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Study on the inclusion interactions of β-cyclodextrin and its derivative with dyes by spectrofluorimetry and its analytical application
Talanta 72 (2007) 237–242
Study on the inclusion interactions of -cyclodextrin and its derivative with dyes by spectrofluorimetry and its analytical application Xiashi Zhu ∗ , Jing Sun, Jun Wu College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China Received 25 August 2006; received in revised form 28 September 2006; accepted 17 October 2006 Available online 15 November 2006
Abstract The inclusion interactions of -cyclodextrin (-CD) and hydroxypropyl--cyclodextrin (HP--CD) with dyes were developed by spectrofluorimetry, and the inclusion constants of inclusion complexes were determined by direct fluorescence technique. The main factors (the host molecule, the guest molecule, and the pH) for the inclusion interaction were discussed in detail. At the same time, the inclusion interaction of HP--CD and vitamin B6 (VB6 ) was investigated with the competitive fluorescence inclusion method and the inclusion constant of HP--CD and vitamin B6 (VB6 ) was obtained by indirect fluorescence technique. On the basis of the linear relationship between the change of fluorescence intensity (F) and the concentration of VB6 , a competitive fluorescence inclusion method was used to the determination of VB6 . The method has been successfully applied to the analysis of VB6 in synthetic samples, tablets and injections with satisfactory results. © 2006 Elsevier B.V. All rights reserved. Keywords: Cyclodextrin; Dye; Spectrofluorimetry; Competitive inclusion; VB6
1. Introduction -Cyclodextrin and its various derivatives (-CDs) are well known, which structure is that of a truncated cone with the hydrophilic outer surface and a hydrophobic internal cavity [1] (Fig. 1). This peculiar molecular structure could include guest molecules to form inclusion complexes. The photochemical and photophysical properties of the guest molecules would be altered due to the formation of a super-molecular complex (guest-CDs). Thus, the physical, chemical and biochemical characters of guest molecules are modified, and the application capability of those guest molecules could be improved. So, -CDs with inclusion properties had been widely used in various fields such as medicine [2–5], chemistry [6,7], agriculture [8], and so on. Investigation of the inclusion mechanism of inclusion complexes plays an important part in supramolecular chemistry. So far, the method, which included spectrum (UV–vis and fluorescence), HPLC, surface tension, electrochemistry, calorimetry, dynamics and competition method [8], were used for determining inclusion constants [2,3,5,9]. Fluorescence quantum yield
∗
Corresponding author. Tel.: +86 514 7975244; fax: +86 514 7975244. E-mail addresses: [email protected], [email protected] (X. Zhu).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.10.016
[10], thermodynamic parameters of inclusion process [11], fluorescence probes [12,13], competitive inclusion method [14,15] and 1 H NMR spectroscopy [16] were applied to investigate the inclusion mechanism of inclusion complexes. Vitamins are necessary for normal growth and activity of the body, and obtained naturally from plant, animal foods or medicines. The medicines contained vitamins are generally labile for light, heat and oxygen. While through the formation of inclusion complexes with -CDs the stability, solubility and bioavailability of those medicines could be enhanced. At present, the method of investigation inclusion interactions of vitamin-CDs mainly includes: (1) direct methods which are established based upon the remarkable changes of the observed signals (such as photochemistry, electrochemistry signals) when the vitamin molecules were included into the -CDs; (2) indirect method of determination inclusion constant could be established, namely competitive inclusion method [17] for other vitamins which signals were not changed with -CDs-included. In this paper, six dyes (salicylfluorone (SAF), butyl rhodamine B (BRB), methylene blue (MB), erythrosine sodium (ES), tetrachlorotetraiodo-fluorescein disodium (TCTIF) and tetrachlorofluorescein (TCF)) were chosen to study the inclusion interactions of them with -CD and hydroxypropyl-cyclodextrin (HP--CD) by fluorescence spectroscopy and 1 H
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X. Zhu et al. / Talanta 72 (2007) 237–242
sion complexes were estimated assuming a 1:1 inclusion model, and the inclusion constants could be obtained from fluorescence data by the modified Benesi–Hildebrand equation (double reciprocal plot) [18,19]. 2.4. Determination of inclusion constants by indirect fluorescence method
Fig. 1. Structure of CD oligosaccharide.
NMR spectroscopy technique. The influence factors of inclusion interactions (the host molecule, the guest molecule, and the pH) were discussed in detail. In addition, the inclusion interaction of VB6 -HP--CD was investigated with the competitive fluorescence inclusion method and the inclusion constant of the inclusion complex (VB6 -HP--CD) was obtained by indirect fluorescence technique. On the basis of the linear relationship between the change fluorescence intensity (F) and the concentration of VB6 , a new method for the analysis of VB6 was developed. The method has been successfully used to determine VB6 in synthetic samples, tablets and injections with satisfactory results.
The concentration of ES and TCTIF were 1.0 × 10−5 mol/L, and HP--CD concentration was 3.2 × 10−3 mol/L, whereas changed the concentration of VB6 . Buffer solution was used to control the pH value of the solutions (ES: pH 5.0; TCTIF: pH 7.6). The fluorescence intensities of these solutions were measured at the optimal excitation wavelength as to evaluate the inclusion constant of VB6 -HP--CD. 2.5. Sample preparation
2. Experimental
Took 30 pieces of VB6 tablets (10 mg/piece), crushed them into powder. Then took 0.2 g powder and dissolved it into water, then filtrated it. The filtrate was transferred into a 50 mL volumetric flask, and diluted to final volume with distilled water. Took five tubes of VB6 injections (0.1 g/2 mL) and mixed them. 0.8 mL the mixed solution was transferred into a 25 mL volumetric flask.
