Study on the supramolecular interaction of astemizole with cucurbit[7]uril and its analytical application

Abstract

Astemizole (AST) is non-fluorescent in aqueous solution. This property makes its determination through direct fluorescence methods difficult. Reaction and supramolecular interaction mechanisms, between AST and palmatine (PAL) as they compete for occupancy of the cucurbit[7]uril (CB[7]) cavity, were studied using spectrofluorimetry, ¹H NMR, and molecular modeling calculations. The association constants of the complexes formed between the host and the guest were determined. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescent probe method of high sensitivity and selectivity was developed to determine AST in its pharmaceutical dosage forms and in urine samples with good precision and accuracy. The linear range of the method was from 0.02 μg mL⁻¹ to 2.2 μg mL⁻¹. The detection limit was 0.007 μg mL⁻¹. This shows that the proposed method has promising potential for therapeutic monitoring and pharmacokinetics and for clinical application.

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Introduction

Cucurbit[n]uril (CB[n], n = 5–8, 10) is a family of cyclic host molecules comprised of n glycoluril units bridged by 2n methylene groups.^1–3 The symmetrical CB[n] hosts possess hydrophobic cavities and restrictive polar portals lined with ureido carbonyl groups.^2,3 CB[n] can form inclusion complexes with guest molecules that possess suitable polarity and dimensions.^4–7 A variety of organic drugs and biologically relevant molecules has been encapsulated in CB[n].^7–13 The formation of inclusion complexes often enhances or disturbs the photophysical and photochemical properties of the included guest molecules.¹⁴ The cucurbit[7]uril (CB[7]) (Fig. 1) host has been of particular interest in recent years because of its superior solubility in aqueous solution compared with other CB[n] members and its remarkable capability to form host–guest complexes with organic guest molecules.^4–13 Despite the widespread interest in supramolecular systems containing CB[n]s, however, little attention has been devoted to their fluorescent properties and potential analytical applications.

Fig. 1 Structure of CB[7], PAL, and AST.

Palmatine (PAL) (Fig. 1) is a natural isoquinoline alkaloid. In previous studies,^10,11 we observed that the fluorescence of PAL in aqueous solution is greatly enhanced in the presence of CB[n].

Astemizole (AST) (Fig. 1) is a very potent and long-acting histamine H₁ receptor antagonist and has broader clinical applications because of its fast oral absorption, high serum concentration, and lasting efficacy, among others. AST can be used to cure perennial and seasonal allergic rhinitis, allergic conjunctivitis, chronic urticaria, and other allergic symptoms.¹⁵ Because of the aqueous solution of AST has no native fluorescence, AST cannot be determined directly by the normal fluorimetric method. Therefore, development of a rapid, simple, and highly sensitive spectrofluorimetric method for the determination of AST in aqueous solution is highly desirable. AST has been determined by a variety of methods, including spectrophotometry,^16–19 HPLC,^20,21 TLC,²² voltammetry,²³ and radioimmunoassay.²⁴ Spectrophotometric method has been used most commonly. However, the method is not sensitive enough and has poor selectivity. HPLC method generally requires complicated and expensive equipment, provision for use and disposal of solvents, and labour-intensive sample preparation procedure. Since spectrofluorimetry has the advantages of both sensitivity and simplicity, it has found extensive use in the determination of inorganic, organic and bioactive materials.^25–27 Kelani et al.¹⁸ developed a fluorimetric method to determine AST based on the ternary complex formation with eosin and Pb(II). The detection limit was 0.31 μg mL⁻¹. Karam et al.²⁸ reported a fluorimetric method to determine AST using eosin B. The detection limit was 0.05 μg mL⁻¹. However, these methods do not possess sufficient sensitivity, so they cannot easily be popularized.

