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Analysis and Characterization of Designer
Tryptamines using
KEYWORDS: Tryptamines, Analogues, Designer Drugs, Electrospray Ionization-Mass Spectrometry, ESI-MS, Pseudo-molecular Ion, Fragmentation, Tandem Mass Spectrometry, Collision-Induced Dissociation, GC/MS, Forensic Chemistry.
“Designer drugs” are compounds with structures that are very similar to controlled substances, but that are not specifically controlled. Also known as “analogues,” most of these compounds have never been previously encountered or characterized, and so are not present in commercial spectral libraries; therefore, they can represent unusual challenges for forensic laboratories. Definitive identification of such drugs usually requires in-depth analysis using multiple and complementary techniques, including infrared spectroscopy (IR), gas chromatography/mass spectrometry (GC/MS), and nuclear magnetic resonance (NMR). For decades, the use of electron ionization (EI) mass spectrometry as a detector for gas chromatography (GC) instruments has been a main step in the structure elucidation process used by analytical and synthetic chemists [1]. Electron ionization mass spectra of many thousands of compounds, normally collected under 70 eV of energy, are readily available from reference databases [2] and instrument manufacturers [3]. The capabilities of mass spectrometry, however, have greatly expanded with the more recent development of specialized ionization techniques like electrospray ionization (ESI) [4] and atmospheric pressure chemical ionization (APCI) [5], making possible the analysis of many polar and thermally labile compounds that were not amenable to GC/MS analyses. Through the ESI process, ions in solution are transported into the gas phase by a series of solvent evaporation and Coulomb explosion steps, preserving the original intactions and introducing them into the vacuum-housed mass analyzer without significant fragmentation. As a result, this process produces singly and/or multiply charged ions which, upon mass spectrometric analysis, provide direct molecular weight information - a critical step in identification. In addition, the interface of an ESI source to an ion-trap mass spectrometer provides for not only molecular weight determinations, but also for tandem and MSn fragmentation analysis of intact gas-phase ions via collision-induced dissociation (CID) experiments using a target gas [5]. By performing these experiments under controlled conditions, further structural elucidation can be accomplished, and the additional spectra generated can be collected and stored as part of a laboratory-generated library. Certain synthetic tryptamines produce hallucinogenic effects in humans [6,7]. These properties can be expected based on the structural similarities between these tryptamines and some naturally occurring hallucinogenic tryptamines such as psilocybin, psilocin, dimethyltryptamine, bufotenine, and ibogaine. By slightly modifying the structures of these latter substances, synthetic chemists have developed novel “designer” tryptamines with very similar, new, modified, unusual, and/or otherwise desirable psychedelic properties. Synthetic details for the preparation of some designer tryptamines have been available to the public for many years [8]. However, the scientific literature provides only very limited information regarding their analysis and characterization [9-13]. Most of the available literature focuses on the extraction of the naturally available tryptamines from their source [14], or the analysis and detection of their metabolites in animal biological fluids [15-18]. This paper presents the analysis of various designer tryptamines using ESI-MS. Figure 1 illustrates the core structure of a tryptamine-type molecule, while Table 1 lists the 12 compounds investigated in this work, along with details of their structure. Some of these compounds were recently acquired during a law enforcement investigation targeting their open sale on the internet. Initially, these compounds were identified at this laboratory using IR and NMR spectroscopy, GC, and GC/MS techniques. Analysis by ESI-MS provides complementary information that is valuable for the full characterization of these compounds. For most of the compounds, the generation of pseudo-molecular ions via ESI provides molecular weight information that is not available via GC/MS analysis. MSn fragmentation experiments further complement the structural information obtained from GC/MS, and allow the additional characterization of thermally labile compounds.
Solutions
for each of the analogues were prepared by dissolving the appropriate
amount in methanol to obtain a final concentration of approximately
10 µg/mL. Solutions were introduced into the mass spectrometer
using a ThermoFinnigan Surveyor autosampler. Sample injections (10 µL)
were loaded into a methanol constant flow (200 µL/min) provided
by a ThermoFinnigan Surveyor solvent pump. Experimental data were analyzed using the qualitative analysis program provided within the Xcalibur software suite. In addition, all MS2 fragmentation data were incorporated into an in-house spectral library. For the GC/MS data (displayed in the Appendices), solutions of the 12 analogues were prepared in methanol at a concentration of 1 mg/mL. Samples were introduced into the gas chromatograph using 1.0 µL injections. The GC oven program used was: initial temperature: 90 °C (1 minute hold), ramp to 300 °C (20 °C/minute); final temperature: 300 °C (5 minute hold). Helium was used as the carrier gas at a constant flow of 1 mL/minute. Mass spectra were obtained using a ThermoFinnigan PolarisQ ion-trap mass spectrometer controlled by the Xcalibur software.
