kratom alkaloids an historical perspective - arndt - fsi 208 (2011)

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Kratom alkaloids and O-desmethyltramadol in urine of a Krypton herbal

mixture consumer

Torsten Arndt a,*, Ulrich Claussen b, Brunhilde Gussregen a, Stefanie Schrofel a, Birgit Sturzer b,Annika Werle a, Gerald Wolfb

a Bioscientia, Institut fur Medizinische Diagnostik GmbH, Konrad-Adenauer-Strae 17, D-55218 Ingelheim, Germanyb Therapeutische Einrichtung Auf der Lenzwiese, Annelsbacher Weg 1, 64739 Hoechst-Hassenroth, Germany

1. Introduction

Mitragyna speciosa is a tree of the Southeast Asian region. It is

called Kratom in Thailand or BiakBiak in Malaysia. Theleaves of

this tree contain more than 20 alkaloids, varying in their

composition e.g. between the geographic variants. For example,

M. speciosa leaves from Thailand have been reported to contain

approximately 66% mitragynine, those from Malaysia only 12% [1].

Somealkaloidsfrom M. speciosa have opiate- and cocaine-like effects

whichis whyKratom hasbeen traditionallyused as a stimulant or

as a substitute for opiate addicts. Because of the narcotic effects, the

use ofM. speciosa drugs became illegal in Thailand in 1946 and in

Australia in 2005[14]. It is still not illegal in most of the European

Countries and in the USA. As a consequence, a broad range of

Kratom products is available e.g. viathe Internet. During thelast 5

years, a herbal mixture called Krypton, said to contain Kratom

leaves, has beenquite popular. Validdata regarding the constituents

ofthis productare missing.There is only a shortnotein the scientific

literature that Krypton contains also . . . a metabolite of the

synthetic opioid analgesic tramadol. . . (footnote 17 in[5]).

We have been asked by a drug and alcohol withdrawal

rehabilitation centre for Krypton analysis in urine of a former

Forensic Science International 208 (2011) 4752

A R T I C L E I N F O

Article history:

Received 18 February 2010

Received in revised form 12 October 2010

Accepted 30 October 2010

Available online 26 November 2010

Keywords:

Kratom

Krypton

LCMS/MS

Speciociliatine

Mitragynine

O-desmethyltramadol

A B S T R A C T

Aim: A drug andalcohol withdrawal rehabilitation centre requestedan analysis forKryptonin urine of

a former opiate-addictive woman. She showed an altered clinical picture and behaviour with miosis,

itchiness, agitation, and moderate euphoria after 3 months of until than successful treatment. Literature

search revealed that Krypton is said to contain Kratom (leaves ofMitragyna speciosa), but could also

contain O-desmethyltramadol (European Monitoring Centre for Drugs and Drug Addiction thematic

paper Spice).

Methods: Immunological drug screenings were done with test strips (nal von minden, Regensburg,

Germany) and with cloned enzyme donor immunoassay (Microgenics, Passau, Germany). Kratom

alkaloids and tramadol (metabolites) were analyzed by LCMS/MS (ThermoFisher Scientific Quantum

Ultra Triple Quadrupole mass spectrometer).

Results: Immunoassays were negative for amphetamines, barbiturates, benzodiazepines, benzoylecgo-

nine, buprenorphine, ethylglucuronide, methadone (metabolite), opiates, oxycodone, and THC-COOH,

and test strips were negative for tramadol and its metabolites (cut-off 10 mg/L for O-desmethyl-

tramadol). LCMS/MS detected the Kratom alkaloids mitragynine, speciociliatine, speciogynine,

mitraciliatine, and paynantheine and approximately 9 mg/L O-desmethyltramadol, but no tramadol and

N-desmethyltramadol.Discussion: The detection of M. speciosa alkaloids is a proof of Kratom abuse. Confronted with the

analysis data, the patient admitted to have consumed 34 infusions of Krypton. The origin of the O-

desmethyltramadol is unclear. Tramadol abuse is unlikely since tramadol and N-desmethyltramadol

(physiologically occurring in urine after tramadol intake) were not detectable. Consumption of a

Krypton product spiked with O-desmethyltramadol could explain our findings and the patients

clinical picture. This would be in agreement with a most recent report about spiking apparently natural

herbal mixtures with the synthetic opioid O-desmethyltramadol.

