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MS after Tabernanthe iboga abuse
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Forensic Science International 176 (2008) 58–66 www.elsevier.com/locate/forsciint
Determination of ibogaine and noribogaine in biological fluids and hair by LC–MS/MS after Tabernanthe iboga abuse Iboga alkaloids distribution in a drowning death case Marjorie Che`ze *, Aure´lie Lenoan, Marc Deveaux, Gilbert Pe´pin Laboratoire TOXLAB, 7 rue Jacques Cartier, F-75018 Paris, France Received 4 June 2007; accepted 16 August 2007 Available online 19 November 2007
Abstract Tabernanthe iboga belongs to the Apocynaceae family. In this study, we report the case of a 37-year-old black male working as a security agent in Paris and found dead naked on the beach in Gabon after consumption of iboga. Autopsy revealed a drowning fatality and a myocardial abnormality (myocardial bridging). Samples of blood, urine, bile, gastric content, liver, lungs, vitreous, spleen and hair were taken. Biological fluids were liquid–liquid extracted with saturated NH4Cl pH 9.5 and methylene chloride/isopropanol (95/5, v/v) in presence of clonazepam-d4, used as internal standard. After decontamination with dichloromethane, hair was cut into small pieces then sonicated for 2 h in saturated NH4Cl pH 9.5 before extraction by methylene chloride/isopropanol (95/5, v/v). After evaporation the residues were reconstituted in methanol/ACN/formate buffer pH 3, from which 10 mL were injected into an ODB Uptisphere C18 column (150 mm 2.1 mm, 5 mm) and eluted with a gradient of acetonitrile and formate buffer delivered at a flow rate of 200 mL/min. A Quantum Ultra triple-quadrupole mass spectrometer was used for analyses. Ionization was achieved using electrospray in the positive ionization mode (ESI). For each compound, detection was related to three daughter ions (ibogaine: m/z 311.4 ! 122.1, 174.1 and 188.1; noribogaine: m/z 297.4 ! 122.1, 159.1 and 160.1; clonazepam-d4: m/z 319.9 ! 218.1, 245.1 and 274.1). Ibogaine and noribogaine were detected in all autopsy samples. Hair segmentation was not possible as hair was very short and frizzy. Concentrations of 1.2 and 2.5 ng/mg, respectively were detected. Neither other licit or illicit drugs nor alcohol were found. The presence of ibogaine and noribogaine in all autopsy samples was consistent with the recent absorption of Tabernanthe iboga, which was assumed to be responsible of the drowning fatality. The history of exposure, regarding hair analysis, is discussed. LC–MS/MS appears to be the best method for analyzing complex and poorly volatile alkaloids in autopsy samples and particularly in hair, due to the presence of a nitrogen ring and the relatively low concentrations to be measured. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Iboga; Ibogaine; Noribogaine; Hair; LC–MS/MS
1. Introduction Tabernanthe iboga (Apocynaceae) is an indigenous shrub from central-west Africa, particularly in Gabon, Cameroon and Congo. In small amounts, the root is chewed by Gabonese natives to offset hunger and fatigue, while larger amounts produce profound hallucinations and mental confusion. In Bwiti religious ceremonies, widespread in Gabon, the root bark is pulverized and swallowed with water to produce intense
* Corresponding author. Tel.: +33 1 58 59 28 00; fax: +33 1 58 59 28 01. E-mail address: [email protected] (M. Che`ze). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.08.013
psychoactive effects and it is used to seek information from ancestral spirits in the rite of passage. Iboga shrub reaches about 1.5–2 m, have yellowish or pinkish flowers, and give fruits with sweet pulp without psychoactive alkaloids. The root bark contains about 6% of indole alkaloids: ibogaine (Fig. 1a) is the main one, with tabernanthine, ibogaline, ibogamine, and others less abundant [1]. Ibogaine, an indole alkaloid, was first isolated from Tabernanthe iboga in 1901 by Dybowski and Landrin [2] and independently by Haller and Heckel [3] in the same year. In recent years, it has been increasingly noted for its ability to treat both illicit drugs and alcohol addiction. The use of ibogaine has
M. Che`ze et al. / Forensic Science International 176 (2008) 58–66
Fig. 1. Structures of ibogaine (a) and noribogaine (b).
consequently increased and is not only promoted by some private clinics in the treatment of addiction but also by some active groups in seminars for some kind of ‘‘behavioral rehabilitation’’ and ‘‘psychological experience’’. In addition, ibogaine, either in pure form or as a plant extract, has become available from some lay sources on the Internet. The ibogaine mechanism of action is complex, affecting many different neurotransmitter systems simultaneously [4,5]. The ibogaine weak agonist action on 5HT2A receptor [6,7] may play a role for the hallucinogenic effects [8]. The use of ibogaine in treating substance abuse in human subjects was first proposed by Lotsof in 1985 [9]. A few studies finding that ibogaine helped to diminish opioid withdrawal, morphine self-administration, cocaine self-administration and also to treat alcohol dependence in the rat were published [10– 14]. Very few studies have been done in human subjects and only outside of traditional clinical settings, as a result, until further trials, serious concerns about the safety and efficacy of ibogaine will remain. Ibogaine (12-methoxyibogamine) is metabolized in the human body by cytochrome P450, and the major metabolite is noribogaine (12-hydroxyibogamine; Fig. 1b). Noribogaine is most potent as a serotonin reuptake inhibitor and acts as moderate k- and weak m-opioid receptor full agonist and has therefore also an aspect of an opiate replacement similar to compounds like methadone [15–18]. A single oral dose of 20 mg/kg (1400 mg/70 kg) given to two adult males produced average blood concentrations of ibogaine and noribogaine of 0.91 and 0.67 mg/L, respectively at 4 h, 0.27 and 1.0 mg/L at 12 h, and 0.15 and 0.80 at 19 h [19]. Ibogaine has a blood half-life in humans ranging approximately between 5 and 8 h. Noribogaine throws higher plasma levels than ibogaine and may also be detected for longer periods of time than ibogaine (longer half-life). It is established that ibogaine is largely deposited in fat and in a lesser extent in brain [20]. Because of its fairly low potency at any of its target sites, pure ibogaine is generally used in doses between 5 mg/kg of body weight for minor effect to 30 mg/kg in the cases of strong
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polysubstance addiction. It is unknown whether doses greater than 30 mg/kg in humans produce effects that are therapeutically beneficial, medically risky, or simply prolonged in duration. At therapeutic doses, ibogaine has an active window of 24– 48 h, is often physically and mentally exhausting and produces nausea and ataxia for as long as 12 h, in some cases even longer. Such unpleasant symptoms tend to reduce the attractiveness of ibogaine as a recreational drug. There are 11 documented fatalities that have been associated with ibogaine ingestion [19,21–26] but autopsies have sometimes failed to implicate ibogaine as the sole cause of death due to some patients having significant pre-existing medical problems, and some patients surreptitiously consuming other drugs such as heroin against medical indications during or after ibogaine treatment. Nevertheless, consecutively to the fatalities reported all over the world, the potential of ibogaine abuse and its toxicity, French agency of health (AFSSAPS) has classified Tabernanthe iboga, ibogaine and analogs in the Schedule of illicit substances in March 2007. First determinations of ibogaine and noribogaine in biological fluids by GC–MS have been published between 1995 and 1996 [27–30]. More recently, the determination of ibogaine and noribogaine in blood has been published by Kontrimaviciute et al. by LC-fluorimetry, then in body fluids and tissues by LC–MS [24–26,31]. We present here an original method for the determination of both ibogaine and noribogaine (O-desmethylibogaine) by LC– MS/MS in post-mortem samples focused on hair analysis and its application to a Tabernanthe iboga poisoning. To our knowledge, this is the first report of ibogaine and noribogaine determination in hair. 2. Case report A 37-year-old black male (1.75 m, 85 kg) working in Paris was found dead, naked on a beach in Gabon after consumption of iboga root. Autopsy revealed a drowning fatality and a myocardial abnormality (myocardial bridging). Samples of blood, urine, bile, gastric content, liver, lungs, vitreous, spleen and hair were taken at the autopsy. 3. Materials and methods 3.1. Chemicals and reagents Ammonium formate (minimum 98%) for analysis, formic acid (99%) for analysis, ammonium chloride RPE for analysis, ammonia solution 30% RPE for analysis, methanol RPE for analysis, and acetonitrile HPLC grade RS Plus were purchased from Carlo Erba (Val de Reuil, France). Isopropanol LiChroSolv from Merck and Methylene chloride Chromanorm for HPLC were purchased from VWR International (Fontenay sous bois, France). Clonazepam-d4 was purchased from Cerilliant (Promochem, Molsheim France), ibogaine from Sigma (Saint Quentin Fallavier, France), while noribogaine was obtained from Prof. F.M.M. Bressole (Faculty of pharmacy, University of Montpellier, France). Polytetrafluoroethylene (PTFE) filters (0.2 mm 25 mm) were purchased from Alltech, (Templemars, France).