ESI-MS Experiments Figures 2a and 2b present the positive and negative ion mode full-scan electrospray mass spectra obtained for 4-acetoxy-N,N- diisopropyltryptamine (9). The singly protonated and singly deprotonated pseudo-molecular ions are clearly represented at m/z 303 and 301, respectively, indicating a molecular weight of 302 for this compound. Similar spectra were obtained for the other 11 analogues. The molecular weights determined from all of these experiments are included in Table 1. Under the experimental conditions utilized, the signal intensity for the negative ion spectra was observed to be somewhat dependent on the structure of the compounds, specifically the presence of methoxy, acetoxy, or hydroxy groups. Molecules containing such functionalities were observed to produce more intense deprotonated pseudo-molecular ions. Full-scan electrospray analysis was especially useful for the identification and differentiation of 4 hydroxy-N,N-diisopropyltryptamine (7) and 4-acetoxy-N,N-diisopropyltryptamine (9). The latter compound is thermally unstable, and standard GC/MS analyses gave highly similar spectra (see Appendices). However, ESI-MS provided accurate molecular weight information, allowing a easy identification. ESI-MS2 Experiments In
addition to collecting full-scan ESI spectra for these molecules,
ESI-MS/MS (ESI-MS2) fragmentation experiments were also
performed in
order to obtain structural information that would complement the information
previously obtained via GC/MS experiments. ESI-MS2 fragmentation
experiments were performed in the positive ion mode at various normalized
collision
energies. The spectra generated at 35 eV were considered to be the
most useful, and so were exported and compiled into an in-house library
for future use in the identification of unknowns. For compounds with the same molecular weights, the fragmentation patterns obtained from the ESI-MS2 experiments were useful for elucidating structures. For example, 4-acetoxy-N,N-methylisopropyltryptamine (8) and 5-methoxy-N,N-diisopropyltryptamine (10) have the same molecular weight of 274. In both cases, the protonated and deprotonated pseudo-molecular ions are observed at m/z 275 and 273, respectively, not allowing for their distinction. However, ESI-MS2 analysis produced different fragmentation patterns for these compounds (see Appendices 8 and 10). For 4-acetoxy-N,N-methylisopropyltryptamine (8), the major fragment is observed at m/z 202, with additional fragments at m/z 160 and 86. Whereas for 5-methoxy-N,N-diisopropyltryptamine (10), the major fragment is observed at m/z 114, with additional fragments at m/z 102 and 174. The usefulness of ESI-MS2 analyses can also be illustrated by again comparing 4-hydroxy-N,N-diisopropyltryptamine (7) and 4- acetoxy-N,N-diisopropyltryptamine (9). As mentioned before, these compounds are virtually undistinguishable by GC/MS analysis, due to the thermal instability of the 4-acetoxy group. In addition to providing direct molecular weight information, ESI-MS2 analysis produces distinctive fragments that allow their specific characterization. For 4-acetoxy-N,N-diisopropyltryptamine (9), fragments are produced at m/z 202, 160, 114, and 102. Whereas for 4-hydroxy-N,N-diisopropyltryptamine (7), fragments are produced at m/z 160, 114, and 102. While the fragment at m/z 202 is the predominant fragment produced from the former compound, it is absent from the latter, providing a marker ion for differentiation. GC/MS Experiments The Appendices also include the standard EI spectra obtained using a GC/MS system (bottom panels). Not surprisingly, most of the spectra are characterized by the presence of one major peak (base peak) corresponding to fragmentation of the sigma bond between the alpha and beta carbons. For 10 of the compounds (tryptamines 2-11), alpha cleavage [19] results in retention of the charge by the amine group. For alpha-methyltryptamine (1) and 5-methoxy-alpha-methyltryptamine (12), the most favorable fragmentation, inductive cleavage [19], results in charge migration to the indole-containing fragment, producing peaks at m/z 130 and 160, respectively. An additional hydrogen rearrangement process is also involved, resulting in major peaks at m/z 131 and 161, respectively. GC/MS experiments also produce characteristic signatures for the different amine substituents. For example, m/z 86 is characteristic of the N,N-methylisopropyl and N,N-diethyl groups, while m/z 114 is characteristic of the N,N-dipropyl or N,N-diisopropyl functionalities. For these two latter isomeric species, the additional presence of ions at m/z 72 and 86, respectively, provides a useful distinguishing factor. Additional
bond cleavages in the tryptamine molecule result in the generation
of specific fragmentation signatures. These can be of great
use when interpreting data generated by either GC/MS or ESI-MS2 techniques.
