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Determination of the binding constant for the inclusion complex between procaine hydrochloride and β-cyclodextrin by capillary electrophoresis
Talanta 59 (2003) 493 /499 www.elsevier.com/locate/talanta
Determination of the binding constant for the inclusion complex between procaine hydrochloride and b-cyclodextrin by capillary electrophoresis Nianbing Li a, Jianping Duan b, Hongqing Chen b, Guonan Chen a,* a
b
Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350002, PR China Analytical and Testing Center, Fuzhou University, Fuzhou, Fujian 350002, PR China
Received 5 June 2002; received in revised form 30 September 2002; accepted 30 September 2002
Abstract The apparent electrophoretic mobilities of procaine hydrochloride (mi) in a series of concentration of b-cyclodextrin were measured directly by capillary electrophoresis technology. A new mathematical treatment method is proposed, which based on the fact that the molar ratio of the inclusion complex was 1:1 established by spectrophotometry. Using the proposed method, the binding constant of the inclusion complex of procaine hydrochloride with b-cyclodextrin can be obtained easily. The determination result was in correspondence with those of the spectrophotometric and fluorescence methods. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cyclodextrin; Procaine hydrochloride; Binding constant; Capillary electrophoresis
1. Introduction In recent years, the method of molecular complexation with artificial receptors becomes more and more useful in many technological and research fields [1,2]. Besides, highly specific biological processes make extensive use of molecular complexation, with noncovalent interactions playing an important role [3]. Many researchers working in the field of molecular recognition processes
* Corresponding author. Tel.: /86-591-7893315; fax: /86591-3713866 E-mail address: [email protected] (G. Chen).
have focused their studies on a number of therapeutic molecules, whose bioavailability is often affected by problems such as limited solubility or stability etc. Recently, molecular encapsulation has been successfully used in many technological fields [3,4]. Particularly, pharmaceutical industry have made use of it to improve the bioavailabilities of drugs, to protect them from decomposition, to convert liquids to free-flowing power, and to mask unfavorable odors and tastes. Cyclodextrins, which have become a new family of pharmaceutical excipients, are cyclic oligosaccharides consisting of 6, 7, or 8 (a, b or g cyclodextrins, respectively) glucose units connected by a-1,4glycoside bonds [5]. They are molecules with a
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 5 3 2 - 5
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N. Li et al. / Talanta 59 (2003) 493 /499
shape of truncated cone, having hydrophobic cavity [5]. Many hydroxy groups are situated on the outer part of the ring, which make the CDs both hydrophilic and soluble in water. The unique ability of CDs to entrap certain molecules in their molecular cavities offers remarkable effects in stabilizing and solubilizing lipophilic, unstable substances without the formation of chemical bonds and without changing their structure [6]. This physical interaction can therefore be considered as encapsulation of substances on a molecular scale [7]. Procaine hydrochloride is a kind of local anaesthetic, which produces a reversible loss of sensation by diminishing the conduction of sensory nerve impulses, near to the site of application or injection [8]. However, local anaesthetics often show a short duration of action and adverse side effects. It is well known that the inclusion complex between procaine hydrochloride and b-CD can show a better bioavailability, and can mask or abolish all or some of these undesirable effects. The binding forces between cyclodextrin and guest molecule have been attributed to weak interactions such as hydrogen bonding, van der Waals and hydrophobic interactions [9,10]. As the quantitative description of the inclusion equilibrium between CD and guest molecule, the binding constant reflects the strength of the binding force between them. So, the binding constant is an important and basic parameter to the application of CD. Up to the present, the reported determination methods for the CD binding constant were spectroscopic method [11], surface tension method [12], phase-solubility technique [13], nuclear magnetic resonance method [14], spectrofluorimetry [15], constant current coulometric titration method [16], high-pressure liquid chromatography [17], electrochemistry [18] and resonance Rayleigh scattering technology [19]. For analytes involved in dynamic equilibrium processes, capillary electrophoresis (CE) can provide an alternative method for determining binding constants through the determination of the electrophoretic mobility as a function of the concentration of the host molecule. In recent years, some researchers have been used CE as a
new technology to determine the cyclodextrin binding constant. However, most of them used a nonlinear least-squares method to fit the data and then obtained the binding constant [20 /22]. Bellini et al. attempted to obtained apparent association constant of procaine hydrochloride and b-cyclodextrin by three different linearization plots (double reciprocal, x reciprocal and y reciprocal), but the results did not satisfying [23]. In the present paper, a new mathematical treatment method is proposed using the capillary electrophoresis technology. The binding constant of the inclusion complex of procaine hydrochloride with b-cyclodextrin can be obtained easily by the proposed method. The determination result was in correspondence with those of the spectrophotometric and fluorescence methods.