2.1. Apparatus
3. Results and discussion
The fluorescence measurements were performed with a F4500 spectrofluorimeter (Hitachi, Japan). The pH measurements were made with a model pHS-25 pH meter (Shanghai, China). 1 H NMR experiments were performed on Bruker AV 600 instrument.
3.1. Fluorescence spectra of CDs-dye inclusion complex
2.2. Reagents All chemicals were of analytical-reagent grade. -CD (China Medicine Group Chemical Reagent Corporation), HP--CD (average MW = 1425, D.S. = 5.0, Yi Ming Fine Chemical Corporation, China) were used without prior purification. The stock solutions of all dyes were prepared by directly dissolving the crystal into distilled water or ethanol. A standard stock solution of Vitamin B6 (Nanjing Pharmacy Factory, China) was prepared. HAc-NaAc and NH3 -NH4 Cl buffer solutions were used to control the pH value. D2 O was used as the solvent in the 1 H NMR experiments.
The fluorescence emission spectra of dyes were scanned. Fig. 2 was shown the fluorescence emission spectra of SAF in different media. The effects of the concentration of -CDs on the fluorescence intensity of dyes were determined. The results showed that with an increase of -CD concentration (1) the fluorescence intensities of the SAF and MB were increased; (2) the fluorescence intensity of the BRB was decreased; (3) the fluorescence intensity of the TCF, ES and TCTIF was not obvi-
2.3. Determination of inclusion constants by direct fluorescence method A quantitative solution of dyes was transferred into a 25 mL volumetric flask, whereas added different amount of 0.01 mol/L -CD or HP--CD. Then buffer solution was used to control the pH value of the solutions. The mixed solutions were diluted to final volume with distilled water and shaken thoroughly, then it was thermostated at 20.0 ± 1 ◦ C and the fluorescence intensities were measured at the optimal excitation wavelength. The inclu-
Fig. 2. Fluorescence emission spectra of SAF (1.0 × 10−5 mol/L) in different media: (a) H2 O, (b) -CD, (c) HP--CD.
X. Zhu et al. / Talanta 72 (2007) 237–242 Table 1 Fluorescence spectrum characteristics of SAF, BRB, MB, ES, TCTIF and TCF System SAF--CD SAF-HP--CD BRB--CD BRB-HP--CD MB--CD MB-HP--CD ES--CD ES-HP--CD TCTIF--CD TCTIF-HP--CD TCF--CD TCF-HP--CD
pH
λex (nm)
3.2
486
3.2
556
5.0
664
5.0
520
7.6
546
7.6
538
239
Table 2 Chemical shifts δ of protons in -CD, BRB--CD and MB--CD
λem (nm)
fa
-CD
511 510 584
0.32 0.51 −0.30 −0.18 0.27 0.30 0.02 0.18 0.01 0.17 −0.03 0.01
H1
H2
H3
H4
H5
H6
4.92 4.89 4.91 −0.03 −0.01
3.43 3.41 3.42 −0.02 −0.01
3.73 3.57 3.69 −0.16 −0.04
3.51 3.47 3.50 −0.04 −0.01
3.82 3.65 3.77 −0.17 −0.05
3.73 3.50 3.69 −0.23 −0.04
688 688 550 549 563 564 562 562
-CD: 8.0 × 10−4 mol/L, HP--CD: 8.0 × 10−4 mol/L. a Fluorescence sensitive factor f = (F − F )/F , where F and F represent the 0 0 0 fluorescence intensity of the dyes in the presence and absence of CDs.
ous change. While with an increase of HP--CD concentration (1) the fluorescence intensities of the SAF, MB, ES and TCTIF were increased; (2) the fluorescence intensity of the BRB was decreased; (3) the fluorescence intensity of the TCF was not obvious change. In this paper, fluorescence sensitive factor (f) was used to show the influence degree of CDs on the fluorescence intensity of the dyes (Table 1). The uncertainty budget for f was less than 0.01%. 3.2. The inclusion interactions of dyes and β-CDs The photochemical and photophysical properties of the guest molecules could be altered due to the formation of a host-guest complex (dyes--CDs). The changes of fluorescence intensities of the dyes in the presence of -CDs might be due to the dye molecules entered the hydrophobic cavity of CDs and the hydrophobic cavity of CDs provided the different microenvironment for the guest (dye molecules). But, the changes of fluorescence intensities might be also due to a solvent effect caused by a higher concentration of -CDs. So the fluorescence intensities changes could only show the possibility of inclusion interactions. To demonstrate the formation of a host-guest complex (dyes-CDs), several experimental techniques were used: (1) -CDs are the cyclic oligosaccharides consisting of six or more d-(+)glucopyranose units (Fig. 1), the effect of glucose on the fluorescence spectra of the dyes was tested, which could prove whether fluorescence spectra changes were a solvent effect caused by a higher concentration of -CD or HP--CD [16]; (2) The polarity of the hydrophobic cavity of -CD and HP--CD was similar to that of alcohols (R–OH), the effect of different alcohols on the fluorescence spectra of dyes were investigated, which could reflect the inclusion interactions between dyes and -CDs; (3) The chemical shifts of CDs protons in different system was measured by 1 H NMR spectroscopy, which could be observed the deep of guest molecule entered the cavity of CDs. To prove that these spectral changes were not a solvent effect caused by a high concentration of CDs, the effect of glucose on the spectra of dyes was tested. When the addition of glucose (in
δ-CD δBRB--CD δMB--CD δ1 a δ2 b a b
δ1 = δBRB--CD − δ-CD . δ2 = δMB--CD − δ-CD .