We have previously reported the determination of histamine H₂ receptor antagonist¹¹ and sotalol,¹⁰ where the CB[7]–PAL complex was utilized, but the mechanisms of supramolecular interaction between the host and the guests had not been studied using ¹H NMR and molecular modeling calculation. To our knowledge, however, usage of the fluorescent probe for the determination of AST has not been reported. The aim of this study is to develop a fluorescent probe method with high sensitivity for the determination of AST based on the competition between AST and PAL for occupancy of the CB[7] cavity. This is the first proposed procedure for the determination of AST by a fluorescent probe method. Our proposed method is simple, rapid, accurate and sensitive. The detection limit is 0.007 μg mL⁻¹, which is lower than that of any other spectral method reported in the literature.^16–19,28

Experimental

Apparatus

Fluorescence spectra and intensity measurements were obtained with a Hitachi F-4500 spectrofluorimeter equipped with a 150 W xenon lamp (Japan). The slit width of both the excitation and emission monochromator was set at 5 nm. The fluorescence spectra were recorded at a scan rate of 1200 nm min⁻¹. All measurements were performed in a standard 10 mm path-length quartz cell set to a temperature of 25.0 ± 0.5 °C. The pH values were measured with a pHS-3TC digital precision pH meter (Shanghai, China). ¹H NMR spectra were obtained using a Bruker AV-600 MHz spectrometer (Switzerland).

Reagents and chemicals

The AST, PAL used in the experiment were obtained from the Chinese National Institute for the Control of Pharmaceutical and Biological Products without further purification. AST was dissolved in appropriate amounts of methanol then diluted with double-distilled water to prepare stock solutions with final concentration of 100 μg mL⁻¹. Working solutions were prepared by diluting appropriate amounts of the stock solution before use. Stock solution of PAL was prepared with double-distilled water to a final concentration of 1.0 mM. CB[7] was prepared and characterized according to recently reported procedures.³ A CB[7] stock solution of 1.0 mM was prepared by dissolving CB[7] in double-distilled water. Working solutions were obtained by the dilution of the stock solution. All stock standard solutions were stable for several weeks at room temperature. All other chemicals were of analytical reagent grade, and double-distilled water was used throughout the procedures.

Experimental procedure

A 0.8 mL solution of 0.1 mM CB[7] was poured into a 10 mL colorimetric flask, to which 0.8 mL of the 0.1 mM PAL solution and 1.0 mL of 0.001 M hydrochloric acid were also added. Suitable amounts of AST solution were then sequentially added to the flask. The mixture was diluted to volume with double-distilled water and shaken for 10 min at room temperature. The fluorescence intensity values of the solution (F_{CB[7]–PAL–AST}) and the blank solution (F_CB[7]–PAL) were measured at 495 nm using an excitation wavelength of 343 nm. Finally, the fluorescence quenching values (ΔF = F_CB[7]–PAL − F_{CB[7]–PAL–AST}) were computed.

Analysis of tablets

Ten tablets of AST were carefully pulverized. From this powder, a portion of this powder equivalent to 10 mg AST was accurately weighed, dissolved in 20 mL methanol in a 100 mL volumetric flask, and sonicated for 3 min. The solution was then diluted to the mark with double-distilled water. The first 10 mL of the filtrate was discarded, after which 10 mL of the remaining filtered sample solution was diluted to 100 mL with double-distilled water. Further dilution was made to obtain the sample solution using the same detection method as described in the Experimental procedure.

Analysis of spiked human urine

Urine sample (2 mL) was placed into a centrifuge tube, spiked with 1.0 mL of the drug solution and then centrifuged at 4000 rpm for 5 min. The clear supernatant of the spiked urine sample was extracted according to the literature.²¹ The combined organic layers were finally evaporated to dryness under a gentle stream of nitrogen in a water bath at 55 °C. The residue was dissolved in appropriate amounts of methanol. Further dilution was made to obtain the sample solution using the same detection method as described in the Experimental procedure.

Results and discussion

Fluorescence quenching of CB[7]–PAL complex by AST

Fig. 2 shows the fluorescence spectra of PAL in the presence of different concentrations of CB[7]. Fluorescence intensity increased with increased CB[7] concentration. However significant quenching of fluorescence intensity of the CB[7]–PAL complex with the addition of AST was observed. Fluorescence suppression of the fluorescence of the CB[7]–PAL complex, in the presence of different concentrations of AST, are shown in Fig. 3. Fluorescence intensity decreased with increased AST concentration, which is likely to be due to the competition between the AST and PAL molecules for occupancy of the CB[7] cavity. Parts of the PAL molecule can be expelled from CB[7] cavities by the introduction of the drug, thereby reducing the fluorescence intensity of CB[7]–PAL because of the formation of a new inclusion complex between CB[7] and AST.

