Derivatisation followed by iodine azide reaction was employed for detection of amphetamines and its analogues in

TLC. The derivatisation reaction with phenyl isothiocyanate took place directly on the TLC plate before the developing

step. Afterwards, the plate was sprayed with a mixture of sodium azide and starch solution and then exposed to iodine

vapour. The obtained limits of detection were compared with other commonly visualization techniques: UV, iodine vapour, Marquis and Simon’s reagents, ninhydrin, Fast Black K

Ciesielski, W.

Kaźmierczak, D.

Kochana, Jolanta

Parczewski, Andrzej

Zakrzewska, Agnieszka

Acta Chimica Slovenica

English

Jagiellonian Univeristy Repository

106 Acta Chim. Slov. 2007, 54, 106–109Zakrzewska et al.:   Visualisation of Aphetamine and Its Analogues in TLC1. IntroductionAmphetamine and its derivatives: methampheta-mine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine MDA, p-metoxyamp-hetamine (PMA), p-metoxymethamphetamine (PMMA)are stimulants of the central nervous system. All of theabove substances are abused. EMCDDA (European Moni-toring Centre for Drugs and Drug Addiction) estimated to-tal production of ATS (amphetamine type stimulants) in2005 around 520 tons.1Amphetamine (1-phenylpropan-2-amine) also knownas šspeed’ causes strong physical and mental stimulation.The abusers become alert, full of self-confidence, happyand talkative. They may feel like having more energy andbeing stronger. When the effect diminishes, their feelingsevolve into anxiety, fatigue, disinterest or tiredness.2 Amp-hetamine is produced mainly in Europe. The Leuckart reac-tion is the most common method used to obtain ampheta-mine, but in some cases reductive amination of 1-phenyl-propan-2-one (BMK or P2P) is used. The other syntheticmethods such as oxime or phenylnitropropene routes havenot been put into practice in illegal production.3,4Methamphetamine (N-methyl-1-phenyl-propan-2-amine) named šcrystal’, šcrank’, šmeth’, šYaba’ (tabletscontaining mainly methamphetamine and caffeine)5,6 havea more pronounced stimulatory effect on the central ner-vous system than amphetamine. The high doses of thisdrug can result in effects such as hallucinations, paranoiaand mania.2 The main production centres are placed inEast and South Asia.1The drugs known as šecstasy’, šXTC’, šBeans’ andšAdam’5 in 80% cases contain MDMA. However, theircomposition exhibits substantial variability. In tablets, thefollowing substances: MDA, PMMA, PMA, amphetamineand methamphetamine, ephedrine, ketamine and caffeinewere discovered beside MDMA.7–11 Production of ecstasytablets is concentrated in Europe but manufacture spreadsto other parts of the world: North America, East and South-East Asia.1 Reductive amination of 1-(3,4-methylene-dioxyphenyl)-2-propanone (MDP-2-P or PMK) was foundto be the most frequently used synthesis method of MD-MA. Apart from this method the nitropropene route and sa-frole bromination were encountered. The Leuckart reac-tion to prepare MDMA was seldom used.4,12–14 MDMA in-duce feelings such as euphoria, friendliness, closeness andempathy. But when used frequently, especially in high do-ses, it may cause cardiac arrhythmias, hyperthermia, leadto dysfunction of kidney, liver and brain damage.8The analysis of seized drug tablets or powder is usu-ally carried out using chromatographic methods: GC,15–19HPLC,9,20 or TLC.20,17,24 Other methods, e.g. near-infraredspectroscopy and Raman spectroscopy have also been ap-plied.21,22TLC is a low-cost and very versatile technique dueto the availability of a wide range of possible developingsystems. A great variety