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Identification of MitragynaAlkaloids and Metabolites as Biomarkers of Kratom Use in Postmortem Urine Samples
Stephanie Basiliere*, Ph. D and Sarah Kerrigan, Ph. DDepartment of Forensic Science
Sam Houston State University
Huntsville, TX, USA
Disclaimer
• The authors have no commercial disclosures• This project was supported by Award No. 2016-DN-BX-O006 awarded
by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice
• The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect those of the Department of Justice
2
Kratom: Background
• A powder, resin, or leaf cuttings from the leaves of the Korth Tree (Mitragyna speciosa)
• Originally a cultural practice in Southeast Asia, its use is increasing in the western world
• Used recreationally as an opiate replacement and for the non-medically supervised treatment of opiate addiction
• Easily available on the internet and in head shops
3
Scheduling
• Several attempts to pass legislation statewide and federally
• Current legal status:• Not scheduled federally
• Kratom was temporarily placed into schedule I in 2016
• Illegal in 6 states • Restricted use in 11 states• Legislation is pending in 3
states• Legal in 30 states
• 8 of those states failed to pass legislation
4
Chemistry
• Over 40 compounds present in the leaves of Mitragyna speciosa
• Many alkaloids are psychoactive• MG and 7-MG-OH are the most potent
• Mitragynine (60-66%) • Potency is one-fourth that of
morphine
• 7-Hydroxymitragynine (2%)• 13-fold more potent then morphine• 46-fold more potent then MG
• Other prominent alkaloids:• Speciogynine (6.6%)• Speciociliatine (8.6%)• Paynantheine (0.8%)
5
OCH3
HN
NH3C
O
OH3C
O
CH3
Mitragynine
H
OCH3
HN
NH3C
O
OH3C
O
CH3H
SpeciociliatineO
CH3
HN
NH3C
O
OH3C
O
CH3H
Speciogynine
OCH3
N
NH3C
O
OH3C
O
OH
CH3
7-Hydroxymitragynine
H
OCH3
HN
NH2C
O
OH3C
O
CH3H
Paynanthenine
Pharmacology
• Kratom’s effects are dose-dependent • Low doses produce a stimulant effect • High doses produce opiate-like effects
• µ receptor agonist activity
• In rats, doses of over 1000 mg/kg, increased blood pressure and caused severe hepatotoxicity and nephrotoxicity
• Decreased body weights, behavioral changes
• In mice, mitragynine’s opiate like effect for reducing stress has shown potential for addiction and abuse
• ED50 22 mg/kg• LD50 477 mg/kg
6
CDC Report
•152 Kratom-positive deaths – 37 States (Jul 2016-Dec 2017)• 91 Deaths involved kratom• 7 Deaths only detected kratom
•80% Decedents had a history of substance abuse• 90% Decedents had no medically supervised pain treatment• Co-occurring substances:
• Fentanyl (65%)• Heroin (33%)• Benzodiazepines (22%)• Prescription opioids (20%)• Cocaine (18%)
•Kratom deaths likely underreported• May not be included in the screen
7
Deconjugation of Phase II Metabolites
8
•Deconjugation of phase II metabolites relatively unstudied• Conjugated metabolites could make toxicological interpretation difficult
•Philipp et al. 2009 • In vivo study in human and rats• Identified a number of glucuronidated and sulfated metabolites
• Le et al. 2012• Positive kratom urine samples (n=12)• E. coli β-glucuronidase• Determined deconjugation did not improve urine analysis
• Except in high concentrations (> 1000 ng/mL)
• Lee et al. 2018 • Positive kratom urine samples (n=10)• E. coli β-glucuronidase & H. pomatia β-glucuronidase/sulfatase• Successfully hydrolysed glucuronides, but not sulfates
Postmortem Case
Samples
Acid Hydrolysis
20°C
60°C
80°C
Alkali Hydrolysis
20°C
60°C
80°C
Chemical hydrolysis
Experimental Design (Phase II Metabolism)