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3.2. Extraction procedures The determination of ibogaine and noribogaine has been added to our internal screening procedure for the determination of alkaloids in blood [32,33]. Therefore, the chromatographic conditions and extraction procedure were not optimized for these molecules in particular. 3.2.1. Body fluids Biological fluids (1 mL each) were extracted with 1 mL saturated NH4Cl adjusted at pH 9.5 with ammonia 30%, and 5 mL dichloromethane with 5% 2propanol. Ten nanograms of clonazepam-d4 (10 mL at 1 mg/mL) were added as internal standard (IS) before extraction. After 10-min agitation, the tubes were centrifuge for 5 min at 3500 rpm. The organic layer was transferred into glass tubes then evaporated at 45 8C. The residues were reconstituted in 75 mL of methanol/acetonitrile/formate buffer 2 mM pH 3.0 (25/25/50, v/v/v), and transferred into vials for analysis by LC–MS/MS. 3.2.2. Hair Hair was twice decontaminated using methylene chloride (immersion in 10 mL for 5 min). No segmentation was done as hair was too short (about 8 mm length) and frizzy. About 50 mg were sonicated 2 h in 1 mL of saturated ammonium chloride at pH 9.5, in the presence of 5 ng of clonazepam-d4 (5 mL at 1 mg/mL) used as internal standard (IS). After a liquid–liquid extraction with 3 mL of a mixture of methylene chloride/isopropanol (95/5, v/v) the organic layer was filtered with PTFE filters and then evaporated to dryness at 45 8C. The residue was reconstituted in 200 mL of methanol/acetonitrile/formate buffer 2 mM pH 3.0 (25/25/50, v/v/v), before injection.
3.3. LC–MS/MS procedure Liquid chromatography was performed using a Surveyor system (Thermo Fisher Scientific) fitted with an Uptisphere ODB C18 column (150 mm 2.1 mm, 5 mm) Interchim, thermostated at 30 8C. Mobile phase initial conditions were 80% formate buffer 2 mM pH 3.0 and 20% acetonitrile (v/v) delivered at a flow rate of 200 mL/min and held 1 min, then increased to 10/90 up to 10 min and re-equilibrated for 5 min at initial conditions. The injection volume was 10 mL and the total run time was 15 min. The detection was performed on a triple stage quadrupole (TSQ Quantum Ultra, Thermo Fisher Scientific) with an Ion Max ESI probe in positive polarity and selected reaction monitoring (SRM) mode. For the optimization of detection conditions, direct infusions of standard solutions (10 mg/mL) were done using a T connection with mobile phase at a flow of 100 mL/min. To each protonated pseudo-molecular ion [MH]+, three product ions were acquired at a scan time of 0.1 s with a width of 1 a.m.u. and resolutions for Q1 and Q3 at 0.7. The settings of the Ion Max ESI source were as follows: spray voltage, 4900 V; capillary temperature 360 8C; tube lens, near 100 V; sheath gas pressure (spraying), 30 units; auxiliary gas pressure (desolvating), 5 units; ion sweep gas pressure (curtain), 5 units. Collision cell pressure (Q2) was at 1.5 mTorr (0.2 Pa) of argon, and quad MS/MS bias was set Table 1 SRM transitions for ibogaine and noribogaine determination and IS by LC-ESIMS/MS–TSQ Compound
Parent ion (m/z)
Daughter ions (m/z)
Optimized collision energy (V)
Ibogaine
311.4
122.1 174.1 188.1
36 32 35
Noribogaine
297.4
122.1 159.1 160.1
33 45 35
218.1 245.1 274.1
28 35 26
IS (Clonazepam-d4)
319.9
at 3.7. Collision energies were automatically optimized for a maximum detection of each daughter ion (Table 1). The total ion current (TIC) was used for quantification (sum of three daughter ions for each molecule), and the relative abundance of each product ion was checked for identification.
3.4. Method validation As ibogaine determination in autopsy samples is not so usual in forensic laboratories, the method was not fully validated. Partial validation (one day) was done in hair, and in blood for quantification in biological fluids. The limit of detection (LOD) on each matrix was evaluated by decreasing concentrations of ibogaine and noribogaine until a response equivalent to three times the background noise was observed. A variation of less than 10% was expected on relative abundances of daughter ions at the limit of detection. The accuracy accepted for quantification was 100 15% intra-day. 3.4.1. Blood Blood obtained from donors without ibogaine consumption was used for the preparation of calibration points for the validation step. Standard calibration curves were prepared with drug-free blood spiked with methanolic standards solutions of ibogaine and noribogaine freshly prepared, at the following concentrations 0.05, 0.1, 1, 2, 3, 4 and 5 mg/mL, and in the presence of internal standard (IS). Within-batch precision (n = 6) and accuracy (n = 6) were determined using drug-free blood spiked with ibogaine and noribogaine at 1 and 5 mg/mL. Extraction recoveries (n = 6) were determined by comparing the peak area of ibogaine and noribogaine at concentrations of 1 and 5 mg/mL extracted from drug-free blood spiked before extraction with the peak area of ibogaine and noribogaine extracted from drug-free blood spiked after extraction at the same final concentration. The specificity of the method was evaluated by analyzing drug-free blood from six naive subjects. The matrix effect (n = 6) on the ESI response was evaluated by comparing the peak area of the drugs in a methanolic solution directly injected at the equivalent concentration of 1 and 5 mg/mL, and the same amount of the compounds added to preextracted samples. The peak areas of the methanolic solution provide a relative 100% value and the matrix effect was calculated as the percentage of the difference between the two experiments compared. 3.4.2. Hair Drug-free hair samples were obtained from laboratory staff and relatives. Standard calibration curves were prepared with blank hair spiked with drug standards of ibogaine at the following concentrations 0.01, 0.025, 0.1, 0.5, 1, 2, 3 and 4 ng/mg and noribogaine at the following concentrations 0.025, 0.1, 0.5, 1, 2, 3 and 4 ng/mg, in the presence of internal standard (IS). Within-batch precision (%RSD) and accuracy (n = 6) were determined using blank hair spiked with ibogaine and noribogaine at 0.5 and 2 ng/mg. Extraction recoveries (n = 6) were determined by comparing the peak area of ibogaine and noribogaine at concentrations of 0.5 and 2 ng/mg extracted from blank control hair spiked before extraction with the peak area of ibogaine and noribogaine extracted from drug-free hair spiked after extraction at the same final concentration. The specificity of the method was evaluated by analyzing hair from six naive subjects (from the laboratory staff or relatives). The matrix effect (n = 6) on the ESI response was evaluated by comparing the peak area of the drugs in a methanolic solution directly injected at the equivalent concentration of 0.5 and 2 ng/mg, and the same amount of the compounds added to pre-extracted samples.
4. Results and discussion 4.1. Validation results 4.1.1. Blood Curves were linear with equal weighting in the range 0.05– 5 mg/mL for both analytes, with r2 = 0.980 and 0.996 for ibogaine and noribogaine, respectively. Intra-day precision and accuracy and recoveries at 1 and 5 mg/mL are reported in Table 2.