an equivalent mass of 2.00 × 10−3 mol/L -CD or HP--CD) to a 1.00 × 10−5 mol/L of dye solution, the fluorescence spectra of the dyes were neither spectral shifts nor intensity changed, which contrasted with those found when -CD or HP--CD was added. It confirmed that the -CDs-induced changes observed in the fluorescence spectra of dyes indicated the formation of supramolecular complexes between the two molecules, namely there were the conclusion interactions between the -CDs and dyes. Ethanol/water, 2-propanol/water and glycerol/water were used as media to obtain the fluorescence spectra of MB at different alcohol/water ratios. It was found that the emission wavelength of MB was red-shifted, and its fluorescence intensity was gradually enhanced as the percentage of the alcohols in the mixed solvents increased. The facts suggested that the microenvironment around dye molecules in the presence of -CD and HP--CD was similar to that in alcohols, thus it indicated the formation of inclusion complexes between -CDs and MB. According to the 1 H NMR spectra of -CD, BRB--CD, and MB--CD, apparent changes in chemical shifts of different protons could be observed (Table 2): (1) unlike the dramatic upfield shifts of -CD’s interior H-3, H-5, and H-6 protons, which resulted from the shielding effect exerted by the inclusion of the guest molecule (BRB, MB) into -CD’s cavity, the shifts of -CD’s outside H-2 and H-4 protons could be neglected; the apparent shift changes of -CD’s H-3 indicated that the guest molecule entered the cavity along its wide rim; (2) the chemical shifts’ degree of the protons (δ) was BRB--CD > MB--CD, which indicated that BRB entered the cavity more deeply than MB, and the inclusion interaction of BRB with -CD was more strong than MB with -CD. All the facts showed obviously that the guest molecules were included in -CD’s cavity. The inclusion interaction of -CDs and dyes (host-guest) could be influenced by many factors. The inclusion constant K is an important parameter for characterizing the inclusion interactions of -CDs with guest molecules. K value reflects the intensity of binding force of -CDs with guest molecules. The inclusion constants of the dyes with -CDs were determined by directly fluorescence technique [18,19] and shown in Table 3. From the data, the influence factors of inclusion interactions (the host molecule, the guest molecule, and the pH) were discussed.
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Table 3 Inclusion constants of the dyes with -CD and HP--CD Dyes
pH
K (L/mol) -CDa 1069 1720 710 8886
± ± ± ±
HP--CDa 63 60 21 266
11984 21032 13045 5785
± ± ± ±
SAF
3.2 6.0 9.0 3.2
589 1156 578 135
BRB
6.0 11.3 3.2
4528 ± 96 3642 ± 42 353 ± 14
2090 ± 100 1424 ± 34 285 ± 19
MB
6.0 9.0 3.2
410 ± 21 358 ± 16 –
326 ± 15 178 ± 17 141 ± 10
ES
5.0 11.3 5.0
– – –
340 ± 22 189 ± 13 70 ± 5
TCTIF
7.6 11.3
– –
241 ± 15 146 ± 12
–
–
TCF a
–
The confidence level was 95%.
