to present MAB‐CHMINACA metabolites in an authentic human
57
urine specimen.
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9. Minakata K, Yamagishi I, Nozawa H, et al. Sensitive identification and quantification of parent forms of six synthetic cannabinoids in urine samples of human cadavers by liquid chromatography‐tandem mass spectrometry. Forensic Toxicol. 2017. https://doi.org/10.1007/ s11419‐017‐0354‐0 10. Hasegawa K, Wurita A, Minakata K, et al. Postmortem distribution of AB‐CHMINACA, 5‐fluoro‐AMB and diphenidine in body fluids and solid tissues in a fatal poisoning case: Usefulness of the adipose tissue for detection of the drugs in the unchanged forms. Forensic Toxicol. 2015;33:45 11. Hasegawa K, Wurita A, Minakata K, et al. 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 Toxicol. 2015;33:112 12. Hasegawa K, Wurita A, Minakata K, et al. Postmortem distribution of MAB‐CHMINACA in body fluids and solid tissues of a human cadaver. Forensic Toxicol. 2015;33:380 13. Diao X, Huestis MA. Approach, challenges and advantages in metabolism of new synthetic cannabinoids and identification of optimal urinary marker metabolites. Clin Pharmacol Ther. 2016;101:239
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ET AL.
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17. Kudo K, Ishida T, Hikiji W, et al. Construction of calibration‐locking databases for rapid and reliable drug screening by gas chromatography‐mass spectrometry. Forensic Toxicol. 2009;27:21 18. Matsuzewski BK, Constanzer ML, Chevez‐Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC‐MS/MS. Anal Chem. 2003;75:3019 19. Jang M, Shin I, Kim J, Yang W. Simultaneous quantification of 37 synthetic cannabinoid metabolites in human urine by liquid chromatography‐ tandem mass spectrometry. Forensic Toxicol. 2015;33:221
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How to cite this article: Hasegawa K, Minakata K, Gonmori K,
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et al. Identification and quantification of predominant
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metabolites of synthetic cannabinoid MAB‐CHMINACA in an
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authentic human urine specimen. Drug Test Anal. 2017;1–7.
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