2994 Chem. Commun., 2012, 48, 2994–2996 This journal is c The Royal Society of Chemistry 2012

Cite this: Chem. Commun., 2012, 48, 2994–2996

A turn-on fluorogenic probe for detection of MDMA from ecstasy tabletsw

Daniel Moreno, Borja Dıaz de Grenu, Begona Garcıa, Saturnino Ibeas and Tomas Torroba*

Received 14th December 2011, Accepted 20th January 2012

DOI: 10.1039/c2cc17823k

We report a fluorogenic probe that is able to discriminate a

range of primary or secondary biogenic amines and their natural

or synthetic mimics, in water or buffer, by means of the turn-on

transient generation of green fluorescence, with high quantum

yields and low detection limits, thus making the system suitable for

the detection of abuse drugs, such as MDMA, from ecstasy tablets.

Some biogenic amines are neurotransmitters,1 their appropriate

physiological interactions are crucial for a healthy condition and

their synthetic mimics are some of the most widespread abuse

drugs.2 Having in common only amine groups, discrimination

between the structurally diverse biogenic amines and their

synthetic mimics is only possible by lengthy chromatographic

methods that do not allow in-field assays.3 Fluorimetric4 reagents

for fast detection of amines are a good alternative but these

methods suffer from lack of selectivity for biogenic amines. With

this in mind, we have developed a new bis-diarylurea receptor

tagged with two units of a new highly solvatochromic fluorescent

indicator. In this communication, we aim to report our findings

on the selective fluorescent discrimination of biogenic amines and

their natural or synthetic mimics such as ecstasy.

The synthesis of the fluorescent probe is depicted in Scheme 1.

Compound 15 is a pale yellow (lmaxabs = 398 nm, e =

34500 M�1 cm�1, in CH2Cl2), highly solvatochromic fluorescent

indicator (lmaxem= 537 nm, quantum yieldF=0.6 in CH2Cl2).

Reaction of 1 with bis-isocyanate 2 in chloroform for 2 days at

room temperature afforded bis-diarylurea 3 in 77% yield, fully

characterized by spectroscopic and analytical techniques (see

ESIw). It is known that arylureas increase their colour in the

presence of anions6 but, in our case, 3 diminished its yellow

colour (lmaxabs E 400 nm in DMSO) in the presence of very

few anions, showing very little increase of fluorescence.

Although alkylurea-derivatives of 1 are indeed fluorescent,5

bis-diarylurea 3 showed only a slight fluorescence because of

the effective quenching of the fluorescence by the electron-rich

p-substituent arylether group. It is known that, in related

systems, quenching of the luminescence is assigned to a photo-

induced electron transfer (PET) from the electron-rich alkoxy-

benzene unit to the fluorescent unit.7 Therefore compounds that

could get involved in intermolecular photoinduced electron

transfer processes, such as the classic processes between acceptor

dyes and amines,8 could restore the original fluorescence of the

aminoindane group, giving rise to fluorescent probes for amines.

Consequently, we tested solutions of 3 (10�4 M in DMSO) with a

large number of amines of different types, primary, secondary,

tertiary, aliphatic, aromatic or heterocyclic, including diverse

functionalities in their structures, and observed the influence of

the structure of amine on the generation of fluorescence (see

ESIw). Thus, primary or secondary aliphatic amines, including

benzylic or allylic amines, were best suited for the generation of

fluorescence in solutions of 3, but tertiary aliphatic amines,

primary, secondary or tertiary anilines and aromatic amines such

as pyridine, pyrrole, indole or carbazole did not show any effect.

Diamines such as o/m/p-phenylenediamines, proton sponge,

9,10-diaminophenanthrene, or DMAP did not show any effect.

The only exceptions were DBU, DBN and, more weakly,

DABCO. Several amines of natural origin belonged to the class

of amines for which the fluorogenic probe 3 was able to generate

fluorescence, therefore we tested solutions of 3 with 11 common

biogenic amines, selected from the most representative series

of biological importance. Thus, upon adding one or more

equivalents of commercial biogenic amines in water to solutions

of 3 (10�4 M in DMSO), the initial yellow colour of these

solutions slightly lightened and an intense green fluorescence

appeared under the UV lamp (lexc = 366 nm). We took

quantitative measurements from representative examples, thus

the quantum yield for 3 and cadaverine (1 equiv.) in DMSO

was F = 0.57, and for 3 and tryptamine (1 equiv.) QY was

F = 0.43. Some natural ephedrines or synthetic amphetamine

and its analogues, 3,4-methylene-dioxyamphetamine (MDA)

and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy),

are closely related to biogenic amines (Fig. 1). They are

important therapeutic and recreational drugs, and constitute

a large proportion of currently used drugs of abuse (ecstasy

consumption is second only to cannabis in several countries).9

Therefore, we have studied the changes in fluorescence of 3 in

Scheme 1 Synthesis of fluorescent probe 6.

Departamento de Quımica, Facultad de Ciencias,Universidad de Burgos, 09001 Burgos, Spain.E-mail: ttorroba@ubu.es; Fax: +34 947258831; Tel: +34 947258088w Electronic supplementary information (ESI) available: Experimentalprocedures, physical and spectral data of new products, fluorimetricstudies and NMR titrations. See DOI: 10.1039/c2cc17823k

ChemComm Dynamic Article Links

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This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 2994–2996 2995

the presence of ephedrine, pseudoephedrine, amphetamine, MDA

and MDMA,10 in order to detect and differentiate their presence.

Thus, upon adding one or more equivalents of ephedrines or

amphetamines in water to solutions of 3 (10�4 M in DMSO) the

yellow colour of the solutions lightened very little but an intense

green fluorescence appeared under the UV lamp (lexc = 366 nm)

(Fig. 1). MDA andMDMA, obtained as hydrochloride salts, were

studied in neutralized aqueous solutions as well as in neutralized

solutions in HEPES, pH = 8.2, showing comparable results.

The rate of generation, intensity and duration of the fluorescence

were different in every case, which can be seen by the naked eye,

therefore we studied the kinetics of the interaction of probe 3

with 13 representative biogenic amines or their mimics, under the

same experimental conditions, by stopped-flow measurements

(Bio-Logic MOS-450/SFM-400) using both fluorescence and

absorbance detection, because the absorption peak of the probe

3/amine complex blue shifts to UV ranges due to moderate

disruption of conjugation of the chromophore. Conditions for

pseudo-first-order kinetics were always maintained by using

excess of amine in water over the concentration of 3 in DMSO.

Ten-fold repeated runs were averaged and analyzed by non-

linear functions. Two relaxation times (fast and slow) were

observed by fluorescence detection (Fig. 2A) whereas only a

single effect was observed by absorbance detection (Fig. 2B).

The absorbance effect is in fairly good agreement with the

fast relaxation time deduced from fluorescence measurements;

this finding reveals that the fast relaxation time, denoted as kfv,

is related to fluorescent complex formation, whereas the slow

rate constant, kqv, is related to a quenching process. The two

observed rate constants, kfv and kqv, differing by about one

order of magnitude, are shown in the ESI.w It is worth noting

that MDMA had the highest kfv and the lowest kqv values,

which will be important for its practical detection from ecstasy

tablets. Fluorescent titrations were studied by preparing fresh

individual solutions, corresponding to each titration point,

and then measuring them independently at a fixed time. In this

way, the quenching effect was circumvented and we obtained

classic turn-on fluorescence titration curves and titration profiles

that nicely fitted 1 : 1 complexes, fromwhich the binding constants

were calculated (see ESIw). As a typical example, quantitative

fluorescent titration of a 10�4 M solution of 3 in DMSO and

spermine in water (lexc = 288 nm) showed a 14-fold increase of

the intensity of the green emission centred at 517 nm as spermine

was added, the titration profile fitted a 1 : 1 binding model (Fig. 3).

Epimers such as ephedrine and pseudoephedrine gave similar

binding constants.

Titrations were also performed by dissolving the biogenic

amines in buffer (HEPES, pH = 8.2), although in these cases

the binding constants were different to the ones obtained by

dissolving the amines in water. Job0s plot analysis of fluores-

cence titrations of 3 and tryptamine or spermine revealed a

maximum at 50% mole fraction, according to the proposed

1 : 1 binding stoichiometry (Fig. 3, for spermine). It is noticeable

that the formed fluorescent intermediate was always a 1 : 1

complex, independently of the number of amino groups in the

analyte. We performed 1H NMR titrations of 3 (10�1 M in

DMSO-d6) with a simple secondary amine (pyrrolidine in D2O

or DMSO-d6), which showed only coordination to the urea

group but, after addition of one or more equivalents of amine,

the NMR signals enlarged substantially, therefore we performed

the EPR spectrum of the mixture, which showed signals consistent

with the formation of a stable bi-radical complex. Therefore all

experiments pointed to the formation of an initial intermolecular

charge-transfer fluorescent complex between the amine donor

and the bisurea acceptor that evolved by slow formation of

stable bi-radical species (see ESIw). We calculated some detec-

tion limits of 10�4 M solutions of 3 in DMSO, calculated in

fluorescence emission by the blank variability method,11 and

selected amines. According to a turn-on fluorescent process,

the experimental values were low. Thus, the detection limit for

cadaverine in water was 6.13 � 10�8 M, for cadaverine in

HEPES, pH= 8.2 was 9.51� 10�7 M, for dopamine in HEPES,

Fig. 1 Left: structures of ephedrines and amphetamines. Right:

addition of 2 equiv. of ephedrines or amphetamines in water to 10�4 M

solutions of 3 in DMSO: eph (ephedrine), pse (pseudoephedrine),

amp (amphetamine),MDA (3,4-methylenedioxyamphetamine), MDMA

(3,4-methylenedioxymethamphetamine)

Fig. 2 Stopped flow experiments on the 3-spermidine system recorded

by (A) fluorescence and (B) absorbance detection; in the inset, absorption

spectra of (a) 3 and (b) 3/spermidine complex. (A) lexc = 288 nm,

lem = 517 nm, (B) l= 288 nm. Cspd = 8 � 10�5 M, C6 = 8�10�6 M,

16% v/v water/DMSO, T = 25 1C. Continuous lines show the

fitting of experimental data (A) F = FN + afe�k1t + aqe

�k2t and

(B) A = AN + ae�kt, where k1 = kfv, k2 = kqv; F, FN, A and AN, the

fluorescence and absorbance recorded at t= t and t=N respectively;

af, aq and a stand for the amplitudes of the two florescence and one

absorbance kinetic processes.

Fig. 3 Fluorescence titration curves and titration profile of 10�4 M

solutions of 6 in DMSO and aqueous solutions of spermine. lexc =288 nm, T = 25 1C. Inset: Job0s plot analysis of 3 (10�4 M, DMSO)

and spermine. lexc = 288 nm, lem = 517 nm, T = 25 1C.

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2996 Chem. Commun., 2012, 48, 2994–2996 This journal is c The Royal Society of Chemistry 2012

pH = 8.2 was 6.41 � 10�6 M, for tryptamine in water was

8.35 � 10�7 M, for ephedrine in water was 1.90 � 10�7 M, for

pseudoephedrine in water was 1.25� 10�6 M, for MDA in water

was 9.92� 10�7 M and forMDMA in water was 1.27� 10�7 M.

The detection limit for ephedrine in water and a 5 � 10�5 M

solution of 3 in DMSO, by the least median squares regression

method,12 was found to be 5.19 � 0.89 � 10�7 M, which was of

the same order as in the previous method (see ESIw). The

obtained binding constants (logKeq), rate constants (kvf, kv

q)

and the pKa values of the 13 amines, measured in 40% and

80% w/w DMSO/H2O mixtures and calculated for 80% w/w

DMSO/H2O (see ESIw), were used as variable inputs in a

principal components analysis (PCA). The purpose of the

analysis was to obtain a small number of linear combinations

of the four variables, which accounted for most of the variability

in the data. In this case, two components were extracted,

together they accounted for 72% of the variability in the original

data. Separation between data points on the PCA plot described

how unlike they are from one another. Thus, ephedrine and

pseudoephedrine clustered closely, as well as diamines and

polyamines, but amphetamine, MDA andMDMAwere spatially

separated between them, indicating the ability of the probe to

discriminate the diverse analytes from one another (Fig. 4).

To test a practical use of the probe, we prepared a sample of

ecstasy tablet9b containingMDMA (45%) and common additives

(sucrose, chalk dust, caffeine) and tested 20 ml of aqueous solutionof the sample and 3, in comparison to every pure component,

showing that the ecstasy tablet gave a fluorescent response as

good as pure MDMA, thus proving that the system is suitable for

a fast selective fluorescent detection of MDMA from ecstasy

tablets (Fig. 5). The selectivity of 3 for primary or secondary

amines could permit the detection of MDMA in the presence of

other common tertiary amine additives such as methylenedioxy-

pyrovalerone (MDPV, ‘‘bath salts’’) and some synthetic opioids

(such as meperidine) or cocaine analogs.

In summary, we have evaluated a fluorogenic probe that was

able to detect primary or secondary amines selectively from

anilines, tertiary and aromatic amines, and to discriminate a

range of primary or secondary biogenic amines and their natural

or synthetic mimics in water or buffer, by means of the turn-on

transient generation of green fluorescence, with high quantum

yields and low detection limits. Moreover, the sensitivity to the

detected amines was preserved in a large extension by performing

the experiments in the presence of other organic or inorganic

compounds such as those found in commercial formulations. In

this way, the system was suitable for the quantitative detection of

abuse drugs, such as MDMA, from ecstasy tablets.

We gratefully acknowledge financial support from the

Ministerio de Ciencia e Innovacion, Spain (Projects

CTQ2009-12631-BQU and CTQ 2009-13051-BQU), Junta de

Castilla y Leon, Consejerıa de Educacion y Cultura y Fondo

Social Europeo (Projects BU023A09, GR170 and GR 257).

Notes and references

1 D. Lecca and M. P. Abbracchio, Neurochem. Int., 2008, 52, 339.2 E. Turillazzi, I. Riezzo, M. Neri, S. Bello and V. Fineschi,Curr. Pharm. Biotechnol., 2010, 11, 500.

3 (a) W. H. A. de Jong, M. H. L. I. Wilkens, E. G. E. de Vries andI. P. Kema, Anal. Bioanal. Chem., 2010, 396, 2609;(b) E. Dadakova, M. Krizek and T. Pelikanova, Food Chem.,2009, 116, 365; (c) A. Onal, Food Chem., 2007, 103, 1475–1486.

4 (a) B. Bao, L. Yuwen, X. Zheng, L. Weng, X. Zhu, X. Zhan andL. Wang, J. Mater. Chem., 2010, 20, 9628; (b) A. J. Zucchero,P. L. McGrier and U. H. F. Bunz, Acc. Chem. Res., 2010, 43, 397;(c) P. L. McGrier, K. M. Solntsev, S. Miao, L. M. Tolbert,O. R. Miranda, V. M. Rotello and U. H. F. Bunz, Chem.–Eur. J.,2008, 14, 4503; (d) F. Reviriego, P. Navarro, E. Garcia-Espana,M. T. Albelda, J. C. Frias, A. Domenech, M. J. R. Yunta, R. Costaand E. Orti, Org. Lett., 2008, 10, 5099; (e) L.-G. Qiu, Z.-Q. Li,Y. Wu, W. Wang, T. Xu and X. Jiang, Chem. Commun., 2008, 3642;(f) Q. Wang, X. Chen, L. Tao, L. Wang, D. Xiao, X.-Q. Yu andL. Pu, J. Org. Chem., 2007, 72, 97.

5 D. Moreno, J. V. Cuevas, G. Garcia-Herbosa and T. Torroba,Chem. Commun., 2011, 47, 3183.

6 (a) V. Amendola, L. Fabbrizzi and L. Mosca, Chem. Soc. Rev.,2010, 39, 3889; (b) A.-F. Li, J.-H. Wang, F. Wang and Y.-B. Jiang,Chem. Soc. Rev., 2010, 39, 3729; (c) R. M. Duke, E. B. Veale,F. M. Pfeffer, P. E. Kruger and T. Gunnlaugsson, Chem. Soc. Rev.,2010, 39, 3936; (d) C. Caltagirone and P. A. Gale, Chem. Soc. Rev.,2009, 38, 520.

7 L. Flamigni, B. Ventura, A. Barbieri, H. Langhals, F. Wetzel,K. Fuchs and A. Walter, Chem.–Eur. J., 2010, 16, 13406.

8 (a) S. Sarkar, R. Pramanik, C. Ghatak, V. G. Rao and N. Sarkar,J. Chem. Phys., 2011, 134, 074507; (b) A. Chakraborty, D. Seth,P. Setua and N. Sarkarb, J. Chem. Phys., 2008, 128, 204510;(c) M. Kumbhakar, S. Nath, T. Mukherjee and H. Pal, J. Chem. Phys.,2005, 123, 034705.

9 (a) F. Schifano, Psychopharmacology (Berlin), 2004, 173, 242;(b) A. C. Parrott, Psychopharmacology (Berlin), 2004, 173, 234.

10 Synthesis: amphetamine: R. A. Smith, R. L. White and A. Krantz,J. Med. Chem., 1988, 31, 1558; MDAand MDMA: N. Milhazes,P. Martins, E. Uriarte, J. Garrido, R. Calheiros, M. P. M.Marques and F. Borges, Anal. Chim. Acta, 2007, 596, 231.

11 D. L. Massart, B. G. M. Vandeginste, L. M. C. Buydens,S. De Jong, P. J. Lewi and J. Smeyers-Verbeke, Handbook ofChemometrics and Qualimetrics: Part A, Elsevier, Amsterdam,The Netherlands, 1997, ch. 13, 379.

12 M. A. Alonso-Lomillo, J. M. Kauffmann andM. J. Arcos-Martinez,Biosens. Bioelectron., 2003, 18, 1165.

Fig. 4 PCA score plot indicating the separation and discrimination of

13 amines by 3: 1: tryptamine, 2: tyramine, 3: putrescine, 4: cadaverine,

5: spermidine, 6: spermine, 7: dopamine, 8: serotonin, 9: ephedrine,

10: pseudoephedrine, 11: amphetamine, 12: MDA, 13: MDMA.

Fig. 5 Addition of 1 equiv. of MDMA or ecstasy components in water

[sucrose, dispersed chalk, caffeine, ecstasy (20 ml of MDMA (45%) +

sucrose+ chalk+ caffeine in water)] to 10�4M solutions of 3 in DMSO.

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