For example, the presence of an ion at m/z 144 is indicative
of a non-substituted indole moiety, after cleavage of the sigma bond
between
the amine nitrogen and the alpha carbon. The same type of
cleavage leads to the generation of peaks at m/z 174 and 202
for compounds where the indole contains a methoxy or acetoxy functionality,
respectively.
As previously mentioned, cleavage between the alpha and beta carbons
produces a high intensity peak at m/z 86, due to the generation
of the C4H10+N=CH2 fragment,
which can be produced from the diethylamine or methylisopropylamine
group. Through the same process, the generation
of m/z 114 is indicative of the C6H14+N=CH2 fragment,
consistent with either dipropylamine or diisopropylamine. For dimethyltrypamine
compounds,
the analogous peak will appear at m/z 58 due to C2H6+N=CH2.
As observed for alpha-methyltryptamine (1) and
5-methoxy-alpha-methyltryptamine
(12), cleavage between the alpha and beta carbons
also produces signature ions at m/z 130 and 160, characteristic
of an unsubstituted and a ESI MSn Experiments The
use of ESI combined with a quadrupole ion trap as the mass analyzer
provides the enhanced capabilities of MSn experiments. To
illustrate the usefulness of this type of analysis, ESI-MS3 experiments
were performed
on N,N-dipropyltryptamine (5) and N,N-diisopropyltryptamine
(6). As illustrated in the Appendices,
the fragments generated during MS2 analysis
do not allow for the unambiguous differentiation of these two compounds,
although the presence of m/z 86 (5 % intensity) for N,N-dipropyltryptamine
(5) does suggests that they are in fact different.
By isolation and fragmentation of the singly protonated pseudo-molecular
ions at m/z 245 using a collision energy of 25 eV (%), major
ions at m/z 144
and 114 are generated for both compounds. However, further isolation
and fragmentation of the m/z 114 ion, using a collision
energy of 30 eV, leads to significantly different MS3 spectra
(see Figures
4 and 5). Fragmentation of
the m/z 114 ion produces a fragment at
m/z 86 for N,N-dipropyltryptamine and a fragment
at m/z 72 for N,N-diisopropyltryptamine. These
ions correspond to the loss of the neutral
fragments CH2=CH2 and CH2=CHCH3,
respectively. These losses are characteristics of the dipropyl and
diisopropyl groups, respectively, providing a
tool for differentiation. In fact, further fragmentation (MS4)
of the m/z 86 ion, produced from the fragmentation of N,N-dipropyltryptamine,
generates a peak at m/z 58, consistent with the loss of
a second CH2=CH2 molecule from the remaining
dipropyl chain (data not shown). Similar MS4 analysis
of the m/z 72 ion from N,N-diisopropyltryptamine
would produce additional information regarding the second diisopropyl
group; however, the additional loss of 42 would produce an ion at
m/z 30, which is below the observable low mass limit of
50 for this instrument. These ESI-MS3 results are in
agreement with the GC/MS
data obtained for N,N-dipropyltryptamine and The complementary value of the GC/MS and ESI-MS data can be better illustrated using alpha-methyltryptamine (1). The GC/MS spectrum does not provide molecular weight information, but it does indicate the presence of a non-substituted indole (m/z 130), with the presence of a phenyl group further confirmed by m/z 77. Although the ion at m/z 44 is indicative of C2H6N, no confirmation of the -NH2 group is obtained. By direct observation of the pseudo-molecular ion, the ESI-MS data provide a direct determination of molecular weight at 174. The ESI-MS2 spectrum, while simple and only containing one major ion at m/z 158, provides a signature indicating the loss of ammonia from the protonated intact molecule, confirming the presence of a NH2 substituent.
The analysis of 12 tryptamine analogues using ESI-MS has been presented. Full scan analysis in the positive and negative ionization modes allowed the observation of singly protonated and singly deprotonated ions, providing molecular weight information. MS2 experiments allowed the fragmentation of pseudo-molecular ions to be investigated under controlled experimental conditions, providing structural information for each one of the compounds, and making possible the observation of fragmentation signatures. Particularly useful was the generation of ESI full-scan and MS2 spectra for the thermally-labile compound 4-acetoxy-N,N-diisopropyl-tryptamine, which provided a distinction from 4-hydroxy-N,N-diisopropyltryptamine. MS3 experiments were also performed and results provided an additional technique for the differentiation of compounds with the same molecular weight and similar MS2 spectra. The results presented are in agreement with those obtained using standard GC/MS techniques, and show the utility of ESI-MS as a complementary analytical technique that can be used in conjunction with GC/MS, NMR, and IR spectroscopy in the structural characterization of tryptamines.
The author is grateful to DEA Southwest Laboratory Administrative Assistant D. A. Perez, and also to DEA Southwest Laboratory Forensic Chemists J. M. Katz, N. C. Payne, and H. F. Skinner, for useful discussions and review of the manuscript.