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Table 2 Validation results for ibogaine and noribogaine determination in whole blood by LC-ESI-MS/MS–TSQ Concentrations
Ibogaine Noribogaine
Intra-day precision (%RSD) (n = 6)
Intra-day accuracy (%) (n = 6)
Recoveries (%) (n = 6)
1 mg/mL
5 mg/mL
1 mg/mL
5 mg/mL
1 mg/mL
5 mg/mL
14.4 2.1
5.8 3.9
87.8 86.9
95.3 87.2
33 5 30 4
37 4 33 6
Table 3 Validation results for ibogaine and noribogaine determination in hair by LC-ESI-MS/MS–TSQ Concentrations
Ibogaine Noribogaine
Intra-day precision (%RSD) (n = 6)
Intra-day accuracy (%) (n = 6)
Recoveries (%) (n = 6)
0.5 ng/mg
2 ng/mg
0.5 ng/mg
2 ng/mg
0.5 ng/mg
2 ng/mg
11.1 8.8
5.1 8.5
89.8 93.4
92.2 89.2
100 12 68 1
80 3 75 8
Table 4 Case report values compared to published values after ibogaine poisoning Gender and age
Circumstances
Dosage
Concentrations
References
37-year-old black man
Dead, naked on the beach in Gabon
?
Femoral blood: Ibogaine: 3.3 mg/mL Noribogaine: 4.6 mg/mL Cardiac blood: Ibogaine: 2.4 mg/mL Noribogaine: 2.8 mg/mL Urine: Ibogaine: 7.1 mg/mL Noribogaine: 51.1 mg/mL Kidney exudate: Ibogaine: 1.6 mg/mL Noribogaine: 4.1 mg/mL Liver exudate: Ibogaine: 1.7 mg/mL Noribogaine: 6.2 mg/mL Hair: Ibogaine: 1.2 ng/mg Noribogaine: 2.5 ng/mg
Case reported
Young lady
Dead 20 h post ingestion
29 mg/kg
Cardiac blood: Ibogaine: 0.73 mg/mL Noribogaine: 11 mg/mL
[17]
Man
Dead 24 h post ingestion
85 mg/kg
Ibogaine: 0.36 mg/mL
[19]
48-year-old man
Dead about 53 h post ingestion of iboga root (according to police investigation and forensic data)
?
Femoral blood: Ibogaine: 5.4 mg/mL Noribogaine: 5.6 mg/mL Vena cava blood: Ibogaine: 6.6 mg/mL Noribogaine: 15.5 mg/mL Subclavian blood: Ibogaine: 10.8 mg/mL Noribogaine: 20.8 mg/mL Urine: Ibogaine: 83.3 mg/mL Noribogaine: 21.5 mg/mL Kidney: Ibogaine: 7.6 mg/g Noribogaine: 4.93 mg/g Liver: Ibogaine: 40.5 mg/g Noribogaine: 50.5 mg/g
[22] (partial data)
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The low recoveries were accepted in the field of a screening procedure and regarding the concentration range found after iboga ingestion. They show to be independent of the concentration.
The limits of quantitation (LOQ) (n = 6) were determined at 0.05 mg/mL for both ibogaine and noribogaine with precision of 7.8% and 7.4%, and accuracy of 89.6% and 86.2%, respectively.
Fig. 2. Chromatogram of a drug-free hair spiked with clonazepam-d4 as internal standard (IS). (IS) TIC of daughter ions of internal standard; (a1, a2, a3) daughter ions of ibogaine; (b1, b2, b3) daughter ions of noribogaine.
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The limit of detection (LOD) was determined at 1 ng/mL (S/ N = 3) for both analytes. Matrix effect (n = 6) was 47% (8.9%) and 54% (6.5%) for ibogaine at 1 and 5 mg/mL, and 33% (6.7%)
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and 52% (4%) for noribogaine at the same concentrations. These results could be improved by a higher proportion of methanol in the reconstitution solvent, but it reduces the peak symmetry, particularly for noribogaine with less retention.
Fig. 3. Chromatogram of a drug-free hair spiked with clonazepam-d4 as internal standard (IS), ibogaine and noribogaine at 0.1 ng/mg. (IS) TIC of daughter ions of internal standard; (a1, a2, a3) daughter ions of ibogaine; (b1, b2, b3) daughter ions of noribogaine.
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4.1.2. Hair Curves were linear with equal weighting in the range 0.01– 4 ng/mg for ibogaine and in the range 0.025–4 ng/mg for noribogaine, with r2 = 0.998 and 0.994, respectively. Intra-day precision and accuracy and recoveries at 0.5 and 2 ng/mg are reported in Table 3.
The chromatogram obtained after extraction of a drug-free hair and a drug-free hair spiked with both ibogaine and noribogaine at a final concentration of 0.1 ng/mg each are shown Figs. 2 and 3, respectively. LOD was 5 pg/mg (S/N = 3) with a LOQ (n = 6) of 10 pg/ mg (precision 3.9%, accuracy 86.5%) for ibogaine, and of
Fig. 4. Chromatogram of the hair extract from the case report with clonazepam-d4 as internal standard (IS) and ibogaine and noribogaine at 1.2 and 2.5 ng/mg, respectively. (IS) TIC of daughter ions of internal standard; (a1, a2, a3) daughter ions of ibogaine; (b1, b2, b3) daughter ions of noribogaine.
M. Che`ze et al. / Forensic Science International 176 (2008) 58–66
25 pg/mg (precision 10.6%, accuracy 91.4%) for noribogaine. Under the chromatographic conditions used, there was no significant interference with analytes by chemicals or any extractable endogenous materials present in hair (n = 6). Matrix effect (n = 6) was 30% (13.9%) and 27% (7.1%) for ibogaine at 0.5 and 2 ng/mg, and 31% (7.7%) and 17% (9.9%) for noribogaine at the same concentrations. 4.2. Results from case report Ibogaine and noribogaine were detected in all autopsy samples (Table 4). This confirms their large distribution throughout the body [25]. Neither other licit or illicit drugs nor alcohol were found in blood and urine. The large amounts of ibogaine and noribogaine found in all body fluids were consistent with the relatively recent absorption of Tabernanthe iboga, which was assumed to be responsible for the drowning fatality. Considering the blood ratio of ibogaine versus noribogaine, the rapid decreased of ibogaine in blood, and the previous published data, it can be assumed that the deceased had ingested iboga in the latest 35 h at least. This could be confirmed by the ratio of ibogaine versus noribogaine in urine. The elevated concentrations found compared to other reported fatalities (Table 4) and pharmacokinetic study [19] shows that he must have taken a large quantity of root bark of iboga. His myocardial abnormality (seen at the autopsy) may have also played a role in his fatality. Actually, serious toxic effects (even at therapeutic doses) including slowing of the heart, fall in blood pressure, heart failure, seizures, paralysis, and respiratory arrest have been reported following therapy with ibogaine. Furthermore, deaths related to cardiac arrhythmia have been reported in several individuals taking ibogaine [21]. Moreover, the stimulating amphetamine-like effect of ibogaine could have lead in surpassing one’s physical limits during an effort, i.e. swimming, and enhanced the cardiotoxic effects. Hair concentrations for ibogaine and noribogaine were 1.2 and 2.5 ng/mg, respectively (Fig. 4). We did not know if the subject had previously ingested iboga root once or several times. Hair was too short (about 8 mm) and frizzy for hair segmentation to detect repetitive exposure formally. Interpretation of these results is a bit tricky because according to our experience on hair analysis, we think that during the consumption of iboga over a few days of initiation rite and perspiring, iboga alkaloids may be partly incorporated by sweat and sebum in very short and frizzy hair. Moreover, hair taken at autopsy may be partially pulled out, particularly after a drowning fatality. However, since it usually takes at least about one week until hair grows to the skin surface, past ingestion of iboga roots at least one week before death can be assumed. 5. Conclusion Iboga seems to be the latest fashion among psychodysleptics used in sects and has to be searchable in all kind of samples by toxicologists. The presented method is rapid, specific and