of visualizing reagents for detec-AbstractDerivatisation followed by iodine azide reaction was employed for detection of amphetamines and its analogues inTLC. The derivatisation reaction with phenyl isothiocyanate took place directly on the TLC plate before the developingstep. Afterwards, the plate was sprayed with a mixture of sodium azide and starch solution and then exposed to iodinevapour. The obtained limits of detection were compared with other commonly visualization techniques: UV, iodine va-pour, Marquis and Simon’s reagents, ninhydrin, Fast Black K.Keywords: TLC, iodine azide reaction, visualization of amphetamines;Short communicationVisualization of Amphetamine and Its Analogues in TLCA. Zakrzewska,a,* A. Parczewski,a,b D. Kaźmierczak,c W. Ciesielski,c J. Kochanaaa Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, 30-060 Cracow, Ingardena 3, Poland. Tel.: +48(12)6632014, Fax: +48(12)6340515, E-mail: zakrzews@chemia.uj.edu.pl.b Institute of Forensic Research, 31-033 Cracow, Westerplatte 9, Polandc Department of Instrumental Analysis, University of Ĺódź, 90-236 Ĺódź, Pomorska 163, PolandReceived: 26-09-2006Paper based on a presentation at the 12th International Symposium on Separation Sciences, Lipica, Slovenia, September 27–29, 2006.107Acta Chim. Slov. 2007, 54, 106–109Zakrzewska et al.:   Visualisation of Aphetamine and Its Analogues in TLCThe derivatisation solution was prepared by adding20 µL of 98% phenyl isothiocyanate (PTIC) to 200 µL 2-propanol. The mixture was completed with 20 µL distilledwater and 20 µL phosphate buffer pH 12 (3,58 g Na2HPO4were dissolved in 80 mL water and the mixture was adju-sted to pH = 12 with 1 mol L–1 NaOH and filled up by di-stilled water to 100 mL).Marquis agent was prepared by mixing one part of 38%formaldehyde with five parts of 98% sulphuric acid (v/v). Simon agent was composed of two solutions (A andB): 2% aqueous solution of sodium carbonate made solu-tion A; solution B was prepared by dissolving 5 g of so-dium nitroferricyanide in 100 mL of 10% acetaldehydesolution. The plate was first sprayed with solution A andthen solution B. Ehrlich agent was a 1% solution of p-dimethyloami-nobenzaldehyde in mixture of hydrochloric acid and met-hanol (1:1 v/v). FBK solution was prepared by dissolving 0.5 g of FastBlack K salt (C14H12N5O4 ⋅ 0.5ZnCl4) in 100 mL of water.Ninhydrin solution was prepared by dissolving 0.2 g ofninhydrin in 100 mL of ethanol. All solution used in visuali-zation of the spots on a chromatogram were prepared daily. 2.2. Procedure of Derivatisation of Amphetamine and Its Analogues A portion (1 µL) of drug solution was spotted onTLC plate. Then, on the same place 1 µL derivatisationagent (PTIC) was applied. Next, the plate was put in theTLC chamber in order to complete the derivatisation pro-cess. After 20 min, the plate was developed with mixtureof suitable solvents.2.3. TLC ProcedureTLC silica gel 60F254 aluminium sheets (Merck,Germany; 10 × 10 cm, 0.2 mm thin layer) were used fordetermination of detection limits of amphetamine and itsanalogues. The plates were developed in the horizontalchamber (Camag, Switzerland). The developing distancewas 8 cm. After derivatisation, the analytes were sepera-ted using the following mobile phase: n-hexane, toluene,1,4-dioxane (3: 3: 1 v/v/v) which made System I. In casethe derivatisation was not carried out, another eluent wasused: 1,4-dioxane, methanol, chloroform and 25% aque-ous ammonia (6: 2: 2: 1 v/v/v/v) – System II. After deve-lopment the plates were dried at 100 °C for 15 min. Thensuitable spraying agents were applied.2.4. Visualization of Spots on TLC