Fig. 2 Fluorescence spectra of 1.0 μM PAL in the presence of CB[7]. The concentrations of CB[7] (μM): (a) 0; (b) 0.1; (c) 0.5; (d) 1.0; (e) 1.5; (f) 2.5; (g) 4.0; (h) 20.0. pH 4.0, λ_ex = 343 nm.

Fig. 3 Fluorescence spectra of CB[7]–PAL in the presence of AST. Concentrations of AST (μM): (a) 0; (b) 0.22; (c) 0.44; (d) 0.87; (e) 1.31; (f) 4.36. C_CB[7] = 1.0 μM, C_PAL = 1.0 μM. pH 4.0, λ_ex = 343 nm. The inset shows the [AST]_t/P vs. Q plot according to eqn (1) for the titration of a 1.0 μM CB[7] and 1.0 μM PAL solution with AST at pH 4.0, with data taken at 495 nm.

Apparent association constant

The apparent association constant for the 1 : 1 complex of CB[7] with PAL was determined to be 1.13 × 10⁵ M⁻¹.¹⁰ Unfortunately, when CB[7] was titrated into solution of AST there was no significant change in its UV absorbance and fluorescence intensity. As such, association constant between CB[7] and AST was determined based on competitive fluorimetric method, derived from the competitive spectrophotometric method^29–32 (see the ESI†). In our study, this method is based on PAL (acting as an indicator) at pH 4.0, with the result analysed (see ESI†) using equation (1)where Q = (F − F_∞)/(F₀ − F), P = [CB[7]]_t − 1/QK_CB[7]–PAL − [PAL]_t/(Q + 1), K_CB[7]–PAL is the association constant between CB[7] and PAL, K_CB[7]–AST is the association constant between CB[7] and AST, and [AST]_t is concentration of AST. Thus, according to the [AST]_t/P vs Q plot (Fig. 3 inset), we obtained the association constant K_CB[7]–AST = 3.37 × 10⁶ M⁻¹ from the slope and the known K_CB[7]–PAL.¹⁰ This value is so large it indicates very strong host–guest interaction with excellent size and shape matches. A comparison of K_CB[7]–PAL with K_CB[7]–AST obviously results in K_CB[7]–AST > K_CB[7]–PAL. Thus, AST shows stronger binding with CB[7] than PAL. Accordingly, considering the thermodynamic factor only, the PAL molecules can be expelled from the CB[7] cavities by the AST molecules.

Molecular modeling

Molecular modeling calculations were optimized at the B3LYP/6-31 G(d)³³ level of density functional theory³⁴ using the Gaussian 03 program.³⁵ Results confirmed the partial inclusion of PAL in the hydrophobic cavity of CB[7] (Fig. 4A). In the energy-minimized structure, the methoxy-isoquinoline moiety is embedded in CB[7] cavity, and the heterocyclic nitrogen is located in the vicinity of a carbonyl-laced portal. The partial immersion of PAL in the hydrophobic cavity of CB[7] reduces interaction with water. This state results in less of a polar microenvironment which, in turn, leads to fluorescence enhancement.

Fig. 4 Energy-minimized structures of (A) CB[7]–PAL, and (B) CB[7]–AST complexes in the ground state using balls and tubes for the rendering of atoms. Color codes: PAL and AST, green; CB[7], oxygen, red; nitrogen, blue; carbon, gray.

AST has a hydrophobic phenylethyl group. Thus, when AST was added to the host–guest system of CB[7]–PAL, the PAL and the AST compete to occupy the CB[7] cavity. Some parts of the PAL molecules were expelled from the CB[7] cavity with the introduction of the AST. The energy-minimized structure of the CB[7]–AST complex is shown in Fig. 4B, the phenylethyl moiety is embedded in the CB[7] cavity, and the heterocyclic nitrogen is located in the vicinity of a carbonyl-laced portal. The photochemical property of PAL is strongly dependent on its local microenvironment. The addition of the drug caused PAL to lose its protection in the CB[7] hydrophobic cavity, thus resulting in reduced PAL fluorescence intensity.