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sensitive enough for the determination of ibogaine and noribogaine in autopsy samples and hair. To our knowledge, this is the first report of ibogaine and noribogaine determination in hair. LC–MS or MS/MS method appears to be the best way for screening alkaloids in autopsy samples and particularly in hair, due to the presence of a nitrogen ring and the relatively low concentrations to be measured [24–26,32–34]. Serious toxic effects of iboga alkaloids even at therapeutic doses are related to deaths, mainly by cardiac problems (arrhythmia, heart failure, seizures, slowing of the heart) and the case reported in this paper is a good illustration of that risk. Acknowledgements The authors gratefully acknowledge Ms Prof. F.M.M. Bressole (Faculty of pharmacy, University of Montpellier, France) for providing noribogaine standard, and Ms Prof. Lecomte (Director of the Medico Legal Institute of Paris, France) for providing autopsy samples. References [1] J. Bruneton, Pharmacognosie, phytochimie, plantes me´dicinales, third ed., Tec et Doc dicales internationales, Paris, 1999, p. 1027. [2] J. Dybowski, E. Landrin, Sur l’Iboga, sur ses proprie´te´s excitantes, sa composition et sur l’alcaloı¨de nouveau qu’il renferme, C. R. Acad. Sci. 133 (1901) 748–750. [3] A. Haller, E. Heckel, Sur l’ibogaine, principe actif d’une plante du genre Tabernæmontana native du Congo, C. R. Acad. Sci. 133 (1901) 850–853. [4] P. Popik, P. Skolnick, Pharmacology of ibogaine and ibogaine-related alkaloids, in: The Alkaloids, 52th, Academic Press, San Diego, 1998, pp. 197–231. [5] K.R. Alper, Ibogaine: a review, Alkaloids Chem. Biol. 56 (2001) 1–38. [6] S. Helsley, D. Fiorella, R.A. Rabin, J.C. Winter, Behavioral and biochemical evidence for a nonessential 5-HT2A component of the ibogaineinduced discriminative stimulus, Pharmacol. Biochem. Behav. 59 (1998) 419–425. [7] S.D. Glick, I.S. Mainsonneuve, Mechanisms of antiaddictive actions of ibogaine, Ann. N.Y. Acad. Sci. 844 (1998) 214–226. [8] D. Wei, I.M. Maisonneuve, M.E. Kuehne, S.D. Glick, Acute iboga alkaloid effects on extracellular serotonin (5-HT) levels in nucleus accumbens and striatum in rats, Brain Res. 800 (1998) 260–268. [9] H. Lotsof, Rapid method for interrupting the narcotic addiction syndrome, U.S. Patent 4 499 096 (1985). [10] E.D. Dzoljic, C.D. Kaplan, M.R. Dzoljic, Effect of ibogaine on naloxoneprecipitated withdrawal syndrome in chronic morphine-dependent rats, Arch. Int. Pharmacodyn. Ther. 294 (1988) 64–70. [11] S.D. Glick, K. Rossman, S. Steindorf, I.M. Maisonneuve, J.N. Carlson, Effects and aftereffects of ibogaine on morphine self-administration in rats, Eur. J. Pharmacol. 195 (1991) 341–345. [12] S.L.T. Cappendijk, M.R. Dzoljic, Inhibitory effects of ibogaine on cocaine self-administration in rats, Eur. J. Pharmacol. 241 (1993) 261–265. [13] A. Rezvani, D. Overstreet, Y. Lee, Attenuation of alcohol intake by ibogaine in three strains of alcohol preferring rats, Pharmacol. Biochem. Behav. 52 (1995) 615–620. [14] K.R. Alper, H.S. Lotsof, G.M. Frenken, D.J. Luciano, J. Bastiaans, Treatment of acute opioid withdrawal with ibogaine, Am. J. Addict. 8 (1999) 234–242. [15] M.H. Baumann, J.P. Pablo, S.F. Ali, R.B. Rothman, D.C. Mash, Comparative neuropharmacology of ibogaine and its O-desmethyl metabolite, noribogaine, Alkaloids Chem. Biol. 56 (2001) 79–113.
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[16] C. Zubaran, M. Shoaib, I.P. Stolerman, J. Pablo, D.C. Mash, Noribogaine generalization to the ibogaine stimulus: correlation with noribogaine concentration in rat brain, Neuropsychopharmacology 21 (1999) 119–126. [17] M.H. Baumann, R.B. Rothman, J.P. Pablo, D.C. Mash, In vivo neurobiological effects of ibogaine and its O-desmethyl metabolite, 12-hydroxyibogamine (noribogaine), in rats, J. Pharmacol. Exp. Ther. 297 (2001) 531– 539. [18] D.C. Mash, J.K. Staley, M.H. Baumann, Identification of a primary metabolite of ibogaine that targets serotonin transporters and elevates serotonin, Life Sci. 57 (1995) 45–50. [19] M.J. Bogusz, H. Althoff, D.C. Mash, W.L. Hearn, Ibogaine-associated death in a female heroin addict: forensic and toxicological aspects, in: Presented at the Annual Meeting of the Society of Forensic Toxicologists, Albuquerque, NM, October 8, 1998. [20] L.B. Hough, S.M. Pearl, S.D. Glick, Tissue distribution of ibogaine after intraperitoneal and subcutaneous administration, Life Sci. 58 (1996) 119– 122. [21] U. Maas, S. Strubelt, Fatalities after taking ibogaine in addiction treatment could be related to sudden cardiac death caused by autonomic dysfunction, Med. Hypotheses 67 (2006) 960–964. [22] J. Cienki, D. Mash, W. Hearn, Ibogaine fatalities, Clin. Tox. 39 (2001) 547. [23] Internet: table of ibogaine fatalities – http://www.myeboga.com/fatalities. html consulted at 03/30/2007. [24] V. Kontrimaviciute, J.C. Mathieu-Daude´, O. Mathieu, H. Breton, T. Casper, E. Baccino, F.M.M. Bressolle, Ibogaine and noribogaine determination in whole blood, plasma and urine. Application to an intoxication by Tabernanthe iboga root, Ann. Toxicol. Anal. 18 (2006) 162–163 (in French). [25] V. Kontrimaviciute, O. Mathieu, J.C. Mathieu-Daude´, P. Vainauskas, T. Casper, E. Baccino, F.M.M. Bressolle, Distribution of ibogaine and noribogaine in a man following a poisoning involving root bark of the Tabernanthe iboga shrub, J. Anal. Toxicol. 30 (2006) 434–440.
[26] V. Kontrimaviciute, H. Breton, F. Barnay, J.C. Mathieu-Daude´, F.M.M. Bressolle, Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 843 (2006) 131–141. [27] C.A. Gallagher, L.B. Hough, S.M. Keefner, A. Seyed-Mozaffari, S. Archer, S.D. Glick, Identification and quantification of the indole alkaloid ibogaine in biological samples by gas chromatography–mass spectrometry, Biochem. Pharmacol. 49 (1995) 73–79. [28] W.L. Hearn, J. Pablo, G.W. Hime, D.C. Mash, Identification and quantitation of ibogaine and an o-demethylated metabolite in brain and biological fluids using gas chromatography–mass spectrometry, J. Anal. Toxicol. 19 (1995) 427–434. [29] M.E. Alburges, R.L. Foltz, D.E. Moody, Determination of ibogaine and 12hydroxy-ibogamine in plasma by gas chromatography-positive ion chemical ionization-mass spectrometry, J. Anal. Toxicol. 19 (1995) 381–386. [30] F.R. Ley, A.R. Jeffcoat, B.F. Thomas, Determination of ibogaine in plasma by gas chromatography-chemical ionization mass spectrometry, J. Chromatogr. A 723 (1996) 101–109. [31] V. Kontrimaviciute, M. Larroque, V. Briedis, D. Margout, F. Bressolle, Quantitation of ibogaine and 12-hydroxybogamine in human plasma by liquid chromatography with fluorimetric detection, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 822 (2005) 285–293. [32] Y. Gaillard, G. Pe´pin, Poisoning by plant material: review of human cases and analytical determination of main toxins by high performance liquid chromatography-(tandem) mass spectrometry, J. Chromatogr. B 733 (1999) 181–229. [33] M. Che`ze, M. Deveaux, G. Pe´pin, Identification and quantification of plant poisons by liquid chromatography-tandem mass spectrometry (LC–MS/ MS). Bibliographic overview and Toxlab laboratory’s experience, Ann. Toxicol. Anal. 13 (2005) 43–55 (in French). [34] P. Kintz, M. Villain, Y. Barguil, J.Y. Charlot, V. Cirimele, Testing for atropine and scopolamine in hair by LC–MS/MS after Datura inoxia abuse, J. Anal. Toxicol. 30 (2006) 454–457.
