Electrophoresis 2005, 26, 2391–2401 2391 Carolin Huhn1 Determination of tryptamine derivatives in illicit Michael Pütz2 Natalie Martin2 synthetic drugs by capillary electrophoresis and Rainer Dahlenburg2 ultraviolet laser-induced fluorescence detection Ute Pyell1 1Analytical Chemistry, A method based on separation by capillary electrophoresis combined with UV-laser- Philipps-Universität Marburg, induced fluorescence detection (lex = 266 nm) was developed for the determination of Marburg, Germany nine tryptamine derivatives of forensic interest and potential matrix constituents. The 2Bundeskriminalamt, composition of the separation electrolyte was optimized with respect to the resolution Fachbereich Toxikologie, of solutes of interest and to the sensitivity of fluorescence detection. Native a-cyclo- Wiesbaden, Germany dextrin was employed as a complex forming modifier of the electrophoretic separation and fluorescence-enhancing agent. With the help of a stacking procedure, limits of detection of 0.1–6 mg/L for all analytes were obtained. The repeatability for the peak area (at a concentration of the analyte about 100 times the LOD) was less than 2.3% RSD. A second HPLC method was developed, and its analytical parameters were evaluated for an estimation of the accuracy of the CE-LIF method and for method comparison. The results of the determination of tryptamine derivatives in the samples of forensic interest obtained with the two independent methods are in good agreement. Keywords: Capillary electrophoresis / a-Cyclodextrin / Frequency-multiplied diode-pumped solid-state laser / Illicit drugs / Stacking / Tryptamine derivatives / (UV)laser-induced fluorescence detection DOI 10.1002/elps.200410313 1 Introduction (DMT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), N,N-diethyltryptamine (DET), 5-methoxy-N,N-diisopropyl- Recent years show an increase in the consumption of psy- tryptamine (5-MeO-DIPT), a-methyltryptamine, and chotropic biogenic drugs. The use of the so-called “magic a-ethyltryptamine (structures are depicted in Fig. 1), all of mushrooms” (mainly species of Psilocybe, Stropharia, them influencing the serotonergic system and being rele- Conocybe,andPanaeolus), which contain the tryptamine vant to forensic toxicologists as they are controlled sub- derivatives psilocin and psilocybin as the active ingredients, stances under German narcotics act. Among these con- is well known. Bufotenine (5-hydroxy-DMT), a positional trolled tryptamines, DMTand 5-MeO-DMT not only occur in isomer of psilocin (4-hydroxy-DMT), is the active psycho- illicit synthetic drugs, but also in a large number of natural tropic substance in ancient snuff powders of South America sources. Todate, there is a lack of a state-of-the-art method (typically prepared from seeds of the plant of genus Anade- for the determination of tryptamine derivatives in samples nanthera) and in secretions of toads (Bufo marinus, Bufo with complex matrices. vulgaris, and others). Unlike psilocin and psilocybin, bufo- tenine is no controlled substance under German narcotics In the analysis of tryptamine derivatives, a wide range of act, but a controlled Schedule I hallucinogenic substance in analytical techniques has been employed in the past. Tryp- the United States. Their synthetic counterparts are different tamine derivatives being present in intoxicating snuffs used tryptamine derivatives, mainly N,N-dimethyltryptamine by native people in South America were determined by GC with argon ionization detection after derivatization with Correspondence: Prof. Ute Pyell, Fachbereich Chemie, Phi- hexamethyldisilazane [1], by GC-FID [2], or by GC-flame- lipps-Universität Marburg, Hans-Meerwein-Strasse, D-35032 ionization detector (FID) in combination with TLC and GC- Marburg, Germany MS [3]. DMT in ayahuasca (a South American psychotropic E-mail: [email protected] beverage) and in human plasma after dosing with aya- Fax: 149-6421-282-8917 huasca was quantified by TLC and HPLC-UV detector Abbreviations: DAD, diode array detection; DET, N,N-diethyl- (UVD) [4] and by GC-nitrogen-phosphorus detector (NPD) tryptamine; DMT, N,N-dimethyltryptamine; i. st., internal stand- [5, 6]. Tryptamine derivatives in plant and mushroom ard; LSD, lysergic acid diethylamide; MDMA, 3,4-methylene- extracts were identified and quantified by RP-HPLC-UVD dioxymethamphetamine; 5-MeO-DIPT, 5-methoxy-N,N-diiso- propyltryptamine; 5-MeO-DMT, 5-methoxy-N,N-dimethyltryp- [7], RP-HPLC-DAD [8, 9], RP-HPLC-UVD combined with tamine; NMT, N-methyltryptamine amperometric detection [10, 11], or ion-exchange chro- 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2392 C. Huhn et al. Electrophoresis 2005, 26, 2391–2401 Figure 1. Structures of trypta- mine derivatives quantified in this