2. Basic principles There are three criteria that must be met for determining the CD binding constant by CE [24,25]. That is, (1) the time scale of equilibration is faster than that of the separation being performed. (2) There are significant proportions of both selector and selector: analyte complex in the capillary. (3) The mobility of the analyte and complex are different. If a guest molecule, G, forms a 1:1 inclusion complex with cyclodextrin, CD, the complex formation can be described by the following equation CDG?CDG
(1)
The binding constant is defined as Kf [CDG]=[CD][G]
(2)
where [CD], [G], and [CD/G] are the equilibrium concentrations of CD, G, and CD/G, respectively. The apparent electrophoretic mobility of G (mi)can be determined by the electrophoretic mobilities of the free state (mf) and the inclusion complex (mc) [26]. mi [G]mf =f[G][CDG]g [CDG]mc =f[G][CDG]g
(3)
N. Li et al. / Talanta 59 (2003) 493 /499
According to the expression of the binding constant, Eq. (2) can be further transformed into [CDG] Kf [G][CD]
(4)
495
was prepared using MilliQ equipment (Millipore Corp., France). 3.2. Apparatus
Using Eq. (4), Eq. (3) can be transformed into mi fmf mc Kf [CD]g=f1Kf [CD]g
(5)
Therefore, Eq. (5) can be rewritten as (mi mf )=(mc mi )Kf [CD]
(6)
Taking logarithm for the Eq. (6), then log[CD]log Kf logf(mi mf )=(mc mi )g If y /log Kf/log{(mi/mf)/(mc/mi)}, then the first derivative (y?) and the second derivative (yƒ) can be described as follows: y?(mf mc )=f(mi mf )(mc mi )g yƒ(mf mc ) (mc 2mi mf )=f(mi mf )2 (mc mi )2 g Making yƒ /0. That is (mf mc )(mc 2mi mf )0 Since mf "/mc, therefore, mc/2mi/mf /0, i.e., mi (mf mc )=2
(7)
When mi /(mf/mc)/2, (mi, y (mi)) is the inflexion point of the curve y . Substituting Eq. (7) into Eq. (6), yields: log[CD]log Kf
Electrophoretic experiment was carried out with the Beckman MDQ instrument (Beckman Co., US). Electrophoresis was performed in untreated fused silica capillary tubing, 58 cm (effective length 48.6 cm) /75 mm i.d. The applied voltage was 20 kV and the capillary temperature was 25 8C Sample was sucked into the capillary under a reduced pressure of 5 psi for 5 s. The operating buffer employed was 1.0 /103 mol1 phosphate buffer (pH 5.0). All solutions were passed through a membrane filter (0.45 mm) before CE experiment. The data were recorded at 214 nm. The relative viscosity was determined by taking the ratio of i0 and i (h /h0 /i0/i). i0 and i were the current in the absence and presence of b-CD, respectively. A CH-2 electrochemical detector for CE (Jiangshu Electroanalytical Instrument Factory, China) was used to determine i0 and i . UV / visible absorption spectra data were obtained using a UV /VIS 8500 spectrophotometer (Tianmei company, China). Fluorescence measurements were made using a Hitachi F-2500 spectrofluorophotometer (Tokyo, Japan). Slit (EX/EM): 5.0 nm/5.0 nm, photomultiplier tube voltage: 400 V. 3.3. Procedure
(8)
It can be seen from Eq. (8) that Kf can be obtained by the value of [CD] at the inflexion point. That is, plotting mi as a function of the / log[CD], the Kf value for the inclusion complex can be determined at the inflexion point.
3. Experimental 3.1. Chemicals Procaine hydrochloride (Shanghai Biochemistry Research Institute, China) was of pharmaceutical purity grade. b-CD was from Aldrich. The water
The capillary was washed with 0.1 mol1 NaOH for 5 min, deionized water for 5 min and then the run buffer for 5 min. One milliliter of 1.5 /10 3 mol 1 procaine hydrochloride aqueous solution was added to a series of 25 ml volumetric flasks, and then different concentrations of b-CD were added to each volumetric flask. The mixtures were diluted to volume with water and mixed thoroughly. After 5 min the apparent electrophoretic mobility was determined using electrophoretic experiment. In order to confirm that the proposed method can be used as a new method to determine the binding constant of CD, the UV /VIS spectrophotometry and fluorescence spectroscopy were used to determine the binding constant under the
N. Li et al. / Talanta 59 (2003) 493 /499
496
same experimental conditions. Then, the binding constants obtained with different methods were compared. Absorbance was measured at the maximum absorption wavelength of 307 nm. The intensity of fluorescence was measured at the emission wavelength of 350 nm and the excitation wavelength of 276 nm. Job’s method of continuous variation [27] was used to obtain the composition ratio of the inclusion complex. The absorbance at 307 nm was measured.