Forensic Science International 176 (2008) 58–66 www.elsevier.com/locate/forsciint
Determination of ibogaine and noribogaine in biological fluids and hair by LC–MS/MS after Tabernanthe iboga abuse Iboga alkaloids distribution in a drowning death case Marjorie Che`ze *, Aure´lie Lenoan, Marc Deveaux, Gilbert Pe´pin Laboratoire TOXLAB, 7 rue Jacques Cartier, F-75018 Paris, France Received 4 June 2007; accepted 16 August 2007 Available online 19 November 2007
Abstract Tabernanthe iboga belongs to the Apocynaceae family. In this study, we report the case of a 37-year-old black male working as a security agent in Paris and found dead naked on the beach in Gabon after consumption of iboga. Autopsy revealed a drowning fatality and a myocardial abnormality (myocardial bridging). Samples of blood, urine, bile, gastric content, liver, lungs, vitreous, spleen and hair were taken. Biological fluids were liquid–liquid extracted with saturated NH4Cl pH 9.5 and methylene chloride/isopropanol (95/5, v/v) in presence of clonazepam-d4, used as internal standard. After decontamination with dichloromethane, hair was cut into small pieces then sonicated for 2 h in saturated NH4Cl pH 9.5 before extraction by methylene chloride/isopropanol (95/5, v/v). After evaporation the residues were reconstituted in methanol/ACN/formate buffer pH 3, from which 10 mL were injected into an ODB Uptisphere C18 column (150 mm 2.1 mm, 5 mm) and eluted with a gradient of acetonitrile and formate buffer delivered at a flow rate of 200 mL/min. A Quantum Ultra triple-quadrupole mass spectrometer was used for analyses. Ionization was achieved using electrospray in the positive ionization mode (ESI). For each compound, detection was related to three daughter ions (ibogaine: m/z 311.4 ! 122.1, 174.1 and 188.1; noribogaine: m/z 297.4 ! 122.1, 159.1 and 160.1; clonazepam-d4: m/z 319.9 ! 218.1, 245.1 and 274.1). Ibogaine and noribogaine were detected in all autopsy samples. Hair segmentation was not possible as hair was very short and frizzy. Concentrations of 1.2 and 2.5 ng/mg, respectively were detected. Neither other licit or illicit drugs nor alcohol were found. The presence of ibogaine and noribogaine in all autopsy samples was consistent with the recent absorption of Tabernanthe iboga, which was assumed to be responsible of the drowning fatality. The history of exposure, regarding hair analysis, is discussed. LC–MS/MS appears to be the best method for analyzing complex and poorly volatile alkaloids in autopsy samples and particularly in hair, due to the presence of a nitrogen ring and the relatively low concentrations to be measured. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Iboga; Ibogaine; Noribogaine; Hair; LC–MS/MS
1. Introduction Tabernanthe iboga (Apocynaceae) is an indigenous shrub from central-west Africa, particularly in Gabon, Cameroon and Congo. In small amounts, the root is chewed by Gabonese natives to offset hunger and fatigue, while larger amounts produce profound hallucinations and mental confusion. In Bwiti religious ceremonies, widespread in Gabon, the root bark is pulverized and swallowed with water to produce intense
* Corresponding author. Tel.: +33 1 58 59 28 00; fax: +33 1 58 59 28 01. E-mail address: [email protected] (M. Che`ze). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.08.013
psychoactive effects and it is used to seek information from ancestral spirits in the rite of passage. Iboga shrub reaches about 1.5–2 m, have yellowish or pinkish flowers, and give fruits with sweet pulp without psychoactive alkaloids. The root bark contains about 6% of indole alkaloids: ibogaine (Fig. 1a) is the main one, with tabernanthine, ibogaline, ibogamine, and others less abundant [1]. Ibogaine, an indole alkaloid, was first isolated from Tabernanthe iboga in 1901 by Dybowski and Landrin [2] and independently by Haller and Heckel [3] in the same year. In recent years, it has been increasingly noted for its ability to treat both illicit drugs and alcohol addiction. The use of ibogaine has
M. Che`ze et al. / Forensic Science International 176 (2008) 58–66
Fig. 1. Structures of ibogaine (a) and noribogaine (b).
consequently increased and is not only promoted by some private clinics in the treatment of addiction but also by some active groups in seminars for some kind of ‘‘behavioral rehabilitation’’ and ‘‘psychological experience’’. In addition, ibogaine, either in pure form or as a plant extract, has become available from some lay sources on the Internet. The ibogaine mechanism of action is complex, affecting many different neurotransmitter systems simultaneously [4,5]. The ibogaine weak agonist action on 5HT2A receptor [6,7] may play a role for the hallucinogenic effects [8]. The use of ibogaine in treating substance abuse in human subjects was first proposed by Lotsof in 1985 [9]. A few studies finding that ibogaine helped to diminish opioid withdrawal, morphine self-administration, cocaine self-administration and also to treat alcohol dependence in the rat were published [10– 14]. Very few studies have been done in human subjects and only outside of traditional clinical settings, as a result, until further trials, serious concerns about the safety and efficacy of ibogaine will remain. Ibogaine (12-methoxyibogamine) is metabolized in the human body by cytochrome P450, and the major metabolite is noribogaine (12-hydroxyibogamine; Fig. 1b). Noribogaine is most potent as a serotonin reuptake inhibitor and acts as moderate k- and weak m-opioid receptor full agonist and has therefore also an aspect of an opiate replacement similar to compounds like methadone [15–18]. A single oral dose of 20 mg/kg (1400 mg/70 kg) given to two adult males produced average blood concentrations of ibogaine and noribogaine of 0.91 and 0.67 mg/L, respectively at 4 h, 0.27 and 1.0 mg/L at 12 h, and 0.15 and 0.80 at 19 h [19]. Ibogaine has a blood half-life in humans ranging approximately between 5 and 8 h. Noribogaine throws higher plasma levels than ibogaine and may also be detected for longer periods of time than ibogaine (longer half-life). It is established that ibogaine is largely deposited in fat and in a lesser extent in brain [20]. Because of its fairly low potency at any of its target sites, pure ibogaine is generally used in doses between 5 mg/kg of body weight for minor effect to 30 mg/kg in the cases of strong
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polysubstance addiction. It is unknown whether doses greater than 30 mg/kg in humans produce effects that are therapeutically beneficial, medically risky, or simply prolonged in duration. At therapeutic doses, ibogaine has an active window of 24– 48 h, is often physically and mentally exhausting and produces nausea and ataxia for as long as 12 h, in some cases even longer. Such unpleasant symptoms tend to reduce the attractiveness of ibogaine as a recreational drug. There are 11 documented fatalities that have been associated with ibogaine ingestion [19,21–26] but autopsies have sometimes failed to implicate ibogaine as the sole cause of death due to some patients having significant pre-existing medical problems, and some patients surreptitiously consuming other drugs such as heroin against medical indications during or after ibogaine treatment. Nevertheless, consecutively to the fatalities reported all over the world, the potential of ibogaine abuse and its toxicity, French agency of health (AFSSAPS) has classified Tabernanthe iboga, ibogaine and analogs in the Schedule of illicit substances in March 2007. First determinations of ibogaine and noribogaine in biological fluids by GC–MS have been published between 1995 and 1996 [27–30]. More recently, the determination of ibogaine and noribogaine in blood has been published by Kontrimaviciute et al. by LC-fluorimetry, then in body fluids and tissues by LC–MS [24–26,31]. We present here an original method for the determination of both ibogaine and noribogaine (O-desmethylibogaine) by LC– MS/MS in post-mortem samples focused on hair analysis and its application to a Tabernanthe iboga poisoning. To our knowledge, this is the first report of ibogaine and noribogaine determination in hair. 2. Case report A 37-year-old black male (1.75 m, 85 kg) working in Paris was found dead, naked on a beach in Gabon after consumption of iboga root. Autopsy revealed a drowning fatality and a myocardial abnormality (myocardial bridging). Samples of blood, urine, bile, gastric content, liver, lungs, vitreous, spleen and hair were taken at the autopsy. 3. Materials and methods 3.1. Chemicals and reagents Ammonium formate (minimum 98%) for analysis, formic acid (99%) for analysis, ammonium chloride RPE for analysis, ammonia solution 30% RPE for analysis, methanol RPE for analysis, and acetonitrile HPLC grade RS Plus were purchased from Carlo Erba (Val de Reuil, France). Isopropanol LiChroSolv from Merck and Methylene chloride Chromanorm for HPLC were purchased from VWR International (Fontenay sous bois, France). Clonazepam-d4 was purchased from Cerilliant (Promochem, Molsheim France), ibogaine from Sigma (Saint Quentin Fallavier, France), while noribogaine was obtained from Prof. F.M.M. Bressole (Faculty of pharmacy, University of Montpellier, France). Polytetrafluoroethylene (PTFE) filters (0.2 mm 25 mm) were purchased from Alltech, (Templemars, France).