9
Authentic Case Samples
Recombinant
BGTurbo
BGS
ASPC
IMCSzyme
Glucuronidase
E Coli
H. Pomatia
P. vulgata
Sulfatase
A. aerogenes
Abalone entrails
H. pomatia
P. vulgata
Enzymatic hydrolysis• Recombinant enzymes (4)
• Including two with dual β-glucuronidase/sulfatase activity
• Traditional β-Glucuronidases (3)• Including two with dual β-
glucuronidase/sulfatase activity• Traditional Sulfatases (4)
• All have some β-glucuronidase activity• Pooled postmortem urine (n=5) to optimize
conditions• Optimum deconjugation method used on
individual postmortem urine specimens (n=16)
Experimental Design (Phase II Metabolism)
10
LC/Q-TOF-MS Analysis (Phase II Metabolism)
11
Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MSLC Separation
Poroshell 120 EC-C18 Column (2.1x100 mm, 2.7 µm)
Mobile Phase A: 5 mM Ammonium Acetate in DiH20
Mobile Phase B: Acetonitrile Flow Rate: 0.4 mL/min LC Gradient: 10% B (0.5 min); 10-90% B (0.5-10
min) Injection Volume: 2 µl
Q/TOF Parameters Gas Temperature: 350°C Gas Flow Rate: 13 L/min Sheath Gas Temperature: 400°C Sheath Gas Flow Rate: 12 L/min Nebulizer Pressure 45 psig
MS Capillary Voltage: 4000 V Fragmentor Voltage: 150 V Nozzle Voltage: 0 V Collision Energy: 25, 27, and 28 eV
Acquisition Full Scan Auto Preferred list (precursor
ions) Run Time: 10 minutes MS Scan Rate: 8 spectra/sec MS/MS Scan Rate: 3
spectra/sec MS Scan Range: 100-1000
m/z ESI Mode: Positive
Phase I Metabolites generated In-Vitro (rCYPs)
12
4x10
00.5
11.5
22.5
33.5
44.5
55.5
9-ODM-MG16-COOH-MG
1
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.54x10
0
1
2
3
4
5
6
71
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.53x10
00.5
11.5
22.5
33.5
44.5
55.5
6
9-ODM-16-COOH-MG1
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5
MG-D36x10
00.2
0.4
0.6
0.8
11.2
1.4
1.6
1.8 1
0.6 1 1.6 2 2.6 3 3.6 4 4.6 5 5.6 6 6.6 7 7.6 8 8.6 9 9.6
7-MG-OH [M+H]+ 415
[M+H]+ 385
[M+H]+: 371
[M+H]+: 402
HN
NOH
OO
O
HN
NO
OHO
O
N
NO
OO
OH
OH
HN
NOH
OHO
O
Postmortem Samples – Urine (n=16)
13
• 9-ODM-MG & 7-MG-OH• Most prevalent
• 16-Carboxy-MG• Minor
• 16-17 dihydro metabolites (m/z 387)• Multiple products• Complicated by other
alkaloids• SC/SG isomers
• Structural identification• Mass accuracy• Confirmed using in-vitro
generated metabolite (where possible)
13%
69%
13%
56%
75%
63%
0%
10%
20%
30%
40%
50%
60%
70%
80%
Conf
irmat
ion
Rate
(% C
ases
Con
firm
ed)
Chemical Hydrolysis-Pooled Urine (n=2)
14
010000002000000300000040000005000000600000070000008000000
Mea
n Pe
ak A
rea
7-Hydroxymitragynine
0
2000000
4000000
6000000
8000000
10000000
12000000
Mea
n Pe
ak A
rea
9-O-Demethylmitragynine
• Reaffirms stability studies in literature• Metabolites also acid/base labile• Enzymatic hydrolysis preferable due to moderate pH of
reaction mixture
Enzymatic Hydrolysis-Pooled Urine (n=2)
15
• 7-MG-OH• Significant increase in
abundance for all four sulfatases
• Sulfatases were not cost effective
• Statistically significant increase for BGSTM (p=0.06, α=0.05)
• Sulfatase/glucuronidaseactivity
• Overall, increases in abundance were nominal
• 9-ODM-MG• No significant increase• Possible stability issue
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
Mea
n Pe
ak A
rea
7-MG-OG
*
*
*
* *
16-Carboxymitragynine
16
• 16-Carboxymitragynine• Not detected in pooled urine
• Individual urine specimens• BGSTM and H. pomatia
• Glucuronidase/sulfatase activity
• 16-COOH-MG only detected in two unhydrolyzed samples
• Deconjugation was necessary to see 16-COOH-MG in the majority of cases
• 16-COOH-MG extensively conjugated or minor metabolite
0
200000
400000
600000
800000