4. Results and discussion 4.1. Formation of the inclusion complex and its molar ratio Procaine hydrochloride, an ester of the p aminobenzoic acid, may suffer hydrolysis in aqueous media. Besides, its cationic form is in equilibrium with its nonionized form as follows: H2 NPhCOO(CH2 )2 NH(C2 H2 )2 H Cl K
?H2 NPhCOO(CH2 )2 NH(C2 H5 )2 H Cl
(9)
Where K is the equilibrium constant. In order to confirm that the hydrolysis of the ester does not occur in aqueous media, Aicart and his co-worker measured pH of the procaine hydrochloride solution as a function of the drug concentration at 25 8C [28]. They determined K for procaine hydrochloride [29] and obtained the value of 5/ 1010 at 25 8C. From the result, it can be seen that (i) the ester is not hydrolyzed and (ii) equilibrium (Eq. (9)) is almost totally shifted toward the ionized form of the drug, with a negligible contribution of the nonionized form. Therefore, in our experimental condition, procaine hydrochloride exists as an ionized form. The diameter of the cavity of b-CD is estimated ˚ [30], and the diameter of benzene ring in as 6.8 A the procaine hydrochloride molecule is about 6.7 / ˚ , which matches the diameter of the cavity of 6.8 A b-CD. Therefore, procaine hydrochloride can enter the cavity of b-CD and react with b-CD to
Fig. 1. Absorption spectra of b-CD, procaine hydrochloride and the procaine hydrochloride b-CD complex. (1) 4.0/ 10 3mol1 b-CD; (2) 6.0 /10 5 mol 1 procaine hydrochloride; (3) the mixture of 4.0/10 3 mol 1 b-CD and 6.0 /10 5 mol1 procaine hydrochloride.
form a steady inclusion complex. Fig. 1 shows the UV /VIS spectra of aqueous solutions of procaine hydrochloride in the absence and presence of bCD. It can be seen from Fig. 1 that b-CD itself does not contain conjugate system and it has no absorption from near ultra violet to visible region. But procaine hydrochloride has two absorption peaks at 221 and 290 nm. The absorption peak of 221 nm was attributed to f/NH2 (f represents a phenyl group). The absorption peak of 290 nm, corresponding to the p 0/p* transition (k band), was the characteristic absorption peak of bireplacement benzene ring [31]. After procaine hydrochloride was included by b-CD, the formation of the inclusion complex provoked a clear bathochromic shift of the two peaks observed in the spectrum of the procaine hydrochloride. The two absorption peaks shifted to 231 and 307 nm and the intensities of the absorption peaks increased. This phenomenon also indicated that the size of procaine hydrochloride matched the diameter of the cavity of b-CD. When b-CD included the benzene ring, the cavity of b-CD with abundant electron cloud enhanced the electron cloud
N. Li et al. / Talanta 59 (2003) 493 /499
density of the guest and increased the conjugative effect of p electron of the benzene ring. Therefore, the energy of electron transition from p to p* decreased, and the maximum absorption wavelength (lmax)shifted to longer wavelength. The absorbance of procaine hydrochloride was enhanced with an increase in b-CD concentration. The isosbestic points were observed at 243 and 290 nm, indicating an obvious interaction between procaine hydrochloride and b-CD. The molar ratio of the inclusion complex was performed using Job’s method of continuous variation. The results showed that the maximum ratio of [b-CD]/ ([G]/[b-CD]) was 0.5, which indicated that the complex had a 1:1 stoichiometry. 4.2. Determination of the cyclodextrin binding constant by using capillary electrophoresis It is well known that the electrophoretic mobility, m , can be expressed as follows: mq=6phr
(10)
where q is the charge, r the radius, and h the viscosity. The viscosity of the solution containing b-CD affected m, especially when the concentration of b-CD was higher, the effect was more serious, which can result in the misinterpretation of data, as has been previously pointed out. Therefore, the viscosity correction must be carried out. The value of current was used to rectify the viscosity usually through the expression [32]: m?i mi h=h0 mi i0 =i
(11)
where m i? is the apparent electrophoretic mobility after rectified, mi is the apparent electrophoretic mobility without rectification, h0 and h are the viscosity in the absence and presence of b-CD, and i0 and i are the current in the absence and presence of b-CD, respectively. The units of m i?, h and i are m2 V1 s 1, Pa s and A, respectively. According to the basic principle, the apparent electrophoretic mobility of G (mi) can be determined by the electrophoretic experiment in different b-CD concentrations and m i? can be obtained using Eq. (11). The plot of m i? versus /log[b-CD] is shown in Fig. 2. It can be seen from Fig. 2 that a distinct inflexion point appeared at about the
497
Fig. 2. Plot of m i? against /log[b-CD]. Electrophoretic experimental conditions: capillary, 58 cm (effective length 48.6 cm) / 75 mm i.d.; pressure injection, 5 psi for 5 s; applied voltage, 20 kV; temperature: 25 8C; detection, UV at 214 nm.
/log[b-CD] value of 2.10. From this curve, log Kf was estimated to be close to 2.10. In order to illuminate that the proposed mathematics treatment method can be used to determine the binding constant of procaine hydrochloride and b-CD, the UV/VIS spectrophotometry and fluorescence spectroscopy were used to determine the binding constant under the same experimental conditions. The absorbance of procaine hydrochloride in water varied with the addition of b-CD. The change of the absorbance (DA ) was observed as a function of the concentration of b-CD added. The binding constant value, Kf, of the inclusion complex was evaluated by the Benesi /Hildebrand equation (Eq. (12)) from the UV /VIS spectroscopic data [11]. 1=DA 1=(DoCG )1=(DoKf CG [b-CD])
(12)
where CG is the concentration of procaine hydrochloride and [b-CD] represents the equilibrium concentration of b-CD. DA is the change in the absorbance of procaine hydrochloride before and after addition of b-CD, and Do is the difference of the molar absorptivities between complex and free procaine hydrochloride. Plotting 1/DA against 1/ [b-CD] (if [b-CD] /[G], [b-CD] can be replaced with Cb-CD) gives a straight line with slope equal to 1/DoKfCG (Fig. 3). Kf was directly obtained from
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N. Li et al. / Talanta 59 (2003) 493 /499
1=DF 1=(aCG )1=(aKf Cg [b-CD])
(13)
where DF is the fluorescence intensity difference, a is the coefficient. The excitation and emission spectra of procaine hydrochloride and the procaine hydrochloride -b-CD complex are shown in Fig. 4. Procaine hydrochloride has a maximum excitation peak at 276 nm and its emission peak is at 350 nm. When b-CD was added to the procaine Table 1 Binding constant of procaine hydrochloride-b-CD complex with different determination methods
Fig. 3. Plot of 1/DA versus 1/Cb-CD (1) and Plot of 1/DF versus 1/Cb-CD (2).