1. Silverstein RM, Bassler GC, Morrill TC. Spectrometric identification of organic compounds. Third Edition. John Wiley and Sons, New York, 1974. 2. Pfleger K, Maurer HH, Weber A. Mass spectral and GC data of drugs, poisons, pesticides, pollutants and their metabolites. Second Edition. Wiley VCH, Weinheim, 1992. 3. Stein S, Mirokhin Y, Tchekhovskoi D, Mallard G. The NIST/EPA/NIH mass spectral library. Version 2.0a, July 2002. 4. Cole RB. Electrospray ionization mass spectrometry: Fundamentals, instrumentation and applications. John Wiley and Sons, New York, 1997. 5. Johnstone RAW, Rose ME. Mass spectrometry for chemists and biochemists. Second Edition. Cambridge University Press, Cambridge, 1996. 6. Gahlinger P. Illegal drugs: A complete guide to their history, chemistry, use, and abuse. Plume, New York, 2004. 7. Perrine DM. The chemistry of mind altering drugs: History, pharmacology and cultural context; pp. 278 288. American Chemical Society, Washington, D.C., 1996. 8. Shulgin A, Shulgin A. TIHKAL: The continuation. Transform Press, Berkeley, 1997. 9. Clark A. 5-Methoxy-N,N-dimethyltryptamine. Microgram 1974;7(4):42.* 10. Chamakura R. Bufotenine. Microgram 1993;16(8):185.* 11. Vohlken B. Bufotenine and Psilocin: Mass Spectral Distinctions. Microgram 1993;16(10):233.* 12. Roesner P. Mass spectra of designer drugs. John Wiley and Sons, Inc., New York, 2003. 13. Brandt SD, Freeman S, Fleet IA, McGagh P, Alder JF. Analytical chemistry of synthetic routes to psychoactive tryptamines. Part I. Characterisation of the Speeter and Anthony synthetic route to 5-methoxy-N,N-diisopropyltryptamine using ESI-MS-MS and ESI-TOF-MS. Analyst 2004;129:1047. 14. Casale, JF. An aqueous organic extraction method for the isolation and identification of psilocin from hallucinogenic mushrooms. Journal of Forensic Sciences 1985;30:247. 15. Katagi M, Tsutsumi H, Miki A, Nakajima K, Tsuchihashi H. Analyses of clandestine tablets of amphetamines and their designer drugs encountered in recent Japan. Japanese Journal of Forensic Toxicology 2002;20:303. 16. McClean S, Robinson RC, Shaw C, Smyth WF. Characterisation and determination of indole alkaloids in frog skin secretions by electrospray ionisation ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 2002;16:346. 17. Meatherall R, Sharma P. Foxy, a designer tryptamine hallucinogen. Journal of Analytical Toxicology 2003;27:313. 18. Vorce SP, Sklerov JH. A general screening and confirmation approach to the analysis of designer tryptamine and phenethylamine in blood and urine using GC-EI-MS and HPLC-electrospray-MS. Journal of Analytical Toxicology 2004;28:410. 19. McLafferty FW, Turecek F. Interpretation of mass spectra. Fourth Edition; pp. 57 68. University Science Books, Mill Valley, 1993. * Law Enforcement Restricted. * * * * * * * * * * *
Table 1. Designer Tryptamines Investigated (Refer to Figure 1 for Substituent Positions).
Figure 2: (a) Positive and (b) negative ion mode electrospray ionization mass spectra for 4-acetoxy-N,N-diisopropyltryptamine (6).
Figure 3: Chemical diagram illustrating the two main fragmentation processes observed during the MS/MS analysis of tryptamines.
Figure
4. Full Mass, MS2 and MS3 Spectra
Obtained for N,N-Dipropyltryptamine (5).
Figure 5. Full Mass, MS2 and MS3 Spectra Obtained for N,N-Diisopropyltryptamine (6).
Appendix 1. alpha-Methyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix
2. N,N-Dimethyltryptamine: ESI-MS2 (top)
and GC/MS (bottom).
Appendix 3. N,N-Diethyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 4. N,N-Methylisopropyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 5. N,N-Dipropyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 6. N,N-Diisopropyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 7. 4-Hydroxy-N,N-diisopropyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 8. 4-Acetoxy-N,N-methylisopropyltryptamine: ESI-MS2 (top) and GC/MS (bottom)
Appendix 9. 4-Acetoxy-N,N-diisopropyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 10. 5-Methoxy-N,N-diisopropyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 11. 5-Methoxy-N,N-methylisopropyltryptamine: ESI-MS2 (top) and GC/MS (bottom).
Appendix 12. 5-Methoxy-alpha-methyltryptamine: ESI-MS2 (top) and GC/MS (bottom). * * * * * |