PlatesThe UV254 Procedure After drying, the developed plates in System I and IIwere observed under UV lamp (254 nm). The substancesquenched fluorescence of the plate background.tion of amphetamines in TLC was reported. Apart fromobservation of spots under UV light (254 nm), ninhy-drin23,24 and Marquis reagents9,25 are most often applied.Also fluorescamine spray,23 iodoplatinate solution wereused.15,23 Ojanpera et.al. detected amphetamines with FastBlack K (FBK) salt.26In the present paper a new visualization and identifi-cation of selected drugs components (amphetamine, met-hamphetamine, MDMA, MDA, PMMA and PMA) is des-cribed. The procedure is based on iodine azide reaction.Its efficiency (detection limits) was compared with othervisualization methods in which the following reagentswere used: ninhydrin, FBK solutions, Marquis, Simon’sand Ehrlich’s reagents as well as iodine vapour were used.The iodine azide reaction proceeds according to thefollowing scheme:This reaction had been successfully used for detectionof amino acids and biogenic amines on TLC plate.27,28 Wooet. al. had applied the iodine-azide reaction to detect aminoacids in HPLC, as well.29,30 Only sulphur(II) compounds in-duce the reaction. Because the analytes tested in the presentwork do not include sulphur atoms in their molecules, theyneed to be transformed into sulphur derivatives. Phenyl isot-hiocyanate (PTIC) was selected as the derivatisation agent.The derivatisation is a pre-chromatographic step and is per-formed directly on TLC plate. After development, the platewas sprayed with freshly prepared mixture of sodium azide(adjusted to proper pH) and 0.5% starch solution. Thenthe plate was exposed to iodine vapour. The plate back-ground became violet-grey and the spots white.2. Experimental2.1. Solutions and ReagentsThe following amines: MDMA (3,4-methylene-dioxymethamphetamine), amphetamine and methamphe-tamine, MDA (3,4-methylenedioxyamphetamine), PM-MA (p-methoxy-methamphetamine), PMA (p-methox-yamphetamine) used in the examination were synthesizedin our laboratory. The examined drugs were dissolved inphosphate buffer pH = 7 (Merck. Germany).Methanol, chloroform, 1,4-dioxane, n-hexane, 25%aqueous ammonia (all Merck, Germany, HPLC grade), to-luene (Eurochem BGD, Poland, analytical grade) wereused to prepare the mobile phases.The spraying agent (iodine-azide reaction) was pre-pared by dissolving 2 g of sodium azide in 20 mL of distil-led water and next adding 25 mL of 0.5% starch solution.Then, the pH of the mixture was adjusted (5.5–6.0) using36% hydrochloride acid solution and diluted to 50 mLwith distilled water.I2 + 2N3– —————→ 2I– + 3N2.C=S inductorThe Iodine ProcedureAfter drying, the developed plates in System I and IIwere exposed to iodine vapour for 5 min. The drugs beca-me visible as brown spots on yellow background of theplate.The Iodine-Azide ProcedureAfter drying, the developed plates (System I) we-re sprayed with mixture containing sodium azide solu-tion (spraying agent) and then exposed to iodine vapourfor 5 s. Due to catalytic effect of the C=S bond on theiodine azide reaction, the examined compounds werevisible as white spots on the violet-grey background.The colour of the plate has been stable for several mi-nutes. Application of Marquis ReagentAfter drying, the developed plates (System II) wereimmersed in Marquis reagent and washed with distilledwater. The spots were dyed blue-green.Application of Simon and Ehrlich ReagentsAfter drying, the developed plates (System II) weresprayed with Simon or Ehrlich reagents and dried at 100°C. The Simon reagent dyed the spots of drugs