¹H NMR

The formation of CB[7]–PAL and CB[7]–AST inclusion complexes in aqueous solution was also confirmed using ¹H NMR spectroscopy (Fig. 5). The results of the ¹H NMR experiments are consistent with the theoretical structures of the complexes. Fig. 5c shows the ¹H NMR spectra of a 1 : 1 host–guest complex between CB[7] and PAL in aqueous solution. Compared to the proton resonances of the unbound PAL molecules (Fig. 5b), the signals from H₈, H₁₃, H₁₁, H₁₂, H₁₄, and H₁₅ protons of the bound PAL were significantly shifted upfield. This behavior is characteristic of this part of the molecule encapsulated in the CB[7] cavity.³⁶ The signals of H₁, H₄, H₅, H₆, H₁₆, and H₁₇ protons of the bound PAL experienced downfield chemical shifts, which is characteristic of the protons in this part of the molecule located just outside the carbonyl portal of the CB[7] host.³⁶ The presence of PAL also influences the chemical shifts of CB[7] protons in the complex. While the location of the singlet of –CH protons remains unchanged after complexation, the doublets of –CH₂ protons are split into quartets, showing that partial inclusion of PAL significantly affects the signal of these protons in CB[7]. Fig. 5e shows the ¹H NMR spectra of the CB[7]–AST complex. Compared with the proton resonances of the unbound AST molecules (Fig. 5f), the signals from a, b, B, and C protons of the bound AST molecule significantly shifted upfield, indicating this part of the molecule encapsulated in the CB[7] cavity. The signals of D, E, F, and G protons of the bound AST experienced downfield chemical shifts, indicating this part of the molecule located just outside the carbonyl portal of the CB[7] host. The chemical shifts of the c, d, e, f, g, and h protons almost did not change, which suggests that no interaction between these protons and CB[7] exists. The chemical shift of the A protons is practically unchanged, which indicates negligible interaction with CB[7]. In similar results with PAL, the presence of AST also influences the chemical shifts of CB[7] protons in the CB[7]–AST complex. Fig. 5d shows the ¹H NMR spectra of the CB[7]–PAL complex solution with the addition of AST. The proton singles of the bound PAL molecules disappeared. However, the signals from the protons of the bound AST molecules as well as the free PAL molecules were observed clearly (Fig. 5d). These findings indicate that the PAL molecules were expelled from the CB[7] cavity due to the addition of AST. These results are consistent with the foregoing discussion.

Fig. 5 ¹H NMR spectra (600 MHz) of CB[7] (a), PAL (b), CB[7]–PAL complex (c), CB[7]–PAL complex in the presence of AST (d), CB[7]–AST complex (e), and AST (f) in D₂O.

In summary, combination of hydrophobic interaction of the cavity of CB[7] as well as ion–dipole interaction between the carbonyl portal of CB[7] and N⁺ ion (of PAL, and AST) leads to the formation of host–guest inclusion complex. Because of the excellent size and shape matches, AST is enclosed in the cavity of CB[7] more tightly than PAL.

Optimization of experimental conditions

The effect of varying PAL concentrations on the fluorescence intensity of the CB[7]–PAL complex were studied. The concentration of PAL was varied from 4.0 μM to 9.0 μM. The fluorescence intensity of the CB[7]–PAL complex was gradually enhanced as PAL concentration increased until it reached the maximum inclusion equilibrium at CB[7] saturation. In the present paper, PAL served as a fluorescent probe; thus, determining the proper concentration was crucial. If the PAL concentration is too low, the sensitivity of the probe will also be low. Conversely, a very high concentration may not help determine the optimum detection limit of the analyte. Taking everything into consideration, the optimal PAL concentration was 8.0 μM.

The effect of pH on ΔF was studied over the pH range of 1.0 to 12.0. The results indicate that ΔF is maximum and almost constant in the pH range of 2.0 to 7.0. However, ΔF significantly decreased with further increases in pH. The reason is that alkali cations are readily coordinated to the carbonyl-fringed portals of CB[7] in alkaline medium. Binding of the alkali cation lowers the rate constant of the ingress of organic guests.⁸ Results show that the presence of alkali cations in alkaline medium lowers not only the equilibrium constant of PAL binding with CB[7] but also the fluorescence quantum yield. Hence, using hydrochloric acid, the pH was adjusted to 4.0, which was the desired pH for all subsequent experiments.