Conclusion: Analysis of Kratom abuse should not be restricted toM. speciosaalkaloids, but should also

consider synthetic drugs which could be added to the herbal mixtures. Mass spectrometry based drug

screenings will gain importance to keep pace with the dynamic drug market.

2010 Elsevier Ireland Ltd. All rights reserved.

* Corresponding author. Tel.: +49 6132 781 349; fax: +49 6132 781 428.

E-mail address: [email protected](T. Arndt).

Contents lists available atScienceDirect

Forensic Science International

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o r s c i i n t

0379-0738/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2010.10.025
http://dx.doi.org/10.1016/j.forsciint.2010.10.025mailto:[email protected]://www.sciencedirect.com/science/journal/03790738http://dx.doi.org/10.1016/j.forsciint.2010.10.025http://dx.doi.org/10.1016/j.forsciint.2010.10.025http://www.sciencedirect.com/science/journal/03790738mailto:[email protected]://dx.doi.org/10.1016/j.forsciint.2010.10.025


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opiate-addictive woman. Using LCMS/MS, we could detect

Kratom alkaloids and O-desmethyltramadol, but not

tramadol and N-desmethyltramadol. These findings could

confirm that Krypton is not only a herbal mixture with

Kratom leaves, but contains also O-desmethyltramadol as a

synthetic additive.

2. Materials and methods

2.1. Patient

A 33-year-old opiate-addictive woman with heroin, cocaine, benzodiazepine,

and alcohol abuse during buprenorphine substitution therapy was treated in an

in-patient detoxification centre.After successful detoxification,she was admitted

to an in-patient drug and alcohol withdrawal rehabilitation centre. After

approximately 3 months, the patient showed an altered clinical picture and

behaviour with miosis, itchiness, agitation, and moderate euphoria. Extra drug

screens for opiates, tramadol and tramadol metabolites were initiated in our

laboratory.

2.2. Drug screen

The urine sample was analyzed by test strips (nal von minden, Regensburg,

Germany) in the rehabilitation centre and after receipt in our laboratory by CEDIA

(ThermoFisher Scientific Microgenics, Passau, Germany) with an Olympus AU 640

analyzer (Olympus, Bottrop-Kirchhellen, Germany), using the cut-offs recom-

mended by the manufacturers[6,7].

2.3. Quantitative LCMS/MS for tramadol (metabolites)

Sample preparation: 50 mL urine, calibrator or control sample were pipetted into

1.5 mL Eppendorf vials, followedby additionof 100 mL waterand100 mLof250 mg/

L tramadol-13C1D3 (internal standard) and 750mL acetonitrile, mixing, and

centrifugation for 5 min at 13.000 U/min (=16.000 g) (Galaxy 16 DH centrifuge,

VWR, Germany). 200 mL of the clear supernatant was transferred into 1.2 mL 96-

deep-well-microtiter plates.

LC: ThermoFinnigan Surveyor MS Pump Plus; 200 mL/min; isocratic 45% water,

55% acetonitrile both with 0.1% formic acid (all data v/v). Run time: 2.5 min;

column: ThermoFisher Aquasil C18 50 mm 2.1 mm, 3 mm particle size, pre-

column: Phenomenex C (Octyl, MOS) 4 mm 2 mm (Phenomenex, Aschaffenburg,

Germany); column oven: 25 8C (MayLab MistraSwitch, Wien, Austria); autosam-

pler: PAL HTC (CTCAnalytics, Zwingen,Switzerland), 16 8C, full-loop-mode, sample

syringe 3 mL overfilled with 10 mL sample.