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3.2. Extraction procedures The determination of ibogaine and noribogaine has been added to our internal screening procedure for the determination of alkaloids in blood [32,33]. Therefore, the chromatographic conditions and extraction procedure were not optimized for these molecules in particular. 3.2.1. Body fluids Biological fluids (1 mL each) were extracted with 1 mL saturated NH4Cl adjusted at pH 9.5 with ammonia 30%, and 5 mL dichloromethane with 5% 2propanol. Ten nanograms of clonazepam-d4 (10 mL at 1 mg/mL) were added as internal standard (IS) before extraction. After 10-min agitation, the tubes were centrifuge for 5 min at 3500 rpm. The organic layer was transferred into glass tubes then evaporated at 45 8C. The residues were reconstituted in 75 mL of methanol/acetonitrile/formate buffer 2 mM pH 3.0 (25/25/50, v/v/v), and transferred into vials for analysis by LC–MS/MS. 3.2.2. Hair Hair was twice decontaminated using methylene chloride (immersion in 10 mL for 5 min). No segmentation was done as hair was too short (about 8 mm length) and frizzy. About 50 mg were sonicated 2 h in 1 mL of saturated ammonium chloride at pH 9.5, in the presence of 5 ng of clonazepam-d4 (5 mL at 1 mg/mL) used as internal standard (IS). After a liquid–liquid extraction with 3 mL of a mixture of methylene chloride/isopropanol (95/5, v/v) the organic layer was filtered with PTFE filters and then evaporated to dryness at 45 8C. The residue was reconstituted in 200 mL of methanol/acetonitrile/formate buffer 2 mM pH 3.0 (25/25/50, v/v/v), before injection.
3.3. LC–MS/MS procedure Liquid chromatography was performed using a Surveyor system (Thermo Fisher Scientific) fitted with an Uptisphere ODB C18 column (150 mm 2.1 mm, 5 mm) Interchim, thermostated at 30 8C. Mobile phase initial conditions were 80% formate buffer 2 mM pH 3.0 and 20% acetonitrile (v/v) delivered at a flow rate of 200 mL/min and held 1 min, then increased to 10/90 up to 10 min and re-equilibrated for 5 min at initial conditions. The injection volume was 10 mL and the total run time was 15 min. The detection was performed on a triple stage quadrupole (TSQ Quantum Ultra, Thermo Fisher Scientific) with an Ion Max ESI probe in positive polarity and selected reaction monitoring (SRM) mode. For the optimization of detection conditions, direct infusions of standard solutions (10 mg/mL) were done using a T connection with mobile phase at a flow of 100 mL/min. To each protonated pseudo-molecular ion [MH]+, three product ions were acquired at a scan time of 0.1 s with a width of 1 a.m.u. and resolutions for Q1 and Q3 at 0.7. The settings of the Ion Max ESI source were as follows: spray voltage, 4900 V; capillary temperature 360 8C; tube lens, near 100 V; sheath gas pressure (spraying), 30 units; auxiliary gas pressure (desolvating), 5 units; ion sweep gas pressure (curtain), 5 units. Collision cell pressure (Q2) was at 1.5 mTorr (0.2 Pa) of argon, and quad MS/MS bias was set Table 1 SRM transitions for ibogaine and noribogaine determination and IS by LC-ESIMS/MS–TSQ Compound
Parent ion (m/z)
Daughter ions (m/z)
Optimized collision energy (V)
Ibogaine
311.4
122.1 174.1 188.1
36 32 35
Noribogaine
297.4
122.1 159.1 160.1
33 45 35
218.1 245.1 274.1
28 35 26
IS (Clonazepam-d4)
319.9
at 3.7. Collision energies were automatically optimized for a maximum detection of each daughter ion (Table 1). The total ion current (TIC) was used for quantification (sum of three daughter ions for each molecule), and the relative abundance of each product ion was checked for identification.
3.4. Method validation As ibogaine determination in autopsy samples is not so usual in forensic laboratories, the method was not fully validated. Partial validation (one day) was done in hair, and in blood for quantification in biological fluids. The limit of detection (LOD) on each matrix was evaluated by decreasing concentrations of ibogaine and noribogaine until a response equivalent to three times the background noise was observed. A variation of less than 10% was expected on relative abundances of daughter ions at the limit of detection. The accuracy accepted for quantification was 100 15% intra-day. 3.4.1. Blood Blood obtained from donors without ibogaine consumption was used for the preparation of calibration points for the validation step. Standard calibration curves were prepared with drug-free blood spiked with methanolic standards solutions of ibogaine and noribogaine freshly prepared, at the following concentrations 0.05, 0.1, 1, 2, 3, 4 and 5 mg/mL, and in the presence of internal standard (IS). Within-batch precision (n = 6) and accuracy (n = 6) were determined using drug-free blood spiked with ibogaine and noribogaine at 1 and 5 mg/mL. Extraction recoveries (n = 6) were determined by comparing the peak area of ibogaine and noribogaine at concentrations of 1 and 5 mg/mL extracted from drug-free blood spiked before extraction with the peak area of ibogaine and noribogaine extracted from drug-free blood spiked after extraction at the same final concentration. The specificity of the method was evaluated by analyzing drug-free blood from six naive subjects. The matrix effect (n = 6) on the ESI response was evaluated by comparing the peak area of the drugs in a methanolic solution directly injected at the equivalent concentration of 1 and 5 mg/mL, and the same amount of the compounds added to preextracted samples. The peak areas of the methanolic solution provide a relative 100% value and the matrix effect was calculated as the percentage of the difference between the two experiments compared. 3.4.2. Hair Drug-free hair samples were obtained from laboratory staff and relatives. Standard calibration curves were prepared with blank hair spiked with drug standards of ibogaine at the following concentrations 0.01, 0.025, 0.1, 0.5, 1, 2, 3 and 4 ng/mg and noribogaine at the following concentrations 0.025, 0.1, 0.5, 1, 2, 3 and 4 ng/mg, in the presence of internal standard (IS). Within-batch precision (%RSD) and accuracy (n = 6) were determined using blank hair spiked with ibogaine and noribogaine at 0.5 and 2 ng/mg. Extraction recoveries (n = 6) were determined by comparing the peak area of ibogaine and noribogaine at concentrations of 0.5 and 2 ng/mg extracted from blank control hair spiked before extraction with the peak area of ibogaine and noribogaine extracted from drug-free hair spiked after extraction at the same final concentration. The specificity of the method was evaluated by analyzing hair from six naive subjects (from the laboratory staff or relatives). The matrix effect (n = 6) on the ESI response was evaluated by comparing the peak area of the drugs in a methanolic solution directly injected at the equivalent concentration of 0.5 and 2 ng/mg, and the same amount of the compounds added to pre-extracted samples.
4. Results and discussion 4.1. Validation results 4.1.1. Blood Curves were linear with equal weighting in the range 0.05– 5 mg/mL for both analytes, with r2 = 0.980 and 0.996 for ibogaine and noribogaine, respectively. Intra-day precision and accuracy and recoveries at 1 and 5 mg/mL are reported in Table 2.