the intercept/slope ratio. In the same way, the binding constant, Kf also can be determined using fluorescence data by the Benesi /Hildebrand equation (Eq. (13)) [33].
Method
log Kfa
Reference
Constant coulometric titration method Resonance Rayleigh scattering method Capillary electrophoresis method UV /VIS spectrophotometric method Fluorescence method
2.14 2.06 /2.10 2.10 2.03 2.09
[16] [19] This work This work This work
The number of determination (n ) is 4. a The unit of Kf is 1 mol 1.
Fig. 4. The excitation (A) and emission (B) spectra of procaine hydrochloride in the presence of b-CD. Concentration of b-CD: (1) 0 mol 1; (2) 4.0/10 4 mol 1; (3) 6.0/10 4 mol1; (4) 1.0/10 3 mol 1; (5) 2.0 /10 3 mol 1; (6) 3.0/10 3 mol 1. Concentration of procaine hydrochloride: 6.0 /10 5 mol 1. Slit (EX/EM): 5.0 nm/5.0 nm, photomultiplier tube voltage: 400 V.
N. Li et al. / Talanta 59 (2003) 493 /499
hydrochloride solution, the maximum excitation and emission peaks did not distinctly change, but the intensities of the peaks increased with an increase in b-CD concentration. The curve of 1/ DF /1/[b-CD] (when [b-CD] /[G], [b-CD] can be replaced with Cb-CD) was plotted (Fig. 3, curve 2), and Kf was the ratio of the intercept to the slope. Table 1 shows the results of different methods. The results present clearly that the determination value with the proposed method is in correspondence with those of the spectrophotometric and fluorescence methods and of the reports, which used the constant coulometric titration method [16] and resonance Rayleigh scattering method [19].
5. Conclusions Capillary electrophoresis has developed into an excellent method to determine binding constants. In this work, the apparent electrophoretic mobility of procaine hydrochloride (mi) in a series of concentration of b-cyclodextrin were measured directly by capillary electrophoresis technology. mi needs to apply correction for viscosity change in order to avoid the misinterpretation of data. A new mathematical treatment method is proposed, which based on the fact that the molar ratio of the inclusion complex between b-cyclodextrin and procaine hydrochloride was 1:1. The determination value of the proposed method was in correspondence with those of the spectrophotometric and fluorescence methods, which indicated that the proposed method had a good accuracy. This method offered the advantages of automation, small amounts of sample, conciseness as well as easy operation.
Acknowledgements This project was supported by the Foundation of Science and Technology Department of Fujian Province, China. Dr Nianbing Li thanks to the Science and Technology Department Foundation of Fuzhou University.
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References [1] K.A. Connors, Chem. Rev. 97 (1997) 1325. [2] V.T. D’Souza, K.B. Lipkowitz, Chem. Rev. 98 (1998) 1741. [3] X.Q. Sun, Q. Meng, H.B. Yan, Introduction of Supermolecular Chemistry, Chinese Petrochemistry Press, Beijing, 1997, p. 11. [4] T. Loftsson, M.E. Brewster, J. Pharm. Sci. 85 (1996) 1017. [5] F. Djedaini, B. Perly, J. Mol. Struct. 239 (1990) 161. [6] L. Szente, J. Szejtli, Analyst 123 (1998) 735. [7] J. Gu, C. Yan, J.H. Pan, Chin. J. Appl. Chem. 13 (1996) 5. [8] S.X. Peng, Pharmaceutical Chemistry, Chemical Industry Press, Beijing, 1995, p. 16. [9] D. Martin-Davies, J.R. Savage, J. Chem. Soc. Perkin Trans. 2 (1994) 1525. [10] E. Junquera, E. Aicart, J. Phys. Chem. B. 101 (1997) 7163. [11] H.A. Benesi, J. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703. [12] Y.Zh. Hui, J.H. Gu, Acta Chimica Sinica 39 (1981) 309. [13] T. Higuchi, K.A. Connors, Anal. Chem. Instrum. 4 (1965) 117. [14] Y. Inoue, Y. Miyata, Bull. Chem. Soc. Jpn 54 (1981) 809. [15] Y.B. Jiang, Spectrochim. Acta 51A (1995) 275. [16] J. Gu, J.H. Pan, Talanta 50 (1999) 35. [17] K. Uekama, F. Hirayama, T. Irie, Chem. Lett. 7 (1978) 661. [18] P.M. Bersier, T. Bersier, B. Klingert, Electroanalysis 3 (1991) 443. [19] N.B. Li, H.Q. Luo, S.P. Liu, G.N. Chen, Spectrochim. Acta Part A 58 (2002) 501. [20] S.G. Penn, D.M. Goodall, J.S. Loran, J. Chromatogr. 636 (1993) 149. [21] G.L. Atkins, I.A. Nimms, Anal. Biochem. 104 (1980) 1. [22] K.L. Rundlett, D.W. Armstrong, Electrophoresis 18 (1997) 2194. [23] M.S. Bellini, Z. Deyl, G. Manetto, M. Kohlickova, J. Chromatogr. A 924 (2001) 483. [24] F. Lelievre, P. Gareil, J. Chromatogr. A 735 (1996) 311. [25] E. Szoko, J. Gyimesi, L. Barcsa, K. Magyar, J. Chromatogr. A 745 (1996) 181. [26] S.A. Wren, R.C. Rowe, J. Chromatogr. 603 (1996) 235. [27] P. Job, Anal. Chim. 9 (1928) 113. [28] C. Merion, E. Junquera, J. Jimenez-Barbero, E. Aicart, Langmuir 16 (2000) 1557. [29] E. Junquera, E. Aicart, J. Phys. Chem. B 101 (1997) 7163. [30] W.B. Qi, Z.H. Qi, New Synergistic Agents for Analysis, Hangzhou University Press, Hangzhou, 1994, p. 79. [31] J. Gu, L. Chen, H.Y. Xi, J.H. Pan, Spectrosc. Spectral Anal. 19 (1999) 575. [32] S.G. Penn, E.T. Bergstrom, I. Knights, G. Liu, A. Ruddick, D.M. Googall, J. Phys. Chem. 99 (1995) 3875. [33] S. Hamai, Bull. Chem. Soc. Jpn 55 (1982) 2721.
Determination of the binding constant for the inclusion complex between procaine hydrochloride and b-cyclodextrin by capillary electrophoresis Nianbing Li a, Jianping Duan b, Hongqing Chen b, Guonan Chen a,* a
b