grey-green. In the case Ehrlich reagent was applied, the back-ground became yellow and the spots white.108 Acta Chim. Slov. 2007, 54, 106–109Zakrzewska et al.:   Visualisation of Aphetamine and Its Analogues in TLCTable 1. Rf and detection limits (nmol per spot) of amphetamines detected as PTC-derivatives; mobilephase – System I.Compounds Rf Visualization procedureiodine-azide procedure iodine vapour UV254Amphetamine 0.44 0.19 0.19 0.19Methamphetamine 0.45 0.17 0.17 0.17MDMA 0.39 0.08 0.13 0.13MDA 0.37 0.09 0.14 0.14PMMA 0.42 0.15 0.15 0.15PMA 0.40 0.14 0.14 0.14Application of FBK and Ninhydrin SolutionsAfter drying, the developed plates (in System II)were sprayed with ninhydrin or FBK solution and dried attemperature in range 100–110 °C. In the case ninhydrinreagent was used the purple-red spots on slight pink back-ground were observed. The FBK dyed the spots red in thecase of secondary amines and violet in the case of primaryamines.3. Results and DiscussionPTIC used as a derivatisation agent transforms ami-nes to phenyl thiocarbamyl derivatives. This is why a dif-ferent mobile phase has to be used.In Tables 1 and 2, the values of Rf and detection li-mits for amphetamines are compared, corresponding todifferent visualization methods, after derivatisation ofanalytes.The application of iodine azide reaction as the vi-sualization method gave the best results. Apart from thelow detection limits, an advantage of this visualizationsystem over other methods (iodine vapour and UV254),was the quality of the obtained chromatogram. The spotsappeared more visible better shaped, observed as whitespots on a violet-grey background, as compared with ot-her examined methods.Table 2. Rf and detection limits (nmol per spot) of amphetamines (without derivatisation; mobile phase – System II).Compounds Rf Visualization methodsUV254 iodine Ninhydrin FBK Marquis Simon's Ehrlich's vapour reagent reagent reagentAmphetamine 0.51 18.5 12.4 9.3 9.3 - 3.4 18.5Methamphetamine 0.42 16.8 11.2 8.4 8.4 - 1.7 –MDMA 0.41 8.6 8.6 6.5 6.5 2.6 1.3 –MDA 0.52 9.3 9.3 7.0 7.0 2.8 2.8 13.4PMMA 0.37 9.3 9.3 7.0 7.0 - 1.4 –PMA 0.49 10.1 10.1 7.6 7.6 - 3.0 15.2109Acta Chim. Slov. 2007, 54, 106–109Zakrzewska et al.:   Visualisation of Aphetamine and Its Analogues in TLCIn Table 2, the detection limits obtained without deri-vatisation are presented. In some cases, limits of detectionexceeded even by two orders the limits presented in Table1, e.g. amphetamine detected under UV light or MDMAusing ninhydrin as compared with iodine-azide procedure.The iodine-azide procedure was successfully ap-plied in determination of MDMA in samples that weresynthesized in our laboratory according to various re-ceipts which are used in clandestine laboratories.12,14 Thesamples obtained from isosafrol and piperonal were stu-died. On TLC plate MDMA spot (Rf = 0.39) was accom-panied by spots that came from derivatisation agent.4. ConclusionsIt has been shown that derivatisation of amphetamineand its analogues with phenyl isothiocyanate, a pre-chro-matographic step in thin layer chromatography, makes itpossible to decrease detection limits of all tested drugs ascompared with other usually used methods of visualizationof spots on TLC chromatograms, applied without derivati-sation. Also, it was shown that visualization procedure ba-sed on iodine azide reaction belongs among the most sen-sitive methods of detection of amphetamines after their de-rivatisation. Though in the present paper only the most po-pular drugs were tested, it may turn out that the procedureproposed will be effective for other amphetamine analo-gues and other drugs