The effect of temperature on ΔF was examined within 10 °C to 80 °C. All formed complexes were stable up to 35 °C. Above 35 °C, the fluorescence intensity greatly decreased due to the dissociation of the complexes at high temperatures. Hence, all subsequent measurements were performed at room temperature.

In addition, ΔF reached a maximum within a very short time (the complexation of CB[7] is very fast) after the reagents were added and remained constant for at least 5 h. Hence, the standard reaction condition was set to room temperature for 10 min.

Effect of interfering substances

Prior to the application of the proposed fluorescent probe method to real samples, the interferences of commonly used tablet excipients on the determination of 0.2 μg mL⁻¹ of AST were studied under optimum experimental conditions. A 3000-fold mass in excess of each excipient over AST was first tested. When interference occurred, the ratio was progressively reduced until the interference ceased. The criterion for interference was fixed at a ± 5% variation in the average fluorescence intensity calculated for the established level of AST. The results are shown in Table 1. The determination was obviously free of interference from the usual excipients. During the testing of real samples for the determination of pharmaceutical preparations, no other interfering substances having similar structure with AST in excipients were observed. Thus, no background interference was present, indicating good selectivity in the method used to test the drug in both raw material and dosage forms.

Table 1 Effect of interference (tolerance error ± 5.0%)

Tolerance ratio in mass	Interference
3000	Starch, glucose, sucrose, lactose, sorbitol, mannitol, boracic acid, hexane diacid
2000	Methyl cellulose
1500	Gelatin, glycin
1000	Sodium hydroxymethyl cellulose, gum acacia power, tryptophan
500	Sodium carboxymethyl cellulose
100	NH4^+, Na^+, K^+
50	Mg^2+, Zn^2+, Ca^2+, Fe^2+
0.2	Alanine, cysteine, cystine, phenylalanine, valine
5	Cetirizine dihydrochloride, chlorphenamine maleate, ketotifen
3	Loratadine, promethazine hydrochloride
1.5	Cyproheptadine, diphenhydramine hydrochloride

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The interferences of commonly used other antihistamines in our laboratory were also tested in the current work (Table 1). The experimental results showed that the determination was obviously free of interference from these antihistamines. The evidence also indicated that the proposed method had a high selectivity to AST.

However, the components in the urine samples (Na⁺, K⁺, Ca²⁺, cysteine, cystine, alanine, phenylalanine, and valine) may quench the fluorescence intensity of the CB[7]–PAL complex to a certain degree (Table 1). Hence, they should be separated prior to the determination. Accordingly, the drug and the interfering substances were separated through the organic solvent extraction method.²¹

Calibration graph and sensitivity

Under the optimum experimental conditions described, the standard calibration curve was drawn by plotting ΔF versus AST concentration (Fig. 6). The linear regression equation for the method was ΔF = 18.26 + 3088.19 × C_AST (μg mL⁻¹). The correlation coefficient was 0.9993, indicating good linearity. The detection limit was 0.007 μg mL⁻¹. The small variance (0.008) confirmed the small degree of scattering of the experimental data points along the regression line. The proposed method proved to have higher sensitivity than any other spectral method for determining AST reported in the literature,^16–19,28 as presented in Table 2.

Fig. 6 The standard calibration curve ΔF vs. [AST].

Table 2 Comparison with other proposed spectral methods for the determination of AST

Technique	Linear range (μg mL^−1)	Detection limit (μg mL^−1)	Application	Ref.
Spectrophotometry	4.6–45.8	1.53	Tabellae	16
	0.4–4.0	0.025	Tabellae	17
	4.1–37.6	1.37	Tabellae, suspension	18
	0.5–100	0.5	Tabellae	19
Spectrofluorimetry	0.94–7.1	0.31	Tabellae, suspension	18
	0.16–0.96	0.05	Tabellae	28
Proposed method	0.02–2.2	0.007	Tabellae, human urine

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Analysis of pharmaceutical formulations

The proposed fluorescent probe was applied in the determination of AST in pharmaceutical preparations. The results are presented in Table 3. The relative standard deviations obtained from the proposed method were less than 1.00%. Moreover, to check the validity of the proposed method, the standard addition method was applied by adding AST to previously analyzed tablets. The recovery of each drug was calculated by comparing the concentration obtained from the (spiked) mixtures with that of the pure drugs. In the t- and F-tests, no significant differences were found between the calculated and theoretical values (95% confidence) of both the proposed and reference methods,^17,28 indicating similar precision and accuracy between the values.