Department of Chemistry, Fuzhou University, Fuzhou, Fujian 350002, PR China Analytical and Testing Center, Fuzhou University, Fuzhou, Fujian 350002, PR China
Received 5 June 2002; received in revised form 30 September 2002; accepted 30 September 2002
Abstract The apparent electrophoretic mobilities of procaine hydrochloride (mi) in a series of concentration of b-cyclodextrin were measured directly by capillary electrophoresis technology. A new mathematical treatment method is proposed, which based on the fact that the molar ratio of the inclusion complex was 1:1 established by spectrophotometry. Using the proposed method, the binding constant of the inclusion complex of procaine hydrochloride with b-cyclodextrin can be obtained easily. The determination result was in correspondence with those of the spectrophotometric and fluorescence methods. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cyclodextrin; Procaine hydrochloride; Binding constant; Capillary electrophoresis
1. Introduction In recent years, the method of molecular complexation with artificial receptors becomes more and more useful in many technological and research fields [1,2]. Besides, highly specific biological processes make extensive use of molecular complexation, with noncovalent interactions playing an important role [3]. Many researchers working in the field of molecular recognition processes
* Corresponding author. Tel.: /86-591-7893315; fax: /86591-3713866 E-mail address: [email protected] (G. Chen).
have focused their studies on a number of therapeutic molecules, whose bioavailability is often affected by problems such as limited solubility or stability etc. Recently, molecular encapsulation has been successfully used in many technological fields [3,4]. Particularly, pharmaceutical industry have made use of it to improve the bioavailabilities of drugs, to protect them from decomposition, to convert liquids to free-flowing power, and to mask unfavorable odors and tastes. Cyclodextrins, which have become a new family of pharmaceutical excipients, are cyclic oligosaccharides consisting of 6, 7, or 8 (a, b or g cyclodextrins, respectively) glucose units connected by a-1,4glycoside bonds [5]. They are molecules with a
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 5 3 2 - 5
494
N. Li et al. / Talanta 59 (2003) 493 /499
shape of truncated cone, having hydrophobic cavity [5]. Many hydroxy groups are situated on the outer part of the ring, which make the CDs both hydrophilic and soluble in water. The unique ability of CDs to entrap certain molecules in their molecular cavities offers remarkable effects in stabilizing and solubilizing lipophilic, unstable substances without the formation of chemical bonds and without changing their structure [6]. This physical interaction can therefore be considered as encapsulation of substances on a molecular scale [7]. Procaine hydrochloride is a kind of local anaesthetic, which produces a reversible loss of sensation by diminishing the conduction of sensory nerve impulses, near to the site of application or injection [8]. However, local anaesthetics often show a short duration of action and adverse side effects. It is well known that the inclusion complex between procaine hydrochloride and b-CD can show a better bioavailability, and can mask or abolish all or some of these undesirable effects. The binding forces between cyclodextrin and guest molecule have been attributed to weak interactions such as hydrogen bonding, van der Waals and hydrophobic interactions [9,10]. As the quantitative description of the inclusion equilibrium between CD and guest molecule, the binding constant reflects the strength of the binding force between them. So, the binding constant is an important and basic parameter to the application of CD. Up to the present, the reported determination methods for the CD binding constant were spectroscopic method [11], surface tension method [12], phase-solubility technique [13], nuclear magnetic resonance method [14], spectrofluorimetry [15], constant current coulometric titration method [16], high-pressure liquid chromatography [17], electrochemistry [18] and resonance Rayleigh scattering technology [19]. For analytes involved in dynamic equilibrium processes, capillary electrophoresis (CE) can provide an alternative method for determining binding constants through the determination of the electrophoretic mobility as a function of the concentration of the host molecule. In recent years, some researchers have been used CE as a
new technology to determine the cyclodextrin binding constant. However, most of them used a nonlinear least-squares method to fit the data and then obtained the binding constant [20 /22]. Bellini et al. attempted to obtained apparent association constant of procaine hydrochloride and b-cyclodextrin by three different linearization plots (double reciprocal, x reciprocal and y reciprocal), but the results did not satisfying [23]. In the present paper, a new mathematical treatment method is proposed using the capillary electrophoresis technology. The binding constant of the inclusion complex of procaine hydrochloride with b-cyclodextrin can be obtained easily by the proposed method. The determination result was in correspondence with those of the spectrophotometric and fluorescence methods.