study: (1) tryptamine, (2) 5- methyltryptamine, (3) DET, (4) 5- MeO-DMT, (5) DMT, (6) 6-meth- oxytryptamine, (7) serotonin, (8) bufotenine, (9) 5-MeO-DIPT, (10) acridine (i.st.), (11) a-ethyl- tryptamine (a-ET), (12) a-me- thyltryptamine (a-MT), (13) NMT. Substances marked with an asterisk are controlled sub- stances under German narcot- ics act. matography combined with fluorescence detection sitivity of fluorescence detection. The first paper describ- (lex = 267 nm, lem = 335 nm) [12]. In mouse or rat brain, ing the use of CE-(UV-)LIF within a forensic background tryptamine and related substances were quantified by was published by Frost et al. [17] describing the determi- GC-MS [13] or by RP-HPLC combined with fluorescence nation of the structurally related lysergic acid diethyla- detection (lex = 267 nm, lem = 335 nm) [14]. GC-MS was mide (LSD). also used for the determination of bufotenine and related It is well-known that complexation with cyclodextrins tryptamine derivatives in different samples of forensic (CDs) can be employed for an improvement of the sensi- interest [15, 16]. Most of the developed procedures are tivity of fluorescence detection after an HPLC separation restricted to a limited number of tryptamine derivatives, by adding CD to the eluent in a postcolumn mode. This however, in forensic applications analytical methods are effect is based on an enhancement of the fluorescence desired, which make it possible to identify and quantify a quantum yield due to a decrease in the rotation motion of large number of possible tryptamine derivatives within the entrapped molecule and/or decrease in solvent one single procedure. relaxation [18]. In our work, we studied the influence of pH, the presence or absence of SDS micelles, and the In the present work, we develop a method for the deter- influence of the a-CD concentration in the separation mination of nine tryptamine derivatives (tryptamine, electrolyte on the selectivity of the separation and on the 5-methyltryptamine, DET, 5-MeO-DMT, DMT, 6-me- sensitivity of the detection. thoxytryptamine, serotonin, bufotenine, 5-MeO-DIPT) in items of evidence based on the separation of tryptamine In order to elucidate the accuracy of the method, a refer- derivatives and possible matrix constituents by CE com- ence method employing HPLC-diode array detection bined with selective and sensitive laser-induced fluores- (DAD) was developed for the baseline separation of eight cence detection. A low-cost frequency-multiplied diode- tryptamine derivatives. Both methods were applied to the pumped solid-state laser emitting at 266 nm matching analysis of seized samples of illicit synthesis, showing a well the absorbance maxima of the analytes of interest is good correlation of results. The HPLC-DAD method was employed as the excitation source. The composition of based on an HPLC method originally developed for the the separation electrolyte is optimized concerning the determination of indole alkaloids [19] (octadecyl silica gel, resolution of solutes of interest and concerning the sen- identical composition of the mobile phase). 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2005, 26, 2391–2401 CE-(UV)LIF determination of tryptamine derivatives 2393 2 Materials and methods with a run buffer for 10 min. Rinsing with the running buffer for 1 min was used for cleaning the capillary be- 2.1 Chemicals tween runs. A voltage of 15 kV was used for separation. The LIF detection system was a TIDAS LIF 266 system Serotonin, tryptamine, triethylamine, acridine, mono- from J&M Analytische Mess- und Regeltechnik, which sodium dihydrogenphosphate, sodium tetraborate, and was described by Pütz [20]. The excitation radiation hydrochloric acid were from Fluka (Buchs, Switzerland), source was an FQSS266-S laser from Crystal (Berlin, SDS from Roth (Karlsruhe, Germany), a-CD, ammonium Germany), which is a passively Q-switched frequency- acetate, acetonitrile (LiChrosolv hypergrade), sodium hy- converted diode-pumped solid-state microchip neody- droxide, and glacial acetic acid from Merck (Darmstadt, mium:yttrium-aluminum garnet laser with an optical Germany), b-CD from Aldrich, (Milwaukee, WI, USA), and quasi-cw power of about 0.5 mW at 266 nm. A photo- methanol (HPLC grade) from Riedel-de Haën (Seelze, multiplier detector was used for measuring the fluores- Germany). Other tryptamine derivatives and seized sam- cence intensity. Optical fibers connected the excitation ples of synthetic drugs containing tryptamine derivatives source and the photomultiplier detector to the in-capil- were provided by the Federal Criminal Police Office lary detection cell, placed in a commercially available (Wiesbaden, Germany). capillary holder (J&M, Analytische Mess- und Regel- technik). The optical