Table 3 Determination of AST in pharmaceutical formulation using CB[7]/PAL (n = 5)

Batch no.	Fluorescent probe method			Literature method
	Found (mg/tablet)	Equivalent nominal content (%) ± S.D.^a	Recovery (%)	Found (mg/tablet)	Equivalent nominal content (%) ± S.D.^a
a Average of five determination. (The tabulated values of t and F at the 95% confidence limit are t = 2.31 and F = 6.39.) b Xian–Janssen Pharmaceutical Ltd.
010906827^b	2.95	98.33 ± 0.97	99.70 ± 0.68	2.94	98.02 ± 0.76 (ref. 17)
		F = 1.23 (6.39)
		t = 1.61 (2.31)
030804182^b	2.93	97.67 ± 0.86	98.97 ± 0.88	2.91	97.03 ± 0.95 (ref. 28)
		F = 1.65 (6.39)
		t = 1.42 (2.31)

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Analysis of spiked human urine

The proposed method was applied in the determination of the AST in spiked samples of human urine. Accuracy was assessed by investigating the recovery of AST at five concentration levels covering the specified range (five replicates of each concentration). The results (Table 4) showed that percentage recoveries were 97.20–99.20% with an R.S.D. of less than 2.0 for spiked human urine, indicating both good accuracy and precision.

Table 4 Determination of AST in spiked urine (n = 5)

Samples	Amount added (μg mL^−1)	Amount found (μg mL^−1)	Recovery (%) ± R.S.D.^a
a Average of five determination.
Urine 1	0.10	0.0972	97.20 ± 1.3
Urine 2	0.20	0.197	98.50 ± 1.4
Urine 3	0.40	0.391	97.75 ± 1.2
Urine 4	0.80	0.785	98.12 ± 1.1
Urine 5	1.0	0.992	99.20 ± 0.7

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Conclusions

A fluorescent probe method with high sensitivity and selectivity for the determination of AST in pharmaceutical preparations and biological fluids has been developed. The proposed method is more sensitive than any other spectral methods reported in the literature. The association constants of the complexes formed between the host and the guest were calculated. The interaction mechanism between the fluorescent probe and the analyte was confirmed via the ¹H NMR spectrum. The interaction models of the supramolecular complexes were established through theoretical calculations. This method can also be used in a fluorescence sensor for the detection of non-fluorescent or weakly fluorescent substances. Related studies are in progress in our laboratory.

Acknowledgements

This work was supported by the Research Fund for the Doctoral Program of Higher Education of China (no.20091404110001). Helpful suggestions by anonymous referees are also gratefully acknowledged.