2. Basic principles There are three criteria that must be met for determining the CD binding constant by CE [24,25]. That is, (1) the time scale of equilibration is faster than that of the separation being performed. (2) There are significant proportions of both selector and selector: analyte complex in the capillary. (3) The mobility of the analyte and complex are different. If a guest molecule, G, forms a 1:1 inclusion complex with cyclodextrin, CD, the complex formation can be described by the following equation CDG?CDG
(1)
The binding constant is defined as Kf [CDG]=[CD][G]
(2)
where [CD], [G], and [CD/G] are the equilibrium concentrations of CD, G, and CD/G, respectively. The apparent electrophoretic mobility of G (mi)can be determined by the electrophoretic mobilities of the free state (mf) and the inclusion complex (mc) [26]. mi [G]mf =f[G][CDG]g [CDG]mc =f[G][CDG]g
(3)
N. Li et al. / Talanta 59 (2003) 493 /499
According to the expression of the binding constant, Eq. (2) can be further transformed into [CDG] Kf [G][CD]
(4)
495
was prepared using MilliQ equipment (Millipore Corp., France). 3.2. Apparatus
Using Eq. (4), Eq. (3) can be transformed into mi fmf mc Kf [CD]g=f1Kf [CD]g
(5)
Therefore, Eq. (5) can be rewritten as (mi mf )=(mc mi )Kf [CD]
(6)
Taking logarithm for the Eq. (6), then log[CD]log Kf logf(mi mf )=(mc mi )g If y /log Kf/log{(mi/mf)/(mc/mi)}, then the first derivative (y?) and the second derivative (yƒ) can be described as follows: y?(mf mc )=f(mi mf )(mc mi )g yƒ(mf mc ) (mc 2mi mf )=f(mi mf )2 (mc mi )2 g Making yƒ /0. That is (mf mc )(mc 2mi mf )0 Since mf "/mc, therefore, mc/2mi/mf /0, i.e., mi (mf mc )=2
(7)
When mi /(mf/mc)/2, (mi, y (mi)) is the inflexion point of the curve y . Substituting Eq. (7) into Eq. (6), yields: log[CD]log Kf
Electrophoretic experiment was carried out with the Beckman MDQ instrument (Beckman Co., US). Electrophoresis was performed in untreated fused silica capillary tubing, 58 cm (effective length 48.6 cm) /75 mm i.d. The applied voltage was 20 kV and the capillary temperature was 25 8C Sample was sucked into the capillary under a reduced pressure of 5 psi for 5 s. The operating buffer employed was 1.0 /103 mol1 phosphate buffer (pH 5.0). All solutions were passed through a membrane filter (0.45 mm) before CE experiment. The data were recorded at 214 nm. The relative viscosity was determined by taking the ratio of i0 and i (h /h0 /i0/i). i0 and i were the current in the absence and presence of b-CD, respectively. A CH-2 electrochemical detector for CE (Jiangshu Electroanalytical Instrument Factory, China) was used to determine i0 and i . UV / visible absorption spectra data were obtained using a UV /VIS 8500 spectrophotometer (Tianmei company, China). Fluorescence measurements were made using a Hitachi F-2500 spectrofluorophotometer (Tokyo, Japan). Slit (EX/EM): 5.0 nm/5.0 nm, photomultiplier tube voltage: 400 V. 3.3. Procedure
(8)
It can be seen from Eq. (8) that Kf can be obtained by the value of [CD] at the inflexion point. That is, plotting mi as a function of the / log[CD], the Kf value for the inclusion complex can be determined at the inflexion point.