MS/MS: ThermoFisher Scientific Quantum Ultra Triple Quadrupole mass

spectrometer; software: XCALIBUR 2.0.7. Sheath gas (nitrogen) pressure: 30 a.u.;auxillary gas (nitrogen) pressure: 20 a.u.; collision gas (argon) pressure: 1.5 mTorr;

capillary temperature: 350 8C; spray voltage:5000 V; ion source:positiveESI mode.

MS acquisition time: 2.5 min; scan width: 0.01 m/z; scan time: 0.06 s; peak width

Q1 and Q3: 0.7 FWHM.

Validation data: Calibration: 50, 250, 500, 1000, 2500 mg/L in serum matrix,

calibration function calculated by linear regression, minimum acceptable linear

correlation coefficient: 0.995. Dilution limit: 2500 mg/L, at maximum 1:5 dilution

with0.9% (w/v) NaCl. Detection limit:2 mg/L,lowerlimit of quantification: 50 mg/L;

upper limit of quantification: 2500mg/L; Analytical measurable range: 50

2500 mg/L; clinical reportable range: 5012,500 mg/L. Intra- and Inter-assay CV:


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Krypton. The immunological drug screening at the rehabilitation

centre was negative (test strips nal von minden). The same was

true for CEDIA drug screenings in our laboratory (Table 2).

The subsequent qualitative and quantitative LCMS/MS analy-

ses yielded the following results (Table 3). LCMS/MS ion

chromatograms from mitragynine, speciociliatine, speciogynine,

and mitraciliatine and from tramadol (metabolites) are presented

in Figs. 1 and 2.Confronted with the analysis data, the patient admitted to have

drunk 34 infusions of Krypton herbal mixture during the last

week. She insisted that she has not taken any other drugs or drug

preparations. The patient was discharged in accordance with the

codes of conduct of the rehabilitation centre.

4. Discussion

The presence of M. speciosa alkaloids in blood or urine is

considered to be a proof of Kratom abuse [810]. Thedetection of

the 5 alkaloids, mitragynine, speciociliatine, speciogynine, mitra-

ciliatine, and paynantheine (Table 3) in the patients urine clearly

points to a recent consumption of Kratom, most probably of a

Krypton herbal mixture as admitted by the patient. The fact that

the immunological drug screenings by nal von minden test strips

and CEDIA did not detect this Kratom abuse is not surprising

since Kratom alkaloids or their metabolites are not target

analytes of these drug screening reagents.

Mitragynine, speciogynine, speciociliatine, and mitraciliatine

are diastereomers with an identical constitutional structure and

molecular formula. They differ in the configuration of at least 1 of

the 3 chiralic C-atoms (Fig. 3). Mass spectra or selected reaction

monitoring (SRM) mass transitions are identical for diastereomers

(Table 1). A valid identification of the individual diastereomers can

only be obtained after chromatographic separation, e.g. by gas or

liquid chromatography. However, reference substances which are

needed for establishing the individual retention times and for

standard addition experiments are often missing. Due to this, we

could not identify Kratom alkaloid metabolites like described in

[3,11]. As listed in Table 3 we detected paynantheine in the

patients urine with LCMS/MS. Indeed, we obtained two signals in

the extracted ion chromatogram with different retention times

using the tuned SRM traces for paynantheine. Standard addition

experiments showed that the first and smaller one has to be

assigned to paynantheine. We suppose theother oneresults from a

paynantheine diastereomer[9], but we were not able to identify itdue to missing reference substance.

Inspired by a footnote in [5] regarding the presence of O-

desmethyltramadol in Krypton herbal mixtures, we included in

our analyses a quantitative LCMS/MS for tramadol and its

pharmacologically active metabolites O- and N-desmethyltrama-

dol.We foundO-desmethyltramadol (9 mg/L or 8 mg/g creatinine),

but not tramadol and N-desmethyltramadol in the patients urine

(Table 3 and Fig. 2). Such a tramadol (metabolite) excretion pattern

is uncommon. Tramadol is a synthetic opioid-receptor agonist

which is used as a narcotic analgesic. It is mainly metabolized to O-

desmethyltramadol (showing a 24-fold stronger analgesic effect

than tramadol) and N-desmethyltramadol and O-desmethyl-N-

Table 3

Results of the LCMS/MS for Kratom alkaloids and for tramadol (metabolites).