References

1. W. A. Freeman, W. L. Mock and N. Y. Shih, J. Am. Chem. Soc., 1981, 103, 7367–7368.
2. A. Day, A. P. Arnold, R. J. Blanch and B. Snushall, J. Org. Chem., 2001, 66, 8094–8100.
3. J. Kim, I. S. Jung, S. Y. Kim, E. Lee, J. K. Kang, S. Sakamoto, K. Yamaguchi and K. Kim, J. Am. Chem. Soc., 2000, 122, 540–541.
4. J. W. Lee, S. Samal, N. Selvapalam, H. J. Kim and K. Kim, Acc. Chem. Res., 2003, 36, 621–630.
5. J. Lagona, P. Mukhopadhyay, S. Chakrabarti and L. Isaacs, Angew. Chem., Int. Ed., 2005, 44, 4844–4870.
6. W. M. Nau and J. Mohanty, Int. J. Photoenergy, 2005, 7, 133–141.
7. J. Mohanty, A. C. Bhasikuttan, S. Dutta Choudhury and H. Pal, J. Phys. Chem. B, 2008, 112, 10782–10785.
8. M. Megyesi, L. Biczók and I. Jablonkai, J. Phys. Chem. C, 2008, 112, 3410–3416.
9. M. D. Pozo, L. Hernández and C. Quintanaaa, Talanta, 2010, 81, 1542–1546.
10. H. M. Zhang, J. Y. Yang, L. M. Du, C. F. Li and H. Wu, Anal. Methods, 2011, 3, 1156–1162.
11. Y. X. Chang, Y. Q. Qiu, L. M. Du, C. F. Li and M. Guo, Analyst, 2011, 136, 4168–4173.
12. S. W. Heo, T. S. Choi, K. M. Park, Y. H. Ko, S. B. Kim, K. Kim and H. I. Kim, Anal. Chem., 2011, 83, 7916–7923.
13. S. Liu, C. Ruspic, P. Mukhopadhyay, S. Chakrabarti, P. Y. Zavalij and L. Isaacs, J. Am. Chem. Soc., 2005, 127, 15959–15967.
14. K. A. Connors, Chem. Rev., 1997, 97, 1325–1358.
15. A. M. Al-Obaid and M. S. Mian, in Analytical Profiles of Drug Substances, ed. K. Florey, Academic Press, New York, 1991, vol. 20, p. 173.
16. S. Güngör and F. Onur, J. Pharm. Biomed. Anal., 2001, 25, 511–521.
17. C. S. P. Sastry and P. Y. Naidu, Talanta, 1998, 45, 795–799.
18. K. Kelani, L. I. Bebawy and L. Abdel-Fattah, J. Pharm. Biomed. Anal., 1999, 18, 985–992.
19. A. A. Alwarthan and A. M. Al-Obaid, J. Pharm. Biomed. Anal., 1996, 14, 579–582.
20. M. V. Suryanarayana, S. Venkataraman, M. S. Reddy and B. P. Reddy, Talanta, 1993, 40, 1357–1360.
21. R. Woestenborghs, L. Embrechts and J. Heykants, J. Chromatogr., B, 1983, 278, 359–366.
22. S. Mangalan, R. B. Patel, T. P. Gandhi and B. K. Chakravarthy, J. Chromatogr., B, 1991, 567, 498–503.
23. A. H. Alghamdi, Chem. Pap., 2008, 62, 339–344.
24. R. Woestenborghs, I. Geuens, M. Michiels, R. Hendriks and J. Heykants, Drug Dev. Res., 1986, 8, 63–69.
25. Z. P. Wang, L. L.Shi, G. S. Chen and K. L. Cheng, Talanta, 2000, 51, 315–326.
26. B. Tang, L. Zhang and Y. Geng, Talanta, 2005, 65, 769–775.
27. H. M. Abdel-Wadood, N. A. Mohamed and A. M. Mahmoud, Spectrochim. Acta, Part A, 2008, 70, 564–570.
28. H. Karam, N. E. Kousy and M. Towakkol, Anal. Lett., 1999, 32, 79–96.
29. K. A. Conners, Binding Constants: The Measurement of Molecular Complex Stability, John Wiley and Sons, New York, 1987.
30. Z. Zhong and E. V. Anslyn, J. Am. Chem. Soc., 2002, 124, 9014–9015.
31. A. E. Hargrove, Z. Zhong, J. L. Sessler and E. V. Anslyn, New J. Chem., 2010, 34, 348–354.
32. L. Zhu, Z. Z. Zhong and E. V. Anslyn, J. Am. Chem. Soc., 2005, 127, 4260–4269.
33. C. Lee, W. Yang and R. G. Parr, Phys. Rev. B: Condens. Matter Mater. Phys., 1988, 37, 785–789.
34. A. D. Becke, Phys. Rev. A, 1988, 38, 3098–3100.
35. M. J. Frisch, et al., Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford, CT, 2004.
36. W. L. Mock and N. Y. Shih, J. Org. Chem., 1986, 51, 4440–4446.

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Footnote

† Electronic supplementary information (ESI) available: Derivation of eqn (1) and determination of association constant between CB[7] and AST (K_CB[7]–AST). See DOI: 10.1039/c2ay25929j

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