3. Experimental 3.1. Chemicals Procaine hydrochloride (Shanghai Biochemistry Research Institute, China) was of pharmaceutical purity grade. b-CD was from Aldrich. The water
The capillary was washed with 0.1 mol1 NaOH for 5 min, deionized water for 5 min and then the run buffer for 5 min. One milliliter of 1.5 /10 3 mol 1 procaine hydrochloride aqueous solution was added to a series of 25 ml volumetric flasks, and then different concentrations of b-CD were added to each volumetric flask. The mixtures were diluted to volume with water and mixed thoroughly. After 5 min the apparent electrophoretic mobility was determined using electrophoretic experiment. In order to confirm that the proposed method can be used as a new method to determine the binding constant of CD, the UV /VIS spectrophotometry and fluorescence spectroscopy were used to determine the binding constant under the
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same experimental conditions. Then, the binding constants obtained with different methods were compared. Absorbance was measured at the maximum absorption wavelength of 307 nm. The intensity of fluorescence was measured at the emission wavelength of 350 nm and the excitation wavelength of 276 nm. Job’s method of continuous variation [27] was used to obtain the composition ratio of the inclusion complex. The absorbance at 307 nm was measured.
4. Results and discussion 4.1. Formation of the inclusion complex and its molar ratio Procaine hydrochloride, an ester of the p aminobenzoic acid, may suffer hydrolysis in aqueous media. Besides, its cationic form is in equilibrium with its nonionized form as follows: H2 NPhCOO(CH2 )2 NH(C2 H2 )2 H Cl K
?H2 NPhCOO(CH2 )2 NH(C2 H5 )2 H Cl
(9)
Where K is the equilibrium constant. In order to confirm that the hydrolysis of the ester does not occur in aqueous media, Aicart and his co-worker measured pH of the procaine hydrochloride solution as a function of the drug concentration at 25 8C [28]. They determined K for procaine hydrochloride [29] and obtained the value of 5/ 1010 at 25 8C. From the result, it can be seen that (i) the ester is not hydrolyzed and (ii) equilibrium (Eq. (9)) is almost totally shifted toward the ionized form of the drug, with a negligible contribution of the nonionized form. Therefore, in our experimental condition, procaine hydrochloride exists as an ionized form. The diameter of the cavity of b-CD is estimated ˚ [30], and the diameter of benzene ring in as 6.8 A the procaine hydrochloride molecule is about 6.7 / ˚ , which matches the diameter of the cavity of 6.8 A b-CD. Therefore, procaine hydrochloride can enter the cavity of b-CD and react with b-CD to
Fig. 1. Absorption spectra of b-CD, procaine hydrochloride and the procaine hydrochloride b-CD complex. (1) 4.0/ 10 3mol1 b-CD; (2) 6.0 /10 5 mol 1 procaine hydrochloride; (3) the mixture of 4.0/10 3 mol 1 b-CD and 6.0 /10 5 mol1 procaine hydrochloride.
form a steady inclusion complex. Fig. 1 shows the UV /VIS spectra of aqueous solutions of procaine hydrochloride in the absence and presence of bCD. It can be seen from Fig. 1 that b-CD itself does not contain conjugate system and it has no absorption from near ultra violet to visible region. But procaine hydrochloride has two absorption peaks at 221 and 290 nm. The absorption peak of 221 nm was attributed to f/NH2 (f represents a phenyl group). The absorption peak of 290 nm, corresponding to the p 0/p* transition (k band), was the characteristic absorption peak of bireplacement benzene ring [31]. After procaine hydrochloride was included by b-CD, the formation of the inclusion complex provoked a clear bathochromic shift of the two peaks observed in the spectrum of the procaine hydrochloride. The two absorption peaks shifted to 231 and 307 nm and the intensities of the absorption peaks increased. This phenomenon also indicated that the size of procaine hydrochloride matched the diameter of the cavity of b-CD. When b-CD included the benzene ring, the cavity of b-CD with abundant electron cloud enhanced the electron cloud
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density of the guest and increased the conjugative effect of p electron of the benzene ring. Therefore, the energy of electron transition from p to p* decreased, and the maximum absorption wavelength (lmax)shifted to longer wavelength. The absorbance of procaine hydrochloride was enhanced with an increase in b-CD concentration. The isosbestic points were observed at 243 and 290 nm, indicating an obvious interaction between procaine hydrochloride and b-CD. The molar ratio of the inclusion complex was performed using Job’s method of continuous variation. The results showed that the maximum ratio of [b-CD]/ ([G]/[b-CD]) was 0.5, which indicated that the complex had a 1:1 stoichiometry. 4.2. Determination of the cyclodextrin binding constant by using capillary electrophoresis It is well known that the electrophoretic mobility, m , can be expressed as follows: mq=6phr
(10)
where q is the charge, r the radius, and h the viscosity. The viscosity of the solution containing b-CD affected m, especially when the concentration of b-CD was higher, the effect was more serious, which can result in the misinterpretation of data, as has been previously pointed out. Therefore, the viscosity correction must be carried out. The value of current was used to rectify the viscosity usually through the expression [32]: m?i mi h=h0 mi i0 =i
(11)
where m i? is the apparent electrophoretic mobility after rectified, mi is the apparent electrophoretic mobility without rectification, h0 and h are the viscosity in the absence and presence of b-CD, and i0 and i are the current in the absence and presence of b-CD, respectively. The units of m i?, h and i are m2 V1 s 1, Pa s and A, respectively. According to the basic principle, the apparent electrophoretic mobility of G (mi) can be determined by the electrophoretic experiment in different b-CD concentrations and m i? can be obtained using Eq. (11). The plot of m i? versus /log[b-CD] is shown in Fig. 2. It can be seen from Fig. 2 that a distinct inflexion point appeared at about the
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Fig. 2. Plot of m i? against /log[b-CD]. Electrophoretic experimental conditions: capillary, 58 cm (effective length 48.6 cm) / 75 mm i.d.; pressure injection, 5 psi for 5 s; applied voltage, 20 kV; temperature: 25 8C; detection, UV at 214 nm.