that contain amine group as well.5. References1. Annual report on the state of drugs problem in Europe, Euro-pean Monitoring Centre for drugs and Drugs Addiction,2005, http://ar2005.emcdda.europa.eu/en/home-en.html,(accessed: 30.07.2006).2. S. Wills, Drug of abuse, The Pharmaceutical Press, London,1997, 62–75.3. Sinnema, A. M. A. Verweij, Bulletin on Narcotics 1981, 3,37–54.4. W. Krawczyk, Profiling of drugs, Central Forensic Labora-tory of Police, Warszawa, 1998, (in Polish).5. http://www.erowid.org/psychoactives/psychoactives.shtml,(accessed: 30.07.2006).6. V. Puthaviriyakorn, N. Siriviriyasomboon, J. Phorachata, W.Pan-ox, T. Sasaki, K. Tanaka, Forensic Sci. Int. 2002, 126,105–113.7. Y. Makino, S. Kurobane, K. Miyasaka, K. Nagano, Micro-gram J. 2003, 1(3–4), 169–176.8. D.E Joseph (Ed.), Drug of abuse, Drug Enforcement Admi-nistration U.S Department of Justice, 2005, 54–56,www.dea.gov., (accessed: 30.07.2006).9. P. Adamowicz, E. Chudzikiewicz, W. Lechowicz, ProblemsForensic Sci. 2003, LVI, 98–106.10. T. A. D. Cason, Forensic Sci. Int. 2001, 119, 168–194.11. M. Lim, K. H. Ng, T. K. Lee, Forensic Sci. Int. 2003, 136Suppl. 1, 9.12. M. A. Verweij, Forensic Sci. Rev. 1992, 4, 137–145.13. M. A. Verweij, Forensic Sci. Int. 1990, 45, 91–96.14. S. Shulgin, A. Shulgin, PIHKAL – A chemical love story,Transform Press, Barkeley, 1991.15. Furnari, V. Ottaviano, F. Rosati, V. Tondi, Forensic Sci Int.1998, 92, 49–58.16. Mitrevski, Z. Zdravkovski, Forensic Sci. Int. 2005,152,199–293.17. S. P. Sharma, B. C. Purkait, S. C. Lahiri, Forensic Sci. Int.2005, 152, 235–240.18. M. Świst, J. Wilamowski, A. Parczewski, Forensic Sci. Int.2005, 152, 175–184.19. M. Świst, J. Wilamowski, A. Parczewski, Forensic Sci. Int.2005, 155, 100–111.20. J. Kulikowska, R. Celiñski, A. Soja, H. Sybirska, ProblemsForensic Sci. 2002, XLIX, 99–113.21. R. C. Schneider, K. A. Kovar, Forensic Sci. Int. 2003, 134,187–195.22. S. E .J. Bell, D. T. Burns, A. C. Dennis, L. J. Matchett, J. S.Speers, Analyst 2000, 125, 1811–1815.23. M. A. Shaw, H. W. Peel, J. Chromatogr. 1975, 104, 201–204.24. C. Moffat (Ed.): Clarke’s isolation and identification ofdrugs, The Pharmaceutical Press, London, 1986, 169–177. 25. K. Madej, M. Ka ĺa, Thin layer chromatography screening ofamphetamine, opiates and canabinoids using fluorescenceand colorimetric detection, in V. Spiehler (Eds.) Proceedingsof the 1998 Joint SOFT/TIAFT International Meeting,DABEFT: Newport Beach, Albuquerque, New Mexico,1998.26. I. Ojanpera, P. Lillsunde, J. Vartiovaara, E. Vuori, J. PlanarChromatogr. 1991, 4, 373–378.27. D. Kaźmierczak, W. Ciesielski, R. Zakrzewski, Liq. Chro-matogr. Rel. Techn. 2006, 29, 2425–2436. 28. D. Kaźmierczak, W. Ciesielski, R. Zakrzewski, M. Z·uber, J.Chromatogr. A 2004, 1059, 171–174.29. K. L Woo, Q. Ch. Hwang, H. S. Kim, J. Chromatogr. A 1996,740, 31–40.30. K. L Woo, Y. K. Ahon, J. Chromatogr. A, 1996, 740, 41–50.PovzetekZa detekcijo amfetaminov in analogov pri tenkoplastni kromatografiji smo uporabili derivatizacijo, ki ji sledi reakcija zjodovim in azidom. Derivatizacijsko reakcijo s fenil izotiocianatom smo izvedli neposredno na plo{~i pred razvijanjem.Naknadno smo plo{~e napr{ili z me{anico natrijevega azida in raztopine {kroba in jih nato izpostavili param joda. Mejedolo~ljivosti smo primerjali z drugimi obi~ajnimi tehnikami vizualizacije: UV, jodove pare, reagenti po Marquisu andSimonu, ninhidrin, Fast Black K.

Visualization of amphetamine and its analogues in TLC

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Visualization of amphetamine and its analogues in TLC

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