3.2.1. Effect of the host molecule on inclusion interaction -Cyclodextrins have the hydrophilic outer surface and a hydrophobic internal cavity. The inclusion interaction of -CDs and the guest molecules were affected by the size of the internal cavity and the hydrophilic, hydrophobic characters of the host. It is generally believed that dipole-dipole, electrostatic, van der Walls forces, hydrogen bonding, hydrophobic interaction, and the release of distortion energy of CD ring upon guest binding cooperatively govern the stability of an inclusion complex. The neutral -CDs are not charged (2.0 < pH < 11.0) and the major inclusion interactions are hydrophobic interactions between the guest and CD cavity, and hydrogen bonding of the guest to OH groups or other introduced groups on CD ring [7]. As a result of additional groups of HP--CD comparative parent -CD, the hydrophilic property of HP--CD was better than that -CD. Meanwhile, the chemical modification enlarged the cavity of the host molecule, thus as a rule its inclusion capability was enhanced. Table 3 showed that the K value of SAF-HP--CD was larger than that SAF--CD, Moreover, compared with HP-CD, the inclusion interactions of ES and TCTIF with -CD were not observed in these experiments. But the inclusion interactions of -CDs with dyes were complicated, besides the size of the internal cavity and the hydrophilic, hydrophobic characters of the host, the other effect factors (charge of host and guest, speciation of guest molecule) were contributing and could result from the difference of K value. 3.2.2. Effect of the guest molecule on inclusion interaction The structure of guest is very important for host-guest inclusion interaction. The shape and size of the guest must match with the cavity of -CDs, and its polarity should be smaller than water. For SAF and BRB, their hydrophobic properties were relatively stronger; they were easy to enter the hydrophobic cavity of CDs and to form the relatively stable inclusion com-
plexes. For MB, its hydrophobic was weaker than that of BRB, so the host-guest inclusion interaction of -CDs-MB was weaker (KBRB-CDs < KMB-CDs ), which could be proved by the chemical shifts’ degree of the protons (δ), the chemical shifts’ degree of the protons (δ) was BRB--CD > MB--CD, which indicated that BRB entered the cavity more deeply than MB, and the inclusion interaction of BRB with -CD was more strong than MB with -CD. ES and TCTIF were easy to dissolve in water for their good hydrophilic property. So their inclusion complexes with -CDs were relatively labile, and the K values were smaller. 3.2.3. Effect of pH on inclusion interaction The effect of pH on host-guest inclusion interaction mostly behaved that the conformation of guest were dissimilar at different pH values, namely the polarity of the guest changed. For example, SAF was a neutral molecule in neutral medium, but in acidic and basic media, it was a charged molecule. So SAF as molecule was easily included into HP--CD in neutral medium. Similarly, the inclusion complexes of ES and TCTIF with HP-CD were more stable in subacid medium than in acidic and basic media. The reason was probably that ES and TCTIF were neutral molecules in subacid medium and the hydrophobic interactions between them and the -CDs cavity were strong; while in acidic and basic media, the guest molecules were charged, so their hydrophilic properties were better than the neutral molecules. Table 3 was shown that the inclusion constants of dyes with -CD were more sensitive to the changes of pH values. In a word, the factors which influence inclusion interaction were complex, and the K value reflected the general outcome about all the factors. 3.3. The inclusion interaction between VB6 and HP-β-CD The VB6 itself could emit fluorescence. The wavelengths of excitation and emission were at 292 and 394 nm, respectively [17]. When VB6 was into the cavity of CDs, its fluorescence intensity did not changed obviously. So, the direct fluorescence method to determine the inclusion constants of VB6 -HP--CD could not be used, the competitive inclusion method should be chosen. Competitive inclusion method was a technique that based on two different guests completing the same CD’s cavity which caused a remarkable change of the observed signal. When VB6 was added into the solution which containing the CDs-probe, the fluorescence intensity of the fluorescence probe was reduced. That was because some VB6 molecules replaced portion of the fluorescence probe molecules from the CD’s cavity. The competitive inclusion process is as follows: CD-G + V = CD-V + G
(1)
where the symbols G, V, CD-G and CD-V represent fluorescence probe molecule, VB6 , and the inclusion complexes, respectively. With KCD-G , KCD-V to represent the inclusion constant of CD-G and CD-V, respectively, then KCD-V could be obtained from the
X. Zhu et al. / Talanta 72 (2007) 237–242
241
Fig. 3. Effect of VB6 on the fluorescence spectra of ES-HP--CD (pH 5.0). (1) 1.0 × 10−5 mol/L ES + 2.0 × 10−3 mol/L HP--CD; (2–3) 1 + VB6 (4.0 × 10−3 , 8.0 × 10−3 mol/L); (4) 1.0 × 10−5 mol/L ES.
Fig. 4. Effect of VB6 on the fluorescence spectra of TCTIF-HP--CD (pH 7.6). (1) 1.0 × 10−5 mol/L TCTIF + 2.0 × 10−3 mol/L HP--CD; (2–3) 1 + VB6 (4.0 × 10−3 , 1.2 × 10−2 mol/L); (4) 1.0 × 10−5 mol/L TCTIF.
following formula:
the inclusion constant of VB6 -HP--CD was determined to be 71(±4) L/mol. The inclusion constant of Dye-HP--CD (Table 3) was larger than that of VB6 -HP--CD, namely the dyes probes included more tightly with HP--CD. So enough VB6 would be added into the system as to make the VB6 molecules replace the probe molecules from the cavity of HP--CD.