Identificationcriteriafor each analyte were retention time,4 or 3 SRMtraces (Table

1) and the corresponding SRM signal ratios.

Analyte Result

Mitragynine Detectable

Speciociliatine Detectable

Speciogynine Detectable

Mitraciliatine Detectable

Paynantheine DetectablePaynantheine-diastereomer? Detectable (identity unknown)

Tramadol Not detectablea

O-desmethyltramadol Detectablea (approximately 9mg/L

or 8 mg/g creatinine)

N-desmethyltramadol Not detectable

a Confirmed in an external reference laboratory by GCMS or LCMS.

[

Fig. 1.LCMS/MS ion chromatograms for the detection of theMitragyna speciosa(Kratom) alkaloids mitragynine, speciociliatine, speciogynine, and mitraciliatine. The 4

diastereomers were identified by standard addition which resulted in altered signal area ratios but no extra signals at different retention times.

T. Arndt et al./ Forensic Science International 208 (2011) 4752 49


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[

Fig. 2. LCMS/MSion chromatograms forthe detection of tramadol(metabolites). Onlysignalswith identical retentiontime, identicalSRM andSRM ratios were considered as

a proof of the presence of the analyte. This was true for O-desmethyltramadol but not for tramadol (no significant signals for SRM 1 and 2 and different retention times for

signal from SRM3) and also not for N-desmethyltramadol (signals with different retention times compared with the standard).

T. Arndt et al./ Forensic Science International 208 (2011) 475250


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[9] A.A. Philipp, D.K. Wissenbach, A.A. Weber, J. Zapp, S.W. Zoerntlein, J. Kanog-sunthornrat, P.G.M. Zweipfennig, H.H. Maurer, Monitoring of Kratom alkaloidsand its metabolites in urine using LC-Linear Ion Trap-MSn and GCMS, Toxichem.Krimtech. 77 (2010) 180.

[10] S. Lu, B.N. Tran, J.L. Nelsen, K.M. Aldous, Quantitative analysis of mitragynine inhuman urine by high performance liquid chromatographytandem mass spec-

trometry, J. Chromatogr. B 877 (2009) 24992505.[11] A.A. Philipp, D.K. Wissenbach, A.A. Weber, J. Zapp, S.W. Zoerntlein, J. Kanog-sunthornrat, H.H.Maurer,Use of liquid chromatographycoupledto low-and highresolution linear ion trap mass spectrometry for studying the metabolism of

paynantheine, analkaloidof theherbal drug Kratomin ratandhumanurine, Anal.Bioanal. Chem. 396 (2010) 23792391.

[12] R.C. Baselt, Disposition of Toxic Drugs and Chemicals in Man, eighth ed.,Biomedical Publications, Foster City, CA, 2008.

[13] S. Dresen, N. Ferreiros, M. Putz, F. Westphal, R. Zimmermann, V. Auwarter,Monitoring of herbal mixtures potentially containing synthetic cannabinoidsas psychoactive compounds, J. Mass Spectrom. 45 (2010) 11861194.

[14] ThermoFisher Scientific Microgenics, Drugs of Abuse CEDIA1 Cross-ReactivityPerformance Guide (Version 2008) incl. Cross-Reactivity Supplementary DataCEDIA1 (Version April 2008).

[15] T. Arndt, U. Claussen, B. Gussregen, S. Schrofel, B. Sturzer, A. Werle, G. Wolf,

Krypton-Konsum als Ursache eines positiven O-Desmethytramadol-Nach-weises im Urin einer Opiatabhangigen in der Entwohnungstherapie, Toxichem.Krimtech. 77 (2010) 2328.

T. Arndt et al./ Forensic Science International 208 (2011) 475252
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kratom alkaloids an historical perspective - arndt - fsi 208 (2011)

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