Spectrofluorimetric Study of the Effects of Cyclodextrins on the Acid–Base Equilibria of Harmine and Harmane† L. Mart�ýn, M. A. Mart�ýn and B. del Castillo* Seccion Departamental de Qu�ýmica Anal�ýtica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040-Madrid, Spain b-Carboline alkaloids are important compounds because they exhibit a variety of pharmacological actions. Their acid–base behaviour can be studied by spectrofluorimetry since these molecules present a remarkable native luminescence.Acid–base equilibria depend on the environment of the molecules and inclusion into cyclodextrin (CD) cavities shifts the acid–base equilibria and alters the apparent pKa values. The influence of CDs on the acid–base equilibria of the model b-carbolines harmine and harmane is described. b-CD and g-CD and the modified b-CDs hydroxypropyl-b-CD (HPb-CD), 2,6-di-O-methyl-b-CD (DMb-CD) and 2,3,6-tri-O-methyl-b-CD (TMb-CD) were used to form the corresponding complexes with harmine and harmane in the pH range 7.8–8.0.In these buffered solutions the complexes with the different CDs exhibit an emission band with resolved peaks at 360 and 380 nm corresponding to the neutral form of harmane and with a remarkable enhancement in the emission intensity compared with aqueous solution. In the case of the complexes with b-CD and g-CD, both the cationic and the neutral emission bands appear. However, for g-CD the cationic band is more intense than the neutral band, the inverse being true for b-CD.In homogeneous aqueous solution at this pH value the cationic band is the only one observed and therefore the presence of the neutral band indicates the formation of inclusion complexes. In the harmane–HPb-CD complexes, the emission bands ascribed to the anionic form are observed after addition of NaOH. This emission is only observed in homogeneous aqueous solution in strongly alkaline media outside the normal pH range.Keywords: Harmane; harmine; cyclodextrin complexes; fluorescence Harmine and harmane (Fig. 1) are b-carboline alkaloids which exhibit a notable native fluorescence and a peculiar acid–base behaviour in the ground and excited states. Thus, the pyridine nitrogen behaves as a base and is easily protonated, and therefore all b-carboline derivatives studied by Bal�on et al.1 present pKa values for this process that vary from 6.2 to 9.5. On the other hand, the pyrrolic nitrogen is acidic and loses its proton in alkaline media, although outside the pH scale (pH > 14).1,2 This behaviour is a typical consequence of the chemical characteristics due to the presence of a p-deficient pyridine ring fused to an electron-excessive indole ring.The acid–base behaviour in the ground state can be easily followed by UV/VIS spectrophotometry, observing the characteristic absorption band which can be attributed to cationic, neutral and anionic species.2,3 However, the acid–base behaviour in the excited state changes remarkably and both the basicity of pyridine and the acidity of pyrrole rings are strongly increased and therefore the pKa* values differ from the pKa.3,4 In the excited state it is also possible to observe another species involved in the acid–base equilibria, which Sakurovs and Ghiggino5 describe as a zwitterion.According to Sakurovs and Ghiggino,5 proton transfer in the excited state is very rapid and excitation of neutral or anionic species formed in the ground state produces the corresponding fluorescent cationic or zwitterionic species in the excited state. Inclusion in cyclodextrin (CD) cavities notably modifies chemical properties such as acid–base or redox behaviour and reactivity.6 A number of groups have studied changes in the acid–base equilibria for several fluorescent molecules included in CDs.Thus, we have shown that modified b-CDs alter proton transfer in carbazole and ellipticine.7 Chattopadhyay8 described differences in the acid–base behaviour of carbazole in b- and g- CD which differ from that observed in homogeneous aqueous solutions.Dissociation processes of 1-naphthol are seriously hampered after inclusion in modified b-CDs,9 a phenomenon which is easily monitored by spectrofluorimetry. The increase or decrease in the observed pKa values after inclusion complex formation depends on the chemical characteristics of the guest molecule and thus the apparent pKa values of nitrophenol derivatives decrease after the inclusion processes.10,11 However, carboxylic acids show the opposite effect and the observed pKa values are increased after inclusion; this is the case for the 1-adamantanecarboxylic acid series,12 cinnamic acid and its analogues13and prostaglandins.14 Considering that b-carbolines are highly fluorescent,15,16 reversed-phase HPLC with fluorimetric detection,17,18 is a very useful technique to determine these compounds in biological fluids.However, problems related to the coexistence of several ionized and neutral species may cause difficulties in the detection. In this present paper we describe the inclusion complexes of harmine and harmane and several CDs as well as the consequences of the complexation processes on the acid– base equilibria of these compounds. We conclude that inclusion into CD allows one to observe in aqueous solution and in the region of neutral pH the emission corresponding to the neutral species of harmine and harmane, an unprecedented and analytically useful observation.Experimental Apparatus and Reagents Uncorrected fluorescence spectra were measured with a Perkin- Elmer (Norwalk, CT, USA) MPF-2A fluorimeter (xenon lamp, † Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17–19, 1996. Fig. 1 Structures of harmine and harmane. Analyst, January 1997, Vol. 122 (45–49) 45150 W). All reagents and solvents were of analytical-reagent grade and were used without further purification. Harmine and harmane (free bases) were purchased from Aldrich (Milwaukee, WI, USA), the cyclodextrins (b-CD, HPb-CD, DMb-CD, TMb- CD and g-CD) from Sigma (St. Louis, MO, USA), and ethanol, sodium bromide and chloride from Merck (Darmstadt, Germany). Water was doubly distilled and de-ionized prior to its use. Procedures Freshly prepared ethanolic solutions of harmine and harmane were prepared at a 0.001 m concentration.Aliquots of 10 ml of these solutions were taken and placed in a round-bottomed flask. The solvent was evaporated under reduced pressure at room temperature and then 10 ml of aqueous solutions of the different CDs at a concentration of 0.01 m were added. The solutions of CDs were prepared in water or buffered aqueous solutions and left to stabilize for 24 h prior to their use in order to ensure complete dissolution.The complexes were prepared at different pH values. Besides de-ionized water (pH 