KCD-V =
([CD]0 − [CD]) [CD]([V]0 − [CD]0 + [CD])
(2)
where [CD]0 and [V]0 represent the initial concentration of CD and VB6 respectively, [CD] was the concentration of the dissociative CD in the solution. [CD] is the only unknown in this formula. It can be obtained from the following formula: [CD] =
F − FG KCD-G (FCD-G − F )
(3)
where FG , FCD-G and F represent the fluorescence intensity of the dissociative G, the fluorescence intensity of the G after it included with the CD, and the fluorescence intensity of the G when G, VB6 and CD was coexisting in the solution, respectively. In this paper, ES and TCTIF were chosen as the fluorescence probes to observe the inclusion interaction betweenVB6 and HP--CD. It was found that the fluorescence intensities of ES and TCTIF were gradually decreased with an increase of VB6 concentration, respectively (Figs. 3 and 4). The inclusion interaction was taken in 5 min, and VB6 -ES(TCTIF)-CDs was stable in 30 min It indicated that the same cavity of HP--CD was competed by the two guests (ES and VB6 , TCTIF and VB6 , respectively) and portion of the probe molecules were replaced out of the cavity by VB6 . According to the formulas (2) and (3),
3.3.1. Applications It was observed from the above experiment that in these systems of ES-HP--CD or TCTIF-HP--CD, the VB6 concentration had a linear relationship with the change fluorescence intensity of the systems (F). The experimental conditions were optimized as follows: the concentration of ES and TCTIF was 1.0 × 10−5 mol/L, HP--CD concentration was 3.2 × 10−3 mol/L, and the pH value of the systems was 5.0 and 7.6, respectively. 3.3.2. Analytical parameters The analytical characteristics for the determination of VB6 were shown in Table 4. 3.3.3. Interferences A study was carried out on the effects of foreign interferences on the determination of 20 mg/25 mL of VB6 (in ES system). With a relative error of less than ±1%, the tolerance limits for the
Table 4 Analytical parameters for the determination of VB6 Analytical characteristic
ES-VB6
TCTIF-VB6
Linear regression equation Linear range (g/mL) Correlation coefficient Limit of detection (g/mL) RSD (%) (n = 5, c = 8.0 × 10−4 g/mL)
F = −3.75 + 104651.98c (g/mL) 4.0 × 10−4 –4.0 × 10−3 0.9987 1.0 × 10−4 0.11
F = 4.79 + 32390.79c (g/mL) 6.4 × 10−4 − 4.8 × 10−3 0.9992 1.5 × 10−4 0.27
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X. Zhu et al. / Talanta 72 (2007) 237–242
Table 5 Determination of VB6 in synthetic samples Synthetic sample
Sample ingredients (mg/mL)
Addition (mg/mL)
Amount found (mg/mL)a
RSD (%)
Recovery (%)
A (ES) A (TCTIF)
VB6 (1.6) + VC (0.6) + VB2 (4.0 × 10−3 ) + VB12 (1.6 × 10−4 )
1.60
1.63 ± 0.02 1.57 ± 0.02
1.1 1.3
100.6–103.1 96.7–99.4
B (ES) B (TCTIF)
VB6 (1.6) + VC (0.4) + VB2 (8.0 × 10−3 ) + VB12 (3.2 × 10−4 )
1.60
1.56 ± 0.02 1.54 ± 0.02
1.5 1.2
96.3–98.8 95.0–97.5
a
Average value of three determination, the confidence level was 95%.
Table 6 The results of determination of the sample Sample Tablet mg/piece (ES) Tablet mg/piece (TCTIF) Injection g/2 mL (ES) Injection g/2 mL (TCTIF) a
Marked content
Proposed methoda
10.0 0.10
10.6 10.7 0.096 0.095
± ± ± ±
0.1 0.1 0.001 0.001
RSD (%) n = 3
HPLC method
0.6 1.0 0.6 0.6
10.9 ± 0.1 0.11 ± 0.01
The confidence level was 95%.
foreign substance were as fallows (mg/25 mL): Serine, leucine, l-cysteine chloride (>300); Vitamin C (25); d-l-tryptophan (20); 3,5-iodogorgoic acid (15); Vitamin B2 (0.3); Vitamin B12 (0.02). It was shown that most of the amino acids did not influence the determination of VB6 . But VB12 could absorb the fluorescence of those systems [20], it would influence the analysis, and should be separated before determination. 3.3.4. Sample analysis The calibration graphs for VB6 were constructed from the results obtained under the optimal conditions, and VB6 in synthetic samples, tablets and injections were determined with standard curve method. The results were compared with the method of pharmacopoeia (HPLC) [21] (Tables 5 and 6). The ttest method was used to do a significant difference test for these data, and it was found that the data of both methods did not have significant difference. 4. Conclusion In this paper, the inclusion interactions of dyes with -CD and HP--CD were investigated, and the inclusion constants of dyes-CDs were determined by fluorescence technique. The related inclusion mechanism was discussed. The inclusion interaction of VB6 with HP--CD was studied by the competitive fluorescence inclusion method. A new method for the analysis of VB6 was developed on the basis of the linear relationship between the change fluorescence intensity of the systems (F) and the concentration of VB6 . The method had been successfully applied to the determination of VB6 in synthetic samples, tablets and injections with satisfactory results.