1000000
1200000
1400000
A B C D E F G H I J K L M N O P
Peak
Are
a
Sample
No Hydrolysis BGS H. Pomatia
16 Postmortem urine specimens
Enzymatic Hydrolysis 16-Carboxymitragynine
17
• 16-COOH-MG more glucuronidated or sulfated?• Case samples were selected using:
• 16-COOH-MG abundance• Sample Volume
• Sample “P” was selected• Replicates (n=2)
• No significant difference between BGS and BGT
• BGT – only glucuronidase activity• 16-COOH-MG most likely
glucuronidated
*
0%
50%
100%
150%
200%
250%
Peak
Are
a (R
elat
ive
to F
ree)
No Hydrolysis BGS BGT
Biomarkers of Kratom Use
18
•SC, SG, and PY-potential biomarkers• Published methods for plant materials often detect many alkaloids• Prominent alkaloids in plant materials
• 1-9% of alkaloid content• Methods for biological matrices primarily target MG and 7-MG-OH
• Information on the prevalence of these compounds is limited• Philipp et al. 2011:
• GC-MS (Qualitative)• Authentic urine specimens (n=120)• SC, SG, and PY not detected consistently
• Arndt et al. 2011:• LC-MS/MS (Qualitative)• Authentic urine specimens (n=1)• Reported the detection of SC, SG, and PY
• SC most prominent
LC/Q-TOF-MS Analysis (Urine)
LC Separation Poroshell 120 EC-C18 Column (2.1x100 mm, 2.7 µm) Mobile Phase A: 5 mM Ammonium Acetate in DiH20 Mobile Phase B: Acetonitrile Flow Rate: 0.4 mL/min LC Gradient: 47% B (0.5 min); 47-90% B (0.5-10 min)
Q/TOF Parameters Gas Temperature: 350°C Gas Flow Rate: 13 L/min Sheath Gas Temperature: 400°C Sheath Gas Flow Rate: 12 L/min Nebulizer Pressure 45 psig
MS Capillary Voltage: 4000 V Fragmentor Voltage: 150 V Nozzle Voltage: 0 V Collision Energy:
Mitragynine: 30 7-Hydroxymitragynine: 27 Paynantheine: 26 Speciociliatine: 30 Speciogynine: 30
Acquisition Targeted Acquisition Run Time: 10 minutes MS Scan Rate: 8 spectra/sec MS/MS Scan Rate: 3 spectra/sec MS Scan Range: 100-1000 m/z ESI Mode: Positive
Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS
19
Chromatographic Separation
20
6x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
Spec
ioci
liatin
e
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8
7-Hy
drox
ymitr
agyn
ine
Spec
iogy
nine
Payn
anth
eine
Mitr
agyn
ine
Postmortem Case Sample Analysis-Urine (n=16)
0
500
1000
1500
2000
2500
3000
3500
1 2 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Con
cent
ratio
n in
Urin
e(n
g/m
L)
Case #
MG SC SG PY
21
• Concentrations: 2 - >3,000 ng/mL• SC, PY, MG, and SG in 100% of
specimens• SC exceeded MG concentration in
75% of specimens• SG exceeded 63%
• PY did not exceed MG • Detectable in all specimens
• 7-MG-OH was detected in 63% of specimens• Phase I metabolite of MG
Conclusions
• Identified optimal deconjugation procedure in urine• Does not improve the detection of 7-MG-OH or 9-ODM-MG• Improved the detection of 16-COOH-MG
• 16-COOH-MG is primarily glucuronidated• Benefits of deconjugation minimal
• Identified SC, PY, 7-MG-OH, and SG as biomarkers of kratomuse
• 100% of specimens had detectable concentrations of SC, PY, and SG• Majority of specimens had concentrations exceeding MG
• 63% of specimens had detectable concentrations of 7-MG-OH• 75% of specimens had detectable phase I metabolites
• Urine from kratom users contain many Mitragyna alkaloids• Separation of isomers must be considered
22
Acknowledgements• This project was supported by Award No. 2016-DN-
BX-O006 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice
• North Carolina Office of the Chief Medical Examiner• Dr. Ruth Winecker• Justin Brower• Laura Friederich
• University of Mississippi, National Center for Natural Products
• Dr. Ikhlas Khan