/log[b-CD] value of 2.10. From this curve, log Kf was estimated to be close to 2.10. In order to illuminate that the proposed mathematics treatment method can be used to determine the binding constant of procaine hydrochloride and b-CD, the UV/VIS spectrophotometry and fluorescence spectroscopy were used to determine the binding constant under the same experimental conditions. The absorbance of procaine hydrochloride in water varied with the addition of b-CD. The change of the absorbance (DA ) was observed as a function of the concentration of b-CD added. The binding constant value, Kf, of the inclusion complex was evaluated by the Benesi /Hildebrand equation (Eq. (12)) from the UV /VIS spectroscopic data [11]. 1=DA 1=(DoCG )1=(DoKf CG [b-CD])
(12)
where CG is the concentration of procaine hydrochloride and [b-CD] represents the equilibrium concentration of b-CD. DA is the change in the absorbance of procaine hydrochloride before and after addition of b-CD, and Do is the difference of the molar absorptivities between complex and free procaine hydrochloride. Plotting 1/DA against 1/ [b-CD] (if [b-CD] /[G], [b-CD] can be replaced with Cb-CD) gives a straight line with slope equal to 1/DoKfCG (Fig. 3). Kf was directly obtained from
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1=DF 1=(aCG )1=(aKf Cg [b-CD])
(13)
where DF is the fluorescence intensity difference, a is the coefficient. The excitation and emission spectra of procaine hydrochloride and the procaine hydrochloride -b-CD complex are shown in Fig. 4. Procaine hydrochloride has a maximum excitation peak at 276 nm and its emission peak is at 350 nm. When b-CD was added to the procaine Table 1 Binding constant of procaine hydrochloride-b-CD complex with different determination methods
Fig. 3. Plot of 1/DA versus 1/Cb-CD (1) and Plot of 1/DF versus 1/Cb-CD (2).
the intercept/slope ratio. In the same way, the binding constant, Kf also can be determined using fluorescence data by the Benesi /Hildebrand equation (Eq. (13)) [33].
Method
log Kfa
Reference
Constant coulometric titration method Resonance Rayleigh scattering method Capillary electrophoresis method UV /VIS spectrophotometric method Fluorescence method
2.14 2.06 /2.10 2.10 2.03 2.09
[16] [19] This work This work This work
The number of determination (n ) is 4. a The unit of Kf is 1 mol 1.
Fig. 4. The excitation (A) and emission (B) spectra of procaine hydrochloride in the presence of b-CD. Concentration of b-CD: (1) 0 mol 1; (2) 4.0/10 4 mol 1; (3) 6.0/10 4 mol1; (4) 1.0/10 3 mol 1; (5) 2.0 /10 3 mol 1; (6) 3.0/10 3 mol 1. Concentration of procaine hydrochloride: 6.0 /10 5 mol 1. Slit (EX/EM): 5.0 nm/5.0 nm, photomultiplier tube voltage: 400 V.
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hydrochloride solution, the maximum excitation and emission peaks did not distinctly change, but the intensities of the peaks increased with an increase in b-CD concentration. The curve of 1/ DF /1/[b-CD] (when [b-CD] /[G], [b-CD] can be replaced with Cb-CD) was plotted (Fig. 3, curve 2), and Kf was the ratio of the intercept to the slope. Table 1 shows the results of different methods. The results present clearly that the determination value with the proposed method is in correspondence with those of the spectrophotometric and fluorescence methods and of the reports, which used the constant coulometric titration method [16] and resonance Rayleigh scattering method [19].
5. Conclusions Capillary electrophoresis has developed into an excellent method to determine binding constants. In this work, the apparent electrophoretic mobility of procaine hydrochloride (mi) in a series of concentration of b-cyclodextrin were measured directly by capillary electrophoresis technology. mi needs to apply correction for viscosity change in order to avoid the misinterpretation of data. A new mathematical treatment method is proposed, which based on the fact that the molar ratio of the inclusion complex between b-cyclodextrin and procaine hydrochloride was 1:1. The determination value of the proposed method was in correspondence with those of the spectrophotometric and fluorescence methods, which indicated that the proposed method had a good accuracy. This method offered the advantages of automation, small amounts of sample, conciseness as well as easy operation.
Acknowledgements This project was supported by the Foundation of Science and Technology Department of Fujian Province, China. Dr Nianbing Li thanks to the Science and Technology Department Foundation of Fuzhou University.
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