Acknowledgement This work was supported by Cultivation and Construction Fund of the State Key Subject of Physical Chemistry. References [1] L. Tong, Cyclodextrin Chemistry, Science Press, Beijing, 2001, p. 10. [2] C. Yanez, R. Salazar, L.J. Nunez, J. Pharm. Biomed. Anal. 35 (2004) 51. [3] S. Rawat, S.K. Jain, Eur. J. Pharm. Biopharm. 57 (2004) 263. [4] J.J. Berzas, A. Alanon, J.A. Lazaro, Talanta 58 (2002) 301. [5] S.O. Fakayode, I.M. Swamidoss, M.A. Busch, Talanta 65 (2005) 838. [6] O. Csernak, A. Buvari-Barcza, L. Barcza, Talanta 69 (2006) 425. [7] G. Zhang, S. Shuang, C. Dong, Spectrochim. Acta, Part A 59 (2003) 2935. [8] N.L. Pacioni, A.V. Veglia, Anal. Chim. Acta 488 (2003) 193. [9] H. Xu, L. Chen, L. Ma, Anal. Test. Technol. Instrum. (Chinese) 8 (2002) 72. [10] X. Zhu, J. Sun, L. Bao, Chin. J. Appl. Chem. 23 (2006) 323. [11] G. Carmen, S. Mignel, Z. Arantza, Talanta 60 (2003) 477. [12] G. Li, J. Li, Y. Wei, Chin. J. Spectrosc. Lab. 22 (2005) 416. [13] T. Wang, H. Feng, S. Li, J. Chromatogr. A 987 (2003) 485. [14] Y. Matsui, Y. Futjie, H. Bull, Chem. Soc. Jpn. 61 (1988) 3409. [15] C. You, Y. Zhao, Y. Liu, Chem. J. Chin. Univ. 22 (2001) 218. [16] B. Tang, X. Wang, H. Liang, J. Agric. Food Chem. 53 (2005) 8452. [17] X. Liu, Y. Yang, G. Zhang, Chin. J. Anal. Chem. 31 (2003) 996. [18] F.V. Bright, T.L. Keimig, L.B. Mcgown, Anal. Chim. Acta 175 (1985) 189. [19] G.C. Ctana, F.V. Bright, Anal. Chim. 61 (1989) 905. [20] B. Liu, J. Gao, G. Yang, Spectrosc. Spectral. Anal. (Chinese) 25 (2005) 1080. [21] Chinese Pharmacopoeia (second part), Chemical Industry Press, Beijing, 2005, p. 667.
Study on the inclusion interactions of -cyclodextrin and its derivative with dyes by spectrofluorimetry and its analytical application Xiashi Zhu ∗ , Jing Sun, Jun Wu College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China Received 25 August 2006; received in revised form 28 September 2006; accepted 17 October 2006 Available online 15 November 2006
Abstract The inclusion interactions of -cyclodextrin (-CD) and hydroxypropyl--cyclodextrin (HP--CD) with dyes were developed by spectrofluorimetry, and the inclusion constants of inclusion complexes were determined by direct fluorescence technique. The main factors (the host molecule, the guest molecule, and the pH) for the inclusion interaction were discussed in detail. At the same time, the inclusion interaction of HP--CD and vitamin B6 (VB6 ) was investigated with the competitive fluorescence inclusion method and the inclusion constant of HP--CD and vitamin B6 (VB6 ) was obtained by indirect fluorescence technique. On the basis of the linear relationship between the change of fluorescence intensity (F) and the concentration of VB6 , a competitive fluorescence inclusion method was used to the determination of VB6 . The method has been successfully applied to the analysis of VB6 in synthetic samples, tablets and injections with satisfactory results. © 2006 Elsevier B.V. All rights reserved. Keywords: Cyclodextrin; Dye; Spectrofluorimetry; Competitive inclusion; VB6
1. Introduction -Cyclodextrin and its various derivatives (-CDs) are well known, which structure is that of a truncated cone with the hydrophilic outer surface and a hydrophobic internal cavity [1] (Fig. 1). This peculiar molecular structure could include guest molecules to form inclusion complexes. The photochemical and photophysical properties of the guest molecules would be altered due to the formation of a super-molecular complex (guest-CDs). Thus, the physical, chemical and biochemical characters of guest molecules are modified, and the application capability of those guest molecules could be improved. So, -CDs with inclusion properties had been widely used in various fields such as medicine [2–5], chemistry [6,7], agriculture [8], and so on. Investigation of the inclusion mechanism of inclusion complexes plays an important part in supramolecular chemistry. So far, the method, which included spectrum (UV–vis and fluorescence), HPLC, surface tension, electrochemistry, calorimetry, dynamics and competition method [8], were used for determining inclusion constants [2,3,5,9]. Fluorescence quantum yield
∗
Corresponding author. Tel.: +86 514 7975244; fax: +86 514 7975244. E-mail addresses: [email protected], [email protected] (X. Zhu).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.10.016
[10], thermodynamic parameters of inclusion process [11], fluorescence probes [12,13], competitive inclusion method [14,15] and 1 H NMR spectroscopy [16] were applied to investigate the inclusion mechanism of inclusion complexes. Vitamins are necessary for normal growth and activity of the body, and obtained naturally from plant, animal foods or medicines. The medicines contained vitamins are generally labile for light, heat and oxygen. While through the formation of inclusion complexes with -CDs the stability, solubility and bioavailability of those medicines could be enhanced. At present, the method of investigation inclusion interactions of vitamin-CDs mainly includes: (1) direct methods which are established based upon the remarkable changes of the observed signals (such as photochemistry, electrochemistry signals) when the vitamin molecules were included into the -CDs; (2) indirect method of determination inclusion constant could be established, namely competitive inclusion method [17] for other vitamins which signals were not changed with -CDs-included. In this paper, six dyes (salicylfluorone (SAF), butyl rhodamine B (BRB), methylene blue (MB), erythrosine sodium (ES), tetrachlorotetraiodo-fluorescein disodium (TCTIF) and tetrachlorofluorescein (TCF)) were chosen to study the inclusion interactions of them with -CD and hydroxypropyl-cyclodextrin (HP--CD) by fluorescence spectroscopy and 1 H
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X. Zhu et al. / Talanta 72 (2007) 237–242
sion complexes were estimated assuming a 1:1 inclusion model, and the inclusion constants could be obtained from fluorescence data by the modified Benesi–Hildebrand equation (double reciprocal plot) [18,19]. 2.4. Determination of inclusion constants by indirect fluorescence method
Fig. 1. Structure of CD oligosaccharide.