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Table 2 Validation results for ibogaine and noribogaine determination in whole blood by LC-ESI-MS/MS–TSQ Concentrations
Ibogaine Noribogaine
Intra-day precision (%RSD) (n = 6)
Intra-day accuracy (%) (n = 6)
Recoveries (%) (n = 6)
1 mg/mL
5 mg/mL
1 mg/mL
5 mg/mL
1 mg/mL
5 mg/mL
14.4 2.1
5.8 3.9
87.8 86.9
95.3 87.2
33 5 30 4
37 4 33 6
Table 3 Validation results for ibogaine and noribogaine determination in hair by LC-ESI-MS/MS–TSQ Concentrations
Ibogaine Noribogaine
Intra-day precision (%RSD) (n = 6)
Intra-day accuracy (%) (n = 6)
Recoveries (%) (n = 6)
0.5 ng/mg
2 ng/mg
0.5 ng/mg
2 ng/mg
0.5 ng/mg
2 ng/mg
11.1 8.8
5.1 8.5
89.8 93.4
92.2 89.2
100 12 68 1
80 3 75 8
Table 4 Case report values compared to published values after ibogaine poisoning Gender and age
Circumstances
Dosage
Concentrations
References
37-year-old black man
Dead, naked on the beach in Gabon
?
Femoral blood: Ibogaine: 3.3 mg/mL Noribogaine: 4.6 mg/mL Cardiac blood: Ibogaine: 2.4 mg/mL Noribogaine: 2.8 mg/mL Urine: Ibogaine: 7.1 mg/mL Noribogaine: 51.1 mg/mL Kidney exudate: Ibogaine: 1.6 mg/mL Noribogaine: 4.1 mg/mL Liver exudate: Ibogaine: 1.7 mg/mL Noribogaine: 6.2 mg/mL Hair: Ibogaine: 1.2 ng/mg Noribogaine: 2.5 ng/mg
Case reported
Young lady
Dead 20 h post ingestion
29 mg/kg
Cardiac blood: Ibogaine: 0.73 mg/mL Noribogaine: 11 mg/mL
[17]
Man
Dead 24 h post ingestion
85 mg/kg
Ibogaine: 0.36 mg/mL
[19]
48-year-old man
Dead about 53 h post ingestion of iboga root (according to police investigation and forensic data)
?
Femoral blood: Ibogaine: 5.4 mg/mL Noribogaine: 5.6 mg/mL Vena cava blood: Ibogaine: 6.6 mg/mL Noribogaine: 15.5 mg/mL Subclavian blood: Ibogaine: 10.8 mg/mL Noribogaine: 20.8 mg/mL Urine: Ibogaine: 83.3 mg/mL Noribogaine: 21.5 mg/mL Kidney: Ibogaine: 7.6 mg/g Noribogaine: 4.93 mg/g Liver: Ibogaine: 40.5 mg/g Noribogaine: 50.5 mg/g
[22] (partial data)
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The low recoveries were accepted in the field of a screening procedure and regarding the concentration range found after iboga ingestion. They show to be independent of the concentration.
The limits of quantitation (LOQ) (n = 6) were determined at 0.05 mg/mL for both ibogaine and noribogaine with precision of 7.8% and 7.4%, and accuracy of 89.6% and 86.2%, respectively.
Fig. 2. Chromatogram of a drug-free hair spiked with clonazepam-d4 as internal standard (IS). (IS) TIC of daughter ions of internal standard; (a1, a2, a3) daughter ions of ibogaine; (b1, b2, b3) daughter ions of noribogaine.
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The limit of detection (LOD) was determined at 1 ng/mL (S/ N = 3) for both analytes. Matrix effect (n = 6) was 47% (8.9%) and 54% (6.5%) for ibogaine at 1 and 5 mg/mL, and 33% (6.7%)
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and 52% (4%) for noribogaine at the same concentrations. These results could be improved by a higher proportion of methanol in the reconstitution solvent, but it reduces the peak symmetry, particularly for noribogaine with less retention.
Fig. 3. Chromatogram of a drug-free hair spiked with clonazepam-d4 as internal standard (IS), ibogaine and noribogaine at 0.1 ng/mg. (IS) TIC of daughter ions of internal standard; (a1, a2, a3) daughter ions of ibogaine; (b1, b2, b3) daughter ions of noribogaine.
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4.1.2. Hair Curves were linear with equal weighting in the range 0.01– 4 ng/mg for ibogaine and in the range 0.025–4 ng/mg for noribogaine, with r2 = 0.998 and 0.994, respectively. Intra-day precision and accuracy and recoveries at 0.5 and 2 ng/mg are reported in Table 3.
The chromatogram obtained after extraction of a drug-free hair and a drug-free hair spiked with both ibogaine and noribogaine at a final concentration of 0.1 ng/mg each are shown Figs. 2 and 3, respectively. LOD was 5 pg/mg (S/N = 3) with a LOQ (n = 6) of 10 pg/ mg (precision 3.9%, accuracy 86.5%) for ibogaine, and of
Fig. 4. Chromatogram of the hair extract from the case report with clonazepam-d4 as internal standard (IS) and ibogaine and noribogaine at 1.2 and 2.5 ng/mg, respectively. (IS) TIC of daughter ions of internal standard; (a1, a2, a3) daughter ions of ibogaine; (b1, b2, b3) daughter ions of noribogaine.
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25 pg/mg (precision 10.6%, accuracy 91.4%) for noribogaine. Under the chromatographic conditions used, there was no significant interference with analytes by chemicals or any extractable endogenous materials present in hair (n = 6). Matrix effect (n = 6) was 30% (13.9%) and 27% (7.1%) for ibogaine at 0.5 and 2 ng/mg, and 31% (7.7%) and 17% (9.9%) for noribogaine at the same concentrations. 4.2. Results from case report Ibogaine and noribogaine were detected in all autopsy samples (Table 4). This confirms their large distribution throughout the body [25]. Neither other licit or illicit drugs nor alcohol were found in blood and urine. The large amounts of ibogaine and noribogaine found in all body fluids were consistent with the relatively recent absorption of Tabernanthe iboga, which was assumed to be responsible for the drowning fatality. Considering the blood ratio of ibogaine versus noribogaine, the rapid decreased of ibogaine in blood, and the previous published data, it can be assumed that the deceased had ingested iboga in the latest 35 h at least. This could be confirmed by the ratio of ibogaine versus noribogaine in urine. The elevated concentrations found compared to other reported fatalities (Table 4) and pharmacokinetic study [19] shows that he must have taken a large quantity of root bark of iboga. His myocardial abnormality (seen at the autopsy) may have also played a role in his fatality. Actually, serious toxic effects (even at therapeutic doses) including slowing of the heart, fall in blood pressure, heart failure, seizures, paralysis, and respiratory arrest have been reported following therapy with ibogaine. Furthermore, deaths related to cardiac arrhythmia have been reported in several individuals taking ibogaine [21]. Moreover, the stimulating amphetamine-like effect of ibogaine could have lead in surpassing one’s physical limits during an effort, i.e. swimming, and enhanced the cardiotoxic effects. Hair concentrations for ibogaine and noribogaine were 1.2 and 2.5 ng/mg, respectively (Fig. 4). We did not know if the subject had previously ingested iboga root once or several times. Hair was too short (about 8 mm) and frizzy for hair segmentation to detect repetitive exposure formally. Interpretation of these results is a bit tricky because according to our experience on hair analysis, we think that during the consumption of iboga over a few days of initiation rite and perspiring, iboga alkaloids may be partly incorporated by sweat and sebum in very short and frizzy hair. Moreover, hair taken at autopsy may be partially pulled out, particularly after a drowning fatality. However, since it usually takes at least about one week until hair grows to the skin surface, past ingestion of iboga roots at least one week before death can be assumed. 5. Conclusion Iboga seems to be the latest fashion among psychodysleptics used in sects and has to be searchable in all kind of samples by toxicologists. The presented method is rapid, specific and