5.5), Britton– Welford titrated solutions (0.2 m KH2PO4 with the desired volume of 0.2 m NaOH) were employed to obtain the required pH values. In the case of complexes prepared at pH 7.8, CD solutions were dissolved in the corresponding buffered aqueous solutions. However, for pH 11.0 this was obtained by addition of a suitable amount of NaOH to the aqueous CD solution. The final concentration of b-carboline in the different solutions was 1.0 3 1026 m.The b-carboline–CD solutions were stirred magnetically for 18–48 h in order to obtain the inclusion complexes. Acid–base equilibria in aqueous and CD solutions were studied using the above-mentioned procedure to prepare the inclusion complexes. When the complexes were obtained, successive aliquots of 10 ml of NaOH (10 m) were added in order to study the shifts in the proton transfer processes. Fluorescence quenching of b-carbolines was studied using bromide ion (NaBr) as quencher.NaCl was added to the complex solutions to achieve a constant ionic strength (1.0 m). Aliquots of 10 ml of the NaBr solution were added to study the quenching effect. The concentration of bromide ion varied in the complex solution from 0.001 to 0.1s and Discussion We studied the influence of CD complexation on the acid–base equilibria of harmine and harmane by spectrofluorimetry, believing that this process could seriously affect the determination of these compounds when CDs are employed.Figs. 2 and 3 show the emission spectra of harmane in ethanolic and aqueous solutions. In ethanolic solution, emission bands appear at 360 and 380 nm, which can be attributed to the neutral form. Addition of small amounts of HCl (1 m) caused the appearance of a band at 430 nm, which can be attributed to the cationic form. Addition of NaOH (1 m) induced a decrease in the intensity of the neutral band, together with a very weak increase in the emission at 480 nm which, according to other workers,1,3,5 is due to the formation of a zwitterionic species.The addition of more concentrated NaOH solutions (10 m) or solid NaOH did not cause the appearance of an anionic band. This happened also when harmine and harmane were dissolved in other organic solvents (hexane or propan-1-ol), and also when triethylamine was added as a base. In agreement with observations by Sakurovs and Ghiggino,5 the excitation spectra were the same for cationic, neutral and zwitterionic species.The same tests were performed in aqueous solutions (Fig. 3) and the behaviour was different, since the cationic band only appeared in the aqueous solution at 430 nm, and the addition of acid (1 m HCl) increased slightly this emission corresponding to the cationic band. Addition of NaOH (1 m) produced the zwitterionic band (480 nm) with a notable fluorescence intensity compared with the ethanolic solution and the neutral (370 nm) band.It is important to note that in all solvents studied the emission intensity of the cationic band was considerably higher than that of the corresponding neutral or zwitterionic bands. Addition of more concentrated NaOH (10 m) did not cause the anionic band to appear, because it is necessary to work outside the pH scale.1,2 Fig. 2 Uncorrected excitation and emission spectra of harmane in ethanolic solution (1).Same sample after addition of small amounts of HCl (2) and NaOH (3). Fig. 3 Uncorrected excitation and emission spectra of harmane in aqueous solution (1). Same sample after addition of small amounts of HCl (2) and NaOH (3). The fluorescence intensity for solution 1 is three times lower than that for solution 2 and that for solution 3 is 81 times lower than that for solution 2. 46 Analyst, January 1997, Vol. 122Inclusion complexes with the different CDs were prepared for both b-carbolines studied.Considering the acid–base behaviour of these compounds, the increase in the emission intensity and the shifts in the emission maxima or changes in the fluorescence lifetime or fluorescence quantum yield are proof of the inclusion processes.19 With this in mind, we tried to prepare the inclusion complexes at different pH values (5.5, 7.8, 11.0). Thus, at pH 5.5 only the cationic form was observed, with a weak (5%) increase in the fluorescence intensity of the complex solution with respect to the aqueous solutions of harmine and harmane.Therefore, the formation of inclusion complexes with cationic species is unlikely. This may be due to the fact that cationic species are water soluble and therefore their tendency to be included is low. In the case of HPb-CD, after several days under magnetic stirring the emission of the neutral form together with the predominant cationic emission were observed. A pH of 7.8 was selected because it is very close to the pKa values of harmine and harmane and under such conditions the same concentration of the cationic and neutral species should be present in the ground state.Therefore, inclusion of the neutral species should be favoured, shifting the acid–base equilibria. Under such conditions we obtained the neutral form with a fluorescence intensity higher than that in water. The spectral shape resembles that observed for the neutral form in ethanol; however, in the presence of the CDs it is better resolved and two peaks appear.When complexes were prepared at pH 11.0, the emission corresponding to the neutral form could be observed, but some differences in the spectral shape with respect to those obtained at pH 7.8 can be noted. Thus, the neutral band is not resolved into two peaks and only a peak at 365 nm appears, together with two shoulders at 380 and 420 nm. These changes in the spectral profile may be associated with the existence of non-complexed zwitterionic or anionic species which are present at such pH values in the ground state. However, the additions of NaOH show that this emission corresponds to the cationic form, which is probably produced in the excited state at this pH value.Excitation produces the cationic form owing to very rapid proton transfer in the excited state, as described by Sakurovs and Ghiggino.5 The excited states of b-carbolines are strong bases and deprotonate water.5 From these results, it can be deduced that pH 8.0 is the most suitable for studying the inclusion complexes. HP-b-CD was selected as a model CD to study the time necessary to obtain the inclusion complexes.The complexes with harmine and harmane were prepared following the experimental procedure described, and at pH 7.8–8.0 the b-carboline–HP-bCD complexes showed the neutral band only after 18–24 h under magnetic stirring. A time span of 24 h was chosen to obtain comparable results for the different complexes.At pH 11.0 the results did not improve compared with those obtained at pH 8.0 and, as these conditions can affect the CDs by hydrolysis,20 they were not used in subsequent experiments. Fig. 4 shows the emission spectra for the different harmane–CD complexes using the optimized procedure to obtain the complexes. After 24 h of magnetic stirring the spectra corresponding to the neutral form are present for modified b-CD (HPb-CD, DMb-CD and TMb-CD). For b- CD, a mixture of cationic and neutral forms exists but the equilibrium is shifted towards the neutral form because the intensity of the cationic band is very small.For g-CD, on the other hand, a mixture was also obtained at the same pH values, but the ratio of the species in equilibrium is the opposite to that observed with b-CD. As can be seen in Fig. 4, the shift in the acid–base equilibria depended on the type of CD since they displaced the acid-base equilibria in a different way because of different efficiencies in including the neutral form.In homogeneous aqueous solution, the fluorescence intensity of the cation is about 10 times higher than that of the neutral form, with a considerable difference in quantum yields1 [FF (cation) = 0.76 and FF (neutral) = 0.17], which makes it difficult to observe in aqueous or alkaline solution owing to the protonation of the excited neutral species by rapid proton exchange with the solvent.5 The formation of complexes at pH 7.8–8.0 induced the appearance of emission bands corresponding to the neutral form.This is an important proof of the formation of an inclusion complex because the excited states are protected inside the cavity of CDs and proton transfer is hampered. The fluorescence intensity for the neutral band of harmane in the complexes is only 2–3 times lower than that of the cation. This enhancement in the neutral emission shows the existence of inclusion complexes and also increases the analytical sensitivity.Fig. 5 shows the titration of harmane–HPb-CD complexes with NaOH. The complexes were prepared according to the experimental procedure at pH 7.8. It can be seen that the emission band for the neutral form is resolved into two peaks, and this does not happen in homogeneous aqueous solution. Increasing amounts of NaOH produced a notable decrease in the intensity of the neutral band with the appearance and increase of the corresponding anionic band; the resolution was better than in 14 m KOH1,2 and consequently the alkaloid remained inside the CD.The isoemissive point at 400 nm shows the existence of only two species (neutral and anionic) in the acid–base equilibria. The formation of an anion was not observed in aqueous solution except outside the pH scale and was a consequence of the deprotonation of the pyrrolic nitrogen. We have described previously the anion emission in micellar aqueous solutions of cetyltrimethylammonium bromide.20 Harmane –HPb-CD complexes were the only examples where the formation of an anionic species was observed and this can be attributed to the effects that the CD environment has on the acid–base properties of the guest molecules.Therefore, HPb- CD can isolate harmane from the aqueous environment, hampering proton transfer from the solvent in the excited state. When the neutral form is included in the different CDs, it is not possible to obtain the cationic form after excitation as in homogeneous aqueous solution. Fig. 6 shows the emission spectra of the harmane–b-CD complex at pH 8.0. In the case of b-CD, it was not possible to shift the inclusion equilibrium to obtain only the neutral form, Fig. 4 Uncorrected excitation and emission spectra of the complexes obtained from harmane and the different CDs studied at pH 7.8: (1) b-CD; (2) HPb-CD; (3) DMb-CD; (4) TMb-CD and (5) g-CD. Analyst, January 1997, Vol. 122 47either for harmane or for harmine. In such cases, when NaOH was added an increase in the fluorescence intensity for the neutral band was initially produced. This was followed by the transformation of the neutral form into the zwitterionic species, as shown by the appearance of the emission corresponding to this form. This behaviour can be explained by considering that the cationic form is solubilized in water and that its acid–base equilibrium is similar to that operating in aqueous solution or, alternatively, considering that the indolyl moiety is included with the pyridine end protruding from the CD cavity.This hypothesis is especially valuable in the case of harmine, where the steric hindrance due to the methoxy group on the indolyl moiety makes the inclusion difficult. The effects of NaOH additions on DMb-CD and TMb-CD complexes are very similar. The inclusion complexes produced the emission band corresponding to the neutral form but a tail was observed in the region of the cationic band (Fig. 4). When NaOH was added, a notable decrease was produced in the neutral band with a very weak increase in the emission at 480 nm (zwitterionic species). The absence of an isoemissive point indicates the presence of cationic, neutral and zwitterionic species in the equilibria. The different behaviour of the complexes with increasing amounts of NaOH causes a decrease in the intensity corresponding to the neutral form and an increase in the emission corresponding to the anionic form (in the case of HPb-CD) or the emission of the zwitterionic form for the other CDs.This means an important change in the acid–base behaviour of the b-carbolines as a consequence of the inclusion and the associated selectivity of CDs to include specific species of harmine or harmane. Table 1 summarizes the spectrofluorimetric characteristics of the inclusion complexes studied. While the emission corresponding to the neutral band is present in all cases, for b-CD and g-CD the maximum corresponding to the cationic band in the emission spectra is also present.The presence of this peak is a consequence of the geometrical characteristics of b-CD and g-CD. Thus, b-CD is too small to accommodate b-carboline completely and the molecules are included but the pyridine moiety protrudes from cavity and is protonated by water. The cavity of g-CD is large enough to accommodate harmane and harmine but water molecules may penetrate into the cavity, producing the cationic band when the neutral species are excited.In order to obtain more detailed information concerning the protection afforded by CDs to the excited states, fluorescence quenching of the b-carbolines–CD complexes was tested. This study was carried out for the neutral, cationic and zwitterionic forms obtained after addition of HCl or NaOH to the neutral complexes. In some complexes the correlation coefficient obtained using the Stern–Volmer treatment was not adequate, showing that other quenching mechanisms (static, collisional, energy transfer, etc.) should be present.However, the presence of bromide ion decreased the fluorescence intensity of the complexes. Table 2 shows that neutral forms are effectively protected in the harmane–CD complexes because the slope obtained was lower than that in homogeneous ethanolic solutions where only the neutral band is present. The microenvironments provided by CDs and homogeneous ethanolic solutions are similar.21 Nevertheless, the slope for these complexes was higher than that obtained in water.The emission Fig. 5 Spectrofluorimetric study of the changes in the acid–base equilibrium for the harmane–HPb-CD inclusion complex prepared in buffered aqueous solution (pH 7.8) with addition of 10 ml of 10 m NaOH. Fig. 6 Spectrofluorimetric study of the changes in the acid–base equilibrium for the harmane–b-CD inclusion complex prepared in buffered aqueous solution (pH 7.8) with addition of 10 ml of 10 m NaOH.The dotted line corresponds to the starting solution of the complex. Table 1 Fluorescence characteristics obtained for the CD inclusion complexes with harmane and harmine. The symbols > and < mean the relative fluorescence intensity of one maximum with respect to another Complex Harmane–HPb-CD Harmane–DMb-CD Harmane–TMb-CD Harmane–b-CD Harmane–g-CD lem/nm 362 > 380 362 > 380 362 > 380 362 > 380 > 430 362, 380 < 430 Complex Harmine–HPb-CD Harmine–DMb-CD Harmine–TMb-CD Harmine–b-CD Harmine–g-CD lem/nm 358, 370 358, 370 358, 370 358, 370 > 416 358 < 370 < 416 48 Analyst, January 1997, Vol. 122of the cationic form is also effectively protected against the quencher because the slopes are lower than in water and consequently it is possible that cationic species are partially included. However, in the case of the zwitterionic species a decrease in the fluorescence intensity was produced in the presence of bromide ion, but this phenomenon does not fit the Stern–Volmer relationship and the slopes for the complexes are higher than in aqueous solution.For the harmine–CD complexes, the neutral form presents a weaker quenching effect than in ethanolic solution. The cationic form is also less quenched compared with the corresponding aqueous solution, although the slopes are higher than for harmane complexes. This is certainly associated with the geometrical characteristics of the inclusion complexes.It can also be considered that a small fraction of cationic harmine is included and that the free cationic harmine is quenched as in aqueous solution. It is remarkable that with b-CD the slope is higher than in water, in contrast to the behaviour of g-CD, where the slope is strongly reduced. The differences in the geometries of harmine and harmane produce different inclusion complexes. Thus g-CD can include harmine by the methoxyindole moiety, but the size of the methoxy group does not allow such an inclusion in b-CD or in modified b-CDs.In conclusion, the existence of the inclusion complexes was verified by the changes in the spectrofluorimetric properties, in the acid–base behaviour and by the protection against the effects of the quenching. Nevertheless, the geometry of the inclusion complexes of harmine and harmane can be different because in the case of harmane the indole moiety can be included with the pyridine moiety protuding from the cavity.For harmine this is not easy and a more difficult penetration into the cavity is expected. Considering this selectivity of CDs, their use in chromatography can contribute to enhancing the chromatographic separation of b-carbolines owing to the frequent use of HPLC with fluorimetric detection in b-carboline determination.17,18 References 1 Bal�on, M., Hidalgo, J., Guardado, P., Mu�noz, M.A., and Carmona, C., J. Chem. Soc. Perkin Trans. 2, 1993, 99. 2 Bal�on, M., Mu�noz, M. A., Hidalgo, J., Carmona, M. C., and S�anchez, M., J. Photochem., 1987, 36, 193.F., Zabala, I., and Olba, A., J. Photochem., 1983, 23, 355. 4 Vander Donckt, E., Prog. React. Kinet., 1970, 5, 274. 5 Sakurovs, R., and Ghiggino, K. P., J. Photochem., 1982, 18, 1. 6 Bender, M. L., and Komiyama, M., Cyclodextrin Chemistry, Springer, Berlin, 1978. 7 Sba�ý, M., Ait Lyazidi, S., Lerner, D.A., del Castillo, B., and Mart�ýn, M. A., Anal. Chim. Acta, 1995, 303, 47. 8 Chattopadhyay, N., J. Photochem. Photobiol. A, 1991, 58, 31. 9 Takahashi, K., J. Chem. Soc. Chem. Commun., 1991, 929. 10 Connors, K. A., and Lipari, J. M., J. Pharm. Sci., 1976, 65, 379. 11 Lin, S. F., and Connors, K. A., J. Pharm. Sci., 1983, 72, 1333. 12 Eftink, M. R., Andy, M. L., Bystrom, K., Perlmutter, M. D., and Kristol, D. S., J. Am. Chem. Soc., 1989, 111, 6765 13 Connors, K. A., and Rosanske, T.W., J. Pharm. Sci., 1980, 69, 173. 14 Uekama, K., Hirayama, F., Nasu, S., and Matsuo, N., Chem. Pharm. Bull., 1978, 26, 3477. 15 Abramovitch, R. A., and Spenser, I. D., Adv. Heterocycl. Chem., 1964, 3, 79. 16 Dillon, J., Spector, A., and Nakanishi, K., Nature (London), 1976, 259, 422. 17 Bossin, T. R., and Faull, K. F., J. Chromatogr., 1988, 428, 229. 18 Moncrieff, J., J. Chromatrogr., 1989, 496, 269. 19 Szejtli, J., Cyclodextrins and Their Inclusion Complexes, Akad�emiai Kiad�o, Budapest, 1982. 20 Mart�ýn, L., Mart�ýn, M.A., and del Castillo, B., J. Fluorescence, in the press. 21 Frankewich, R. P., Thimmaiah, K. N., and Hinze, W. L., Anal. Chem., 1991, 63, 2924. Paper 6/02790C Received April 22, 1996 Accepted October 10, 1996 Table 2 Fluorescence quenching study of b-carboline–CD inclusion complexes b-Carboline–CD lem * r† m† b† Harmane–EtOH x 0.947 4.759 1.035 Harmane–H2O x 0.978 0.558 0.958 y 0.984 1.048 0.98 z 0.996 7.7445 0.9552 Harmane–HPb-CD x ‡ ‡ ‡ y ‡ ‡ ‡ z 0.91 5.4 1.03 Harmane–DMb-CD x 0.95 0.858 1.034 y ‡ ‡ ‡ z 0.97 4.454 1.077 Harmane–TMb-CD x 0.949 4.23 1.021 y ‡ ‡ ‡ z 0.95 5.2 1.04 Harmane–g-CD x 0.96 1.037 0.955 y ‡ ‡ ‡ z 0.94 4.17 1.04 Harmane–b-CD x 0.953 0.916 0.999 y 0.985 1.525 1.019 z 0.994 3.547 1.044 Harmine–EtOH x 0.9873 9.63 1.066 Harmine–H2O x 0.969 0.68 0.98 y 0.986 5.109 0.986 z 0.998 15.768 0.986 Harmine–HPb-CD x 0.974 1.83 1.033 y 0.846 1.23 1.082 z 0.965 9.47 1.085 Harmine–DMb-CD x 0.96 1.737 1.04 y 0.98 0.866 1.0007 z 0.994 11.27 1.034 Harmine–TMb-CD x 0.96 2.69 1.07 y 0.966 1.373 1.066 z 0.958 10.71 1.096 Harmine–g-CD x 0.814 0.32 1.045 y 0.97 2.2 1.052 z 0.83 3.7 1.23 Harmine–b-CD x 0.95 3.7 1.02 y 0.92 1.73 1.097 z 0.977 16.02 1.22 * l = Emission wavelength.x: l = 366 nm (harmane); l = 358 nm (harmine). y: l = 480 nm (harmane; l = 476 nm (harmine). z: l = 430 nm (harmane); l = 416 nm (harmine). † r = correlation coefficient; m = slope; b = intercept inordinate.‡ The values obtained could not be adjusted by the Stern–Volmer treatment: Fo/FA = 1 + K(Q). Analyst, January 1997, Vol. 122 49 Spectrofluorimetric Study of the Effects of Cyclodextrins on the Acid–Base Equilibria of Harmine and Harmane† L. Mart�ýn, M. A. Mart�ýn and B. del Castillo* Seccion Departamental de Qu�ýmica Anal�ýtica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040-Madrid, Spain b-Carboline alkaloids are important compounds because they exhibit a variety of pharmacological actions.Their acid–base behaviour can be studied by spectrofluorimetry since these molecules present a remarkable native luminescence. Acid–base equilibria depend on the environment of the molecules and inclusion into cyclodextrin (CD) cavities shifts the acid–base equilibria and alters the apparent pKa values. The influence of CDs on the acid–base equilibria of the model b-carbolines harmine and harmane is described. b-CD and g-CD and the modified b-CDs hydroxypropyl-b-CD (HPb-CD), 2,6-di-O-methyl-b-CD (DMb-CD) and 2,3,6-tri-O-methyl-b-CD (TMb-CD) were used to form the corresponding complexes with harmine and harmane in the pH range 7.8–8.0.In these buffered solutions the complexes with the different CDs exhibit an emission band with resolved peaks at 360 and 380 nm corresponding to the neutral form of harmane and with a remarkable enhancement in the emission intensity compared with aqueous solution. In the case of the complexes with b-CD and g-CD, both the cationic and the neutral emission bands appear.However, for g-CD the cationic band is more intense than the neutral band, the inverse being true for b-CD. In homogeneous aqueous solution at this pH value the cationic band is the only one observed and therefore the presence of the neutral band indicates the formation of inclusion complexes. In the harmane–HPb-CD complexes, the emission bands ascribed to the anionic form are observed after addition of NaOH.This emission is only observed in homogeneous aqueous solution in strongly alkaline media outside the normal pH range. Keywords: Harmane; harmine; cyclodextrin complexes; fluorescence Harmine and harmane (Fig. 1) are b-carboline alkaloids which exhibit a notable native fluorescence and a peculiar acid–base behaviour in the ground and excited states. Thus, the pyridine nitrogen behaves as a base and is easily protonated, and therefore all b-carboline derivatives studied by Bal�on et al.1 present pKa values for this process that vary from 6.2 to 9.5.On the other hand, the pyrrolic nitrogen is acidic and loses its proton in alkaline media, although outside the pH scale (pH > 14).1,2 This behaviour is a typical consequence of the chemical characteristics due to the presence of a p-deficient pyridine ring fused to an electron-excessive indole ring. The acid–base behaviour in the ground state can be easily followed by UV/VIS spectrophotometry, observing the characteristic absorption band which can be attributed to cationic, neutral and anionic species.2,3 However, the acid–base behaviour in the excited state changes remarkably and both the basicity of pyridine and the acidity of pyrrole rings are strongly increased and therefore the pKa* values differ from the pKa.3,4 In the excited state it is also possible to observe another species involved in the acid–base equilibria, which Sakurovs and Ghiggino5 describe as a zwitterion.According to Sakurovs and Ghiggino,5 proton transfer in the excited state is very rapid and excitation of neutral or anionic species formed in the ground state produces the corresponding fluorescent cationic or zwitterionic species in the excited state. Inclusion in cyclodextrin (CD) cavities notably modifies chemical properties such as acid–base or redox behaviour and reactivity.6 A number of groups have studied changes in the acid–base equilibria for several fluorescent molecules included in CDs.Thus, we have shown that modified b-CDs alter proton transfer in carbazole and ellipticine.7 Chattopadhyay8 described differences in the acid–base behaviour of carbazole in b- and g- CD which differ from that observed in homogeneous aqueous solutions. Dissociation processes of 1-naphthol are seriously hampered after inclusion in modified b-CDs,9 a phenomenon which is easily monitored by spectrofluorimetry. The increase or decrease in the observed pKa values after inclusion complex formation depends on the chemical characteristics of the guest molecule and thus the apparent pKa values of nitrophenol derivatives decrease after the inclusion processes.10,11 However, carboxylic acids show the opposite effect and the observed pKa values are increased after inclusion; this is the case for the 1-adamantanecarboxylic acid series,12 cinnamic acid and its analogues13and prostaglandins.14 Considering that b-carbolines are highly fluorescent,15,16 reversed-phase HPLC with fluorimetric detection,17,18 is a very useful technique to determine these compounds in biological fluids.However, problems related to the coexistence of several ionized and neutral species may cause diffion. In this present paper we describe the inclusion complexes of harmine and harmane and several CDs as well as the consequences of the complexation processes on the acid– base equilibria of these compounds.We conclude that inclusion into CD allows one to observe in aqueous solution and in the region of neutral pH the emission corresponding to the neutral species of harmine and harmane, an unprecedented and analytically useful observation. Experimental Apparatus and Reagents Uncorrected fluorescence spectra were measured with a Perkin- Elmer (Norwalk, CT, USA) MPF-2A fluorimeter (xenon lamp, † Presented at the VIIth International Symposium on Luminescence Spectrometry in Biomedical Analysis, Sophia Antipolis, Nice, France, April 17–19, 1996.Fig. 1 Structures of harmine and harmane. Analyst, January 1997, Vol. 122 (45–49) 45150 W). All reagents and solvents were of analytical-reagent grade and were used without further purification. Harmine and harmane (free bases) were purchased from Aldrich (Milwaukee, WI, USA), the cyclodextrins (b-CD, HPb-CD, DMb-CD, TMb- CD and g-CD) from Sigma (St. Louis, MO, USA), and ethanol, sodium bromide and chloride from Merck (Darmstadt, Germany).Water was doubly distilled and de-ionized prior to its use. Procedures Freshly prepared ethanolic solutions of harmine and harmane were prepared at a 0.001 m concentration. Aliquots of 10 ml of these solutions were taken and placed in a round-bottomed flask. The solvent was evaporated under reduced pressure at room temperature and then 10 ml of aqueous solutions of the different CDs at a concentration of 0.01 m were added.The solutions of CDs were prepared in water or buffered aqueous solutions and left to stabilize for 24 h prior to their use in order to ensure complete dissolution. The complexes were prepared at different pH values. Besides de-ionized water (pH 5.5), Britton– Welford titrated solutions (0.2 m KH2PO4 with the desired volume of 0.2 m NaOH) were employed to obtain the required pH values. In the case of complexes prepared at pH 7.8, CD solutions were dissolved in the corresponding buffered aqueous solutions.However, for pH 11.0 this was obtained by addition of a suitable amount of NaOH to the aqueous CD solution. The final concentration of b-carboline in the different solutions was 1.0 3 1026 m. The b-carboline–CD solutions were stirred magnetically for 18–48 h in order to obtain the inclusion complexes. Acid–base equilibria in aqueous and CD solutions were studied using the above-mentioned procedure to prepare the inclusion complexes.When the complexes were obtained, successive aliquots of 10 ml of NaOH (10 m) were added in order to study the shifts in the proton transfer processes. Fluorescence quenching of b-carbolines was studied using bromide ion (NaBr) as quencher. NaCl was added to the complex solutions to achieve a constant ionic strength (1.0 m). Aliquots of 10 ml of the NaBr solution were added to study the quenching effect. The concentration of bromide ion varied in the complex solution from 0.001 to 0.1 m.Results and Discussion We studied the influence of CD complexation on the acid–base equilibria of harmine and harmane by spectrofluorimetry, believing that this process could seriously affect the determination of these compounds when CDs are employed. Figs. 2 and 3 show the emission spectra of harmane in ethanolic and aqueous solutions. In ethanolic solution, emission bands appear at 360 and 380 nm, which can be attributed to the neutral form. Addition of small amounts of HCl (1 m) caused the appearance of a band at 430 nm, which can be attributed to the cationic form. Addition of NaOH (1 m) induced a decrease in the intensity of the neutral band, together with a very weak increase in the emission at 480 nm which, according to other workers,1,3,5 is due to the formation of a zwitterionic species. The addition of more concentrated NaOH solutions (10 m) or solid NaOH did not cause the appearance of an anionic band.This happened also when harmine and harmane were dissolved in other organic solvents (hexane or propan-1-ol), and also when triethylamine was added as a base.In agreement with observations by Sakurovs and Ghiggino,5 the excitation spectra were the same for cationic, neutral and zwitterionic species. The same tests were performed in aqueous solutions (Fig. 3) and the behaviour was different, since the cationic band only appeared in the aqueous solution at 430 nm, and the addition of acid (1 m HCl) increased slightly this emission corresponding to the cationic band.Addition of NaOH (1 m) produced the zwitterionic band (480 nm) with a notable fluorescence intensity compared with the ethanolic solution and the neutral (370 nm) band. It is important to note that in all solvents studied the emission intensity of the cationic band was considerably higher than that of the corresponding neutral or zwitterionic bands. Addition of more concentrated NaOH (10 m) did not cause the anionic band to appear, because it is necessary to work outside the pH scale.1,2 Fig. 2 Uncorrected excitation and emission spectra of harmane in ethanolic solution (1). Same sample after addition of small amounts of HCl (2) and NaOH (3). Fig. 3 Uncorrected excitation and emission spectra of harmane in aqueous solution (1). Same sample after addition of small amounts of HCl (2) and NaOH (3). The fluorescence intensity for solution 1 is three times lower than that for solution 2 and that for solution 3 is 81 times lower than that for solution 2. 46 Analyst, January 1997, Vol. 122Inclusion complexes with the different CDs were prepared for both b-carbolines studied. Considering the acid–base behaviour of these compounds, the increase in the emission intensity and the shifts in the emission maxima or changes in the fluorescence lifetime or fluorescence quantum yield are proof of the inclusion processes.19 With this in mind, we tried to prepare the inclusion complexes at different pH values (5.5, 7.8, 11.0).Thus, at pH 5.5 only the cationic form was observed, with a weak (5%) increase in the fluorescence intensity of the complex solution with respect to the aqueous solutions of harmine and harmane. Therefore, the formation of inclusion complexes with cationic species is unlikely. This may be due to the fact that cationic species are water soluble and therefore their tendency to be included is low. In the case of HPb-CD, after several days under magnetic stirring the emission of the neutral form together with the predominant cationic emission were observed.A pH of 7.8 was selected because it is very close to the pKa values of harmine and harmane and under such conditions the same concentration of the cationic and neutral species should be present in the ground state. Therefore, inclusion of the neutral species should be favoured, shifting the acid–base equilibria. Under such conditions we obtained the neutral form with a fluorescence intensity higher than that in water.The spectral shape resembles that observed for the neutral form in ethanol; however, in the presence of the CDs it is better resolved and two peaks appear. When complexes were prepared at pH 11.0, the emission corresponding to the neutral form could be observed, but some differences in the spectral shape with respect to those obtained at pH 7.8 can be noted. Thus, the neutral band is not resolved into two peaks and only a peak at 365 nm appears, together with two shoulders at 380 and 420 nm.These changes in the spectral profile may be associated with the existence of non-complexed zwitterionic or anionic species which are present at such pH values in the ground state. However, the additions of NaOH show that this emission corresponds to the cationic form, which is probably produced in the excited state at this pH value. Excitation produces the cationic form owing to very rapid proton transfer in the excited state, as described by Sakurovs and Ghiggino.5 The excited states of b-carbolines are strong bases and deprotonate water.5 From these results, it can be deduced that pH 8.0 is the most suitable for studying the inclusion complexes.HP-b-CD was selected as a model CD to study the time necessary to obtain the inclusion complexes. The complexes with harmine and harmane were prepared following the experimental procedure described, and at pH 7.8–8.0 the b-carboline–HP-bCD complexes showed the neutral band only after 18–24 h under magnetic stirring.A time span of 24 h was chosen to obtain comparable results for the different complexes. At pH 11.0 the results did not improve compared with those obtained at pH 8.0 and, as these conditions can affect the CDs by hydrolysis,20 they were not used in subsequent experiments. Fig. 4 shows the emission spectra for the different harmane–CD complexes using the optimized procedure to obtain the complexes.After 24 h of magnetic stirring the spectra corresponding to the neutral form are present for modified b-CD (HPb-CD, DMb-CD and TMb-CD). For b- CD, a mixture of cationic and neutral forms exists but the equilibrium is shifted towards the neutral form because the intensity of the cationic band is very small. For g-CD, on the other hand, a mixture was also obtained at the same pH values, but the ratio of the species in equilibrium is the opposite to that observed with b-CD. As can be seen in Fig. 4, the shift in the acid–base equilibria depended on the type of CD since they displaced the acid-base equilibria in a different way because of different efficiencies in including the neutral form. In homogeneous aqueous solution, the fluorescence intensity of the cation is about 10 times higher than that of the neutral form, with a considerable difference in quantum yields1 [FF (cation) = 0.76 and FF (neutral) = 0.17], which makes it difficult to observe in aqueous or alkaline solution owing to the protonation of the excited neutral species by rapid proton exchange with the solvent.5 The formation of complexes at pH 7.8–8.0 induced the appearance of emission bands corresponding to the neutral form.This is an important proof of the formation of an inclusion complex because the excited states are protected inside the cavity of CDs and proton transfer is hampered. The fluorescence intensity for the neutral band of harmane in the complexes is only 2–3 times lower than that of the cation.This enhancement in the neutral emission shows the existence of inclusion complexes and also increases the analytical sensitivity. Fig. 5 shows the titration of harmane–HPb-CD complexes with NaOH. The complexes were prepared according to the experimental procedure at pH 7.8. It can be seen that the emission band for the neutral form is resolved into two peaks, and this does not happen in homogeneous aqueous solution.Increasing amounts of NaOH produced a notable decrease in the intensity of the neutral band with the appearance and increase of the corresponding anionic band; the resolution was better than in 14 m KOH1,2 and consequently the alkaloid remained inside the CD. The isoemissive point at 400 nm shows the existence of only two species (neutral and anionic) in the acid–base equilibria. The formation of an anion was not observed in aqueous solution except outside the pH scale and was a consequence of the deprotonation of the pyrrolic nitrogen.We have described previously the anion emission in micellar aqueous solutions of cetyltrimethylammonium bromide.20 Harmane –HPb-CD complexes were the only examples where the formation of an anionic species was observed and this can be attributed to the effects that the CD environment has on the acid–base properties of the guest molecules. Therefore, HPb- CD can isolate harmane from the aqueous environment, hampering proton transfer from the solvent in the excited state.When the neutral form is included in the different CDs, it is not possible to obtain the cationic form after excitation as in homogeneous aqueous solution. Fig. 6 shows the emission spectra of the harmane–b-CD complex at pH 8.0. In the case of b-CD, it was not possible to shift the inclusion equilibrium to obtain only the neutral form, Fig. 4 Uncorrected excitation and emission spectra of the complexes obtained from harmane and the different CDs studied at pH 7.8: (1) b-CD; (2) HPb-CD; (3) DMb-CD; (4) TMb-CD and (5) g-CD.Analyst, January 1997, Vol. 122 47either for harmane or for harmine. In such cases, when NaOH was added an increase in the fluorescence intensity for the neutral band was initially produced. This was followed by the transformation of the neutral form into the zwitterionic species, as shown by the appearance of the emission corresponding to this form. This behaviour can be explained by considering that the cationic form is solubilized in water and that its acid–base equilibrium is similar to that operating in aqueous solution or, alternatively, considering that the indolyl moiety is included with the pyridine end protruding from the CD cavity.This hypothesis is especially valuable in the case of harmine, where the steric hindrance due to the methoxy group on the indolyl moiety makes the inclusion difficult. The effects of NaOH additions on DMb-CD and TMb-CD complexes are very similar.The inclusion complexes produced the emission band corresponding to the neutral form but a tail was observed in the region of the cationic band (Fig. 4). When NaOH was added, a notable decrease was produced in the neutral band with a very weak increase in the emission at 480 nm (zwitterionic species). The absence of an isoemissive point indicates the presence of cationic, neutral and zwitterionic species in the equilibria.The different behaviour of the complexes with increasing amounts of NaOH causes a decrease in the intensity corresponding to the neutral form and an increase in the emission corresponding to the anionic form (in the case of HPb-CD) or the emission of the zwitterionic form for the other CDs. This means an important change in the acid–base behaviour of the b-carbolines as a consequence of the inclusion and the associated selectivity of CDs to include specific species of harmine or harmane.Table 1 summarizes the spectrofluorimetric characteristics of the inclusion complexes studied. While the emission corresponding to the neutral band is present in all cases, for b-CD and g-CD the maximum corresponding to the cationic band in the emission spectra is also present. The presence of this peak is a consequence of the geometrical characteristics of b-CD and g-CD. Thus, b-CD is too small to accommodate b-carboline completely and the molecules are included but the pyridine moiety protrudes from cavity and is protonated by water.The cavity of g-CD is large enough to accommodate harmane and harmine but water molecules may penetrate into the cavity, producing the cationic band when the neutral species are excited. In order to obtain more detailed information concerning the protection afforded by CDs to the excited states, fluorescence quenching of the b-carbolines–CD complexes was tested. This study was carried out for the neutral, cationic and zwitterionic forms obtained after addition of HCl or NaOH to the neutral complexes.In some complexes the correlation coefficient obtained using the Stern–Volmer treatment was not adequate, showing that other quenching mechanisms (static, collisional, energy transfer, etc.) should be present. However, the presence of bromide ion decreased the fluorescence intensity of the complexes. Table 2 shows that neutral forms are effectively protected in the harmane–CD complexes because the slope obtained was lower than that in homogeneous ethanolic solutions where only the neutral band is present.The microenvironments provided by CDs and homogeneous ethanolic solutions are similar.21 Nevertheless, the slope for these complexes was higher than that obtained in water. The emission Fig. 5 Spectrofluorimetric study of the changes in the acid–base equilibrium for the harmane–HPb-CD inclusion complex prepared in buffered aqueous solution (pH 7.8) with addition of 10 ml of 10 m NaOH.Fig. 6 Spectrofluorimetric study of the changes in the acid–base equilibrium for the harmane–b-CD inclusion complex prepared in buffered aqueous solution (pH 7.8) with addition of 10 ml of 10 m NaOH. The dotted line corresponds to the starting solution of the complex. Table 1 Fluorescence characteristics obtained for the CD inclusion complexes with harmane and harmine. The symbols > and < mean the relative fluorescence intensity of one maximum with respect to another Complex Harmane–HPb-CD Harmane–DMb-CD Harmane–TMb-CD Harmane–b-CD Harmane–g-CD lem/nm 362 > 380 362 > 380 362 > 380 362 > 380 > 430 362, 380 < 430 Complex Harmine–HPb-CD Harmine–DMb-CD Harmine–TMb-CD Harmine–b-CD Harmine–g-CD lem/nm 358, 370 358, 370 358, 370 358, 370 > 416 358 < 370 < 416 48 Analyst, January 1997, Vol. 122of the cationic form is also effectively protected against the quencher because the slopes are lower than in water and consequently it is possible that cationic species are partially included.However, in the case of the zwitterionic species a decrease in the fluorescence intensity was produced in the presence of bromide ion, but this phenomenon does not fit the Stern–Volmer relationship and the slopes for the complexes are higher than in aqueous solution. For the harmine–CD complexes, the neutral form presents a weaker quenching effect than in ethanolic solution.The cationic form is also less quenched compared with the corresponding aqueous solution, although the slopes are higher than for harmane complexes. This is certainly associated with the geometrical characteristics of the inclusion complexes. It can also be considered that a small fraction of cationic harmine is included and that the free cationic harmine is quenched as in aqueous solution. It is remarkable that with b-CD the slope is higher than in water, in contrast to the behaviour of g-CD, where the slope is strongly reduced.The differences in the geometries of harmine and harmane produce different inclusion complexes. Thus g-CD can include harmine by the methoxyindole moiety, but the size of the methoxy group does not allow such an inclusion in b-CD or in modified b-CDs. In conclusion, the existence of the inclusion complexes was verified by the changes in the spectrofluorimetric properties, in the acid–base behaviour and by the protection against the effects of the quenching.Nevertheless, the geometry of the inclusion complexes of harmine and harmane can be different because in the case of harmane the indole moiety can be included with the pyridine moiety protuding from the cavity. For harmine this is not easy and a more difficult penetration into the cavity is expected. Considering this selectivity of CDs, their use in chromatography can contribute to enhancing the chromatographic separation of b-carbolines owing to the frequent use of HPLC with fluorimetric detection in b-carboline determination.17,18 References 1 Bal�on, M., Hidalgo, J., Guardado, P., Mu�noz, M.A., and Carmona, C., J. Chem. Soc. Perkin Trans. 2, 1993, 99. 2 Bal�on, M., Mu�noz, M. A., Hidalgo, J., Carmona, M. C., and S�anchez, M., J. Photochem., 1987, 36, 193. 3 Tomas, F., Zabala, I., and Olba, A., J. Photochem., 1983, 23, 355. 4 Vander Donckt, E., Prog. React. Kinet., 1970, 5, 274. 5 Sakurovs, R., and Ghiggino, K. P., J. Photochem., 1982, 18, 1. 6 Bender, M. L., and Komiyama, M., Cyclodextrin Chemistry, Springer, Berlin, 1978. 7 Sba�ý, M., Ait Lyazidi, S., Lerner, D. A., del Castillo, B., and Mart�ýn, M. A., Anal. Chim. Acta, 1995, 303, 47. 8 Chattopadhyay, N., J. Photochem. Photobiol. A, 1991, 58, 31. 9 Takahashi, K., J. Chem. Soc. Chem. Commun., 1991, 929. 10 Connors, K. A., and Lipari, J. M., J. Pharm. Sci., 1976, 65, 379. 11 Lin, S. F., and Connors, K. A., J. Pharm. Sci., 1983, 72, 1333. 12 Eftink, M. R., Andy, M. L., Bystrom, K., Perlmutter, M. D., and Kristol, D. S., J. Am. Chem. Soc., 1989, 111, 6765 13 Connors, K. A., and Rosanske, T. W., J. Pharm. Sci., 1980, 69, 173. 14 Uekama, K., Hirayama, F., Nasu, S., and Matsuo, N., Chem. Pharm. Bull., 1978, 26, 3477. 15 Abramovitch, R. A., and Spenser, I. D., Adv. Heterocycl. Chem., 1964, 3, 79. 16 Dillon, J., Spector, A., and Nakanishi, K., Nature (London), 1976, 259, 422. 17 Bossin, T. R., and Faull, K. F., J. Chromatogr., 1988, 428, 229. 18 Moncrieff, J., J. Chromatrogr., 1989, 496, 269. 19 Szejtli, J., Cyclodextrins and Their Inclusion Complexes, Akad�emiai Kiad�o, Budapest, 1982. 20 Mart�ýn, L., Mart�ýn, M.A., and del Castillo, B., J. Fluorescence, in the press. 21 Frankewich, R. P., Thimmaiah, K. N., and Hinze, W. L., Anal. Chem., 1991, 63, 2924. Paper 6/02790C Received April 22, 1996 Accepted October 10, 1996 Table 2 Fluorescence quenching study of b-carboline–CD inclusion complexes b-Carboline–CD lem * r† m† b† Harmane–EtOH x 0.947 4.759 1.035 Harmane–H2O x 0.978 0.558 0.958 y 0.984 1.048 0.98 z 0.996 7.7445 0.9552 Harmane–HPb-CD x ‡ ‡ ‡ y ‡ ‡ ‡ z 0.91 5.4 1.03 Harmane–DMb-CD x 0.95 0.858 1.034 y ‡ ‡ ‡ z 0.97 4.454 1.077 Harmane–TMb-CD x 0.949 4.23 1.021 y ‡ ‡ ‡ z 0.95 5.2 1.04 Harmane–g-CD x 0.96 1.037 0.955 y ‡ ‡ ‡ z 0.94 4.17 1.04 Harmane–b-CD x 0.953 0.916 0.999 y 0.985 1.525 1.019 z 0.994 3.547 1.044 Harmine–EtOH x 0.9873 9.63 1.066 Harmine–H2O x 0.969 0.68 0.98 y 0.986 5.109 0.986 z 0.998 15.768 0.986 Harmine–HPb-CD x 0.974 1.83 1.033 y 0.846 1.23 1.082 z 0.965 9.47 1.085 Harmine–DMb-CD x 0.96 1.737 1.04 y 0.98 0.866 1.0007 z 0.994 11.27 1.034 Harmine–TMb-CD x 0.96 2.69 1.07 y 0.966 1.373 1.066 z 0.958 10.71 1.096 Harmine–g-CD x 0.814 0.32 1.045 y 0.97 2.2 1.052 z 0.83 3.7 1.23 Harmine–b-CD x 0.95 3.7 1.02 y 0.92 1.73 1.097 z 0.977 16.02 1.22 * l = Emission wavelength. x: l = 366 nm (harmane); l = 358 nm (harmine). y: l = 480 nm (harmane; l = 476 nm (harmine). z: l = 430 nm (harmane); l = 416 nm (harmine). † r = correlation coefficient; m = slope; b = intercept inordinate. ‡ The values obtained could not be adjusted by the Stern–Volmer treatment: Fo/FA = 1 + K(Q). Analyst, Jan