NMR spectroscopy technique. The influence factors of inclusion interactions (the host molecule, the guest molecule, and the pH) were discussed in detail. In addition, the inclusion interaction of VB6 -HP--CD was investigated with the competitive fluorescence inclusion method and the inclusion constant of the inclusion complex (VB6 -HP--CD) was obtained by indirect fluorescence technique. On the basis of the linear relationship between the change fluorescence intensity (F) and the concentration of VB6 , a new method for the analysis of VB6 was developed. The method has been successfully used to determine VB6 in synthetic samples, tablets and injections with satisfactory results.
The concentration of ES and TCTIF were 1.0 × 10−5 mol/L, and HP--CD concentration was 3.2 × 10−3 mol/L, whereas changed the concentration of VB6 . Buffer solution was used to control the pH value of the solutions (ES: pH 5.0; TCTIF: pH 7.6). The fluorescence intensities of these solutions were measured at the optimal excitation wavelength as to evaluate the inclusion constant of VB6 -HP--CD. 2.5. Sample preparation
2. Experimental
Took 30 pieces of VB6 tablets (10 mg/piece), crushed them into powder. Then took 0.2 g powder and dissolved it into water, then filtrated it. The filtrate was transferred into a 50 mL volumetric flask, and diluted to final volume with distilled water. Took five tubes of VB6 injections (0.1 g/2 mL) and mixed them. 0.8 mL the mixed solution was transferred into a 25 mL volumetric flask.
2.1. Apparatus
3. Results and discussion
The fluorescence measurements were performed with a F4500 spectrofluorimeter (Hitachi, Japan). The pH measurements were made with a model pHS-25 pH meter (Shanghai, China). 1 H NMR experiments were performed on Bruker AV 600 instrument.
3.1. Fluorescence spectra of CDs-dye inclusion complex
2.2. Reagents All chemicals were of analytical-reagent grade. -CD (China Medicine Group Chemical Reagent Corporation), HP--CD (average MW = 1425, D.S. = 5.0, Yi Ming Fine Chemical Corporation, China) were used without prior purification. The stock solutions of all dyes were prepared by directly dissolving the crystal into distilled water or ethanol. A standard stock solution of Vitamin B6 (Nanjing Pharmacy Factory, China) was prepared. HAc-NaAc and NH3 -NH4 Cl buffer solutions were used to control the pH value. D2 O was used as the solvent in the 1 H NMR experiments.
The fluorescence emission spectra of dyes were scanned. Fig. 2 was shown the fluorescence emission spectra of SAF in different media. The effects of the concentration of -CDs on the fluorescence intensity of dyes were determined. The results showed that with an increase of -CD concentration (1) the fluorescence intensities of the SAF and MB were increased; (2) the fluorescence intensity of the BRB was decreased; (3) the fluorescence intensity of the TCF, ES and TCTIF was not obvi-
2.3. Determination of inclusion constants by direct fluorescence method A quantitative solution of dyes was transferred into a 25 mL volumetric flask, whereas added different amount of 0.01 mol/L -CD or HP--CD. Then buffer solution was used to control the pH value of the solutions. The mixed solutions were diluted to final volume with distilled water and shaken thoroughly, then it was thermostated at 20.0 ± 1 ◦ C and the fluorescence intensities were measured at the optimal excitation wavelength. The inclu-
Fig. 2. Fluorescence emission spectra of SAF (1.0 × 10−5 mol/L) in different media: (a) H2 O, (b) -CD, (c) HP--CD.
X. Zhu et al. / Talanta 72 (2007) 237–242 Table 1 Fluorescence spectrum characteristics of SAF, BRB, MB, ES, TCTIF and TCF System SAF--CD SAF-HP--CD BRB--CD BRB-HP--CD MB--CD MB-HP--CD ES--CD ES-HP--CD TCTIF--CD TCTIF-HP--CD TCF--CD TCF-HP--CD
pH
λex (nm)
3.2
486
3.2
556
5.0
664
5.0
520
7.6
546
7.6
538
239
Table 2 Chemical shifts δ of protons in -CD, BRB--CD and MB--CD
λem (nm)
fa
-CD
511 510 584
0.32 0.51 −0.30 −0.18 0.27 0.30 0.02 0.18 0.01 0.17 −0.03 0.01
H1
H2
H3
H4
H5
H6
4.92 4.89 4.91 −0.03 −0.01
3.43 3.41 3.42 −0.02 −0.01
3.73 3.57 3.69 −0.16 −0.04
3.51 3.47 3.50 −0.04 −0.01
3.82 3.65 3.77 −0.17 −0.05
3.73 3.50 3.69 −0.23 −0.04
688 688 550 549 563 564 562 562
-CD: 8.0 × 10−4 mol/L, HP--CD: 8.0 × 10−4 mol/L. a Fluorescence sensitive factor f = (F − F )/F , where F and F represent the 0 0 0 fluorescence intensity of the dyes in the presence and absence of CDs.