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sensitive enough for the determination of ibogaine and noribogaine in autopsy samples and hair. To our knowledge, this is the first report of ibogaine and noribogaine determination in hair. LC–MS or MS/MS method appears to be the best way for screening alkaloids in autopsy samples and particularly in hair, due to the presence of a nitrogen ring and the relatively low concentrations to be measured [24–26,32–34]. Serious toxic effects of iboga alkaloids even at therapeutic doses are related to deaths, mainly by cardiac problems (arrhythmia, heart failure, seizures, slowing of the heart) and the case reported in this paper is a good illustration of that risk. Acknowledgements The authors gratefully acknowledge Ms Prof. F.M.M. Bressole (Faculty of pharmacy, University of Montpellier, France) for providing noribogaine standard, and Ms Prof. Lecomte (Director of the Medico Legal Institute of Paris, France) for providing autopsy samples. References [1] J. Bruneton, Pharmacognosie, phytochimie, plantes me´dicinales, third ed., Tec et Doc dicales internationales, Paris, 1999, p. 1027. [2] J. Dybowski, E. Landrin, Sur l’Iboga, sur ses proprie´te´s excitantes, sa composition et sur l’alcaloı¨de nouveau qu’il renferme, C. R. Acad. Sci. 133 (1901) 748–750. [3] A. Haller, E. Heckel, Sur l’ibogaine, principe actif d’une plante du genre Tabernæmontana native du Congo, C. R. Acad. Sci. 133 (1901) 850–853. [4] P. Popik, P. Skolnick, Pharmacology of ibogaine and ibogaine-related alkaloids, in: The Alkaloids, 52th, Academic Press, San Diego, 1998, pp. 197–231. [5] K.R. Alper, Ibogaine: a review, Alkaloids Chem. Biol. 56 (2001) 1–38. [6] S. Helsley, D. Fiorella, R.A. Rabin, J.C. Winter, Behavioral and biochemical evidence for a nonessential 5-HT2A component of the ibogaineinduced discriminative stimulus, Pharmacol. Biochem. Behav. 59 (1998) 419–425. [7] S.D. Glick, I.S. Mainsonneuve, Mechanisms of antiaddictive actions of ibogaine, Ann. N.Y. Acad. Sci. 844 (1998) 214–226. [8] D. Wei, I.M. Maisonneuve, M.E. Kuehne, S.D. Glick, Acute iboga alkaloid effects on extracellular serotonin (5-HT) levels in nucleus accumbens and striatum in rats, Brain Res. 800 (1998) 260–268. [9] H. Lotsof, Rapid method for interrupting the narcotic addiction syndrome, U.S. Patent 4 499 096 (1985). [10] E.D. Dzoljic, C.D. Kaplan, M.R. Dzoljic, Effect of ibogaine on naloxoneprecipitated withdrawal syndrome in chronic morphine-dependent rats, Arch. Int. Pharmacodyn. Ther. 294 (1988) 64–70. [11] S.D. Glick, K. Rossman, S. Steindorf, I.M. Maisonneuve, J.N. Carlson, Effects and aftereffects of ibogaine on morphine self-administration in rats, Eur. J. Pharmacol. 195 (1991) 341–345. [12] S.L.T. Cappendijk, M.R. Dzoljic, Inhibitory effects of ibogaine on cocaine self-administration in rats, Eur. J. Pharmacol. 241 (1993) 261–265. [13] A. Rezvani, D. Overstreet, Y. Lee, Attenuation of alcohol intake by ibogaine in three strains of alcohol preferring rats, Pharmacol. Biochem. Behav. 52 (1995) 615–620. [14] K.R. Alper, H.S. Lotsof, G.M. Frenken, D.J. Luciano, J. Bastiaans, Treatment of acute opioid withdrawal with ibogaine, Am. J. Addict. 8 (1999) 234–242. [15] M.H. Baumann, J.P. Pablo, S.F. Ali, R.B. Rothman, D.C. Mash, Comparative neuropharmacology of ibogaine and its O-desmethyl metabolite, noribogaine, Alkaloids Chem. Biol. 56 (2001) 79–113.
66
M. Che`ze et al. / Forensic Science International 176 (2008) 58–66
[16] C. Zubaran, M. Shoaib, I.P. Stolerman, J. Pablo, D.C. Mash, Noribogaine generalization to the ibogaine stimulus: correlation with noribogaine concentration in rat brain, Neuropsychopharmacology 21 (1999) 119–126. [17] M.H. Baumann, R.B. Rothman, J.P. Pablo, D.C. Mash, In vivo neurobiological effects of ibogaine and its O-desmethyl metabolite, 12-hydroxyibogamine (noribogaine), in rats, J. Pharmacol. Exp. Ther. 297 (2001) 531– 539. [18] D.C. Mash, J.K. Staley, M.H. Baumann, Identification of a primary metabolite of ibogaine that targets serotonin transporters and elevates serotonin, Life Sci. 57 (1995) 45–50. [19] M.J. Bogusz, H. Althoff, D.C. Mash, W.L. Hearn, Ibogaine-associated death in a female heroin addict: forensic and toxicological aspects, in: Presented at the Annual Meeting of the Society of Forensic Toxicologists, Albuquerque, NM, October 8, 1998. [20] L.B. Hough, S.M. Pearl, S.D. Glick, Tissue distribution of ibogaine after intraperitoneal and subcutaneous administration, Life Sci. 58 (1996) 119– 122. [21] U. Maas, S. Strubelt, Fatalities after taking ibogaine in addiction treatment could be related to sudden cardiac death caused by autonomic dysfunction, Med. Hypotheses 67 (2006) 960–964. [22] J. Cienki, D. Mash, W. Hearn, Ibogaine fatalities, Clin. Tox. 39 (2001) 547. [23] Internet: table of ibogaine fatalities – http://www.myeboga.com/fatalities. html consulted at 03/30/2007. [24] V. Kontrimaviciute, J.C. Mathieu-Daude´, O. Mathieu, H. Breton, T. Casper, E. Baccino, F.M.M. Bressolle, Ibogaine and noribogaine determination in whole blood, plasma and urine. Application to an intoxication by Tabernanthe iboga root, Ann. Toxicol. Anal. 18 (2006) 162–163 (in French). [25] V. Kontrimaviciute, O. Mathieu, J.C. Mathieu-Daude´, P. Vainauskas, T. Casper, E. Baccino, F.M.M. Bressolle, Distribution of ibogaine and noribogaine in a man following a poisoning involving root bark of the Tabernanthe iboga shrub, J. Anal. Toxicol. 30 (2006) 434–440.
[26] V. Kontrimaviciute, H. Breton, F. Barnay, J.C. Mathieu-Daude´, F.M.M. Bressolle, Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 843 (2006) 131–141. [27] C.A. Gallagher, L.B. Hough, S.M. Keefner, A. Seyed-Mozaffari, S. Archer, S.D. Glick, Identification and quantification of the indole alkaloid ibogaine in biological samples by gas chromatography–mass spectrometry, Biochem. Pharmacol. 49 (1995) 73–79. [28] W.L. Hearn, J. Pablo, G.W. Hime, D.C. Mash, Identification and quantitation of ibogaine and an o-demethylated metabolite in brain and biological fluids using gas chromatography–mass spectrometry, J. Anal. Toxicol. 19 (1995) 427–434. [29] M.E. Alburges, R.L. Foltz, D.E. Moody, Determination of ibogaine and 12hydroxy-ibogamine in plasma by gas chromatography-positive ion chemical ionization-mass spectrometry, J. Anal. Toxicol. 19 (1995) 381–386. [30] F.R. Ley, A.R. Jeffcoat, B.F. Thomas, Determination of ibogaine in plasma by gas chromatography-chemical ionization mass spectrometry, J. Chromatogr. A 723 (1996) 101–109. [31] V. Kontrimaviciute, M. Larroque, V. Briedis, D. Margout, F. Bressolle, Quantitation of ibogaine and 12-hydroxybogamine in human plasma by liquid chromatography with fluorimetric detection, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 822 (2005) 285–293. [32] Y. Gaillard, G. Pe´pin, Poisoning by plant material: review of human cases and analytical determination of main toxins by high performance liquid chromatography-(tandem) mass spectrometry, J. Chromatogr. B 733 (1999) 181–229. [33] M. Che`ze, M. Deveaux, G. Pe´pin, Identification and quantification of plant poisons by liquid chromatography-tandem mass spectrometry (LC–MS/ MS). Bibliographic overview and Toxlab laboratory’s experience, Ann. Toxicol. Anal. 13 (2005) 43–55 (in French). [34] P. Kintz, M. Villain, Y. Barguil, J.Y. Charlot, V. Cirimele, Testing for atropine and scopolamine in hair by LC–MS/MS after Datura inoxia abuse, J. Anal. Toxicol. 30 (2006) 454–457.