ous change. While with an increase of HP--CD concentration (1) the fluorescence intensities of the SAF, MB, ES and TCTIF were increased; (2) the fluorescence intensity of the BRB was decreased; (3) the fluorescence intensity of the TCF was not obvious change. In this paper, fluorescence sensitive factor (f) was used to show the influence degree of CDs on the fluorescence intensity of the dyes (Table 1). The uncertainty budget for f was less than 0.01%. 3.2. The inclusion interactions of dyes and β-CDs The photochemical and photophysical properties of the guest molecules could be altered due to the formation of a host-guest complex (dyes--CDs). The changes of fluorescence intensities of the dyes in the presence of -CDs might be due to the dye molecules entered the hydrophobic cavity of CDs and the hydrophobic cavity of CDs provided the different microenvironment for the guest (dye molecules). But, the changes of fluorescence intensities might be also due to a solvent effect caused by a higher concentration of -CDs. So the fluorescence intensities changes could only show the possibility of inclusion interactions. To demonstrate the formation of a host-guest complex (dyes-CDs), several experimental techniques were used: (1) -CDs are the cyclic oligosaccharides consisting of six or more d-(+)glucopyranose units (Fig. 1), the effect of glucose on the fluorescence spectra of the dyes was tested, which could prove whether fluorescence spectra changes were a solvent effect caused by a higher concentration of -CD or HP--CD [16]; (2) The polarity of the hydrophobic cavity of -CD and HP--CD was similar to that of alcohols (R–OH), the effect of different alcohols on the fluorescence spectra of dyes were investigated, which could reflect the inclusion interactions between dyes and -CDs; (3) The chemical shifts of CDs protons in different system was measured by 1 H NMR spectroscopy, which could be observed the deep of guest molecule entered the cavity of CDs. To prove that these spectral changes were not a solvent effect caused by a high concentration of CDs, the effect of glucose on the spectra of dyes was tested. When the addition of glucose (in
δ-CD δBRB--CD δMB--CD δ1 a δ2 b a b
δ1 = δBRB--CD − δ-CD . δ2 = δMB--CD − δ-CD .
an equivalent mass of 2.00 × 10−3 mol/L -CD or HP--CD) to a 1.00 × 10−5 mol/L of dye solution, the fluorescence spectra of the dyes were neither spectral shifts nor intensity changed, which contrasted with those found when -CD or HP--CD was added. It confirmed that the -CDs-induced changes observed in the fluorescence spectra of dyes indicated the formation of supramolecular complexes between the two molecules, namely there were the conclusion interactions between the -CDs and dyes. Ethanol/water, 2-propanol/water and glycerol/water were used as media to obtain the fluorescence spectra of MB at different alcohol/water ratios. It was found that the emission wavelength of MB was red-shifted, and its fluorescence intensity was gradually enhanced as the percentage of the alcohols in the mixed solvents increased. The facts suggested that the microenvironment around dye molecules in the presence of -CD and HP--CD was similar to that in alcohols, thus it indicated the formation of inclusion complexes between -CDs and MB. According to the 1 H NMR spectra of -CD, BRB--CD, and MB--CD, apparent changes in chemical shifts of different protons could be observed (Table 2): (1) unlike the dramatic upfield shifts of -CD’s interior H-3, H-5, and H-6 protons, which resulted from the shielding effect exerted by the inclusion of the guest molecule (BRB, MB) into -CD’s cavity, the shifts of -CD’s outside H-2 and H-4 protons could be neglected; the apparent shift changes of -CD’s H-3 indicated that the guest molecule entered the cavity along its wide rim; (2) the chemical shifts’ degree of the protons (δ) was BRB--CD > MB--CD, which indicated that BRB entered the cavity more deeply than MB, and the inclusion interaction of BRB with -CD was more strong than MB with -CD. All the facts showed obviously that the guest molecules were included in -CD’s cavity. The inclusion interaction of -CDs and dyes (host-guest) could be influenced by many factors. The inclusion constant K is an important parameter for characterizing the inclusion interactions of -CDs with guest molecules. K value reflects the intensity of binding force of -CDs with guest molecules. The inclusion constants of the dyes with -CDs were determined by directly fluorescence technique [18,19] and shown in Table 3. From the data, the influence factors of inclusion interactions (the host molecule, the guest molecule, and the pH) were discussed.
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X. Zhu et al. / Talanta 72 (2007) 237–242
Table 3 Inclusion constants of the dyes with -CD and HP--CD Dyes
pH
K (L/mol) -CDa 1069 1720 710 8886
± ± ± ±
HP--CDa 63 60 21 266
11984 21032 13045 5785
± ± ± ±
SAF
3.2 6.0 9.0 3.2
589 1156 578 135
BRB
6.0 11.3 3.2
4528 ± 96 3642 ± 42 353 ± 14
2090 ± 100 1424 ± 34 285 ± 19
MB
6.0 9.0 3.2
410 ± 21 358 ± 16 –
326 ± 15 178 ± 17 141 ± 10
ES
5.0 11.3 5.0
– – –
340 ± 22 189 ± 13 70 ± 5
TCTIF
7.6 11.3
– –
241 ± 15 146 ± 12
–
–
TCF a
–
The confidence level was 95%.
3.2.1. Effect of the host molecule on inclusion interaction -Cyclodextrins have the hydrophilic outer surface and a hydrophobic internal cavity. The inclusion interaction of -CDs and the guest molecules were affected by the size of the internal cavity and the hydrophilic, hydrophobic characters of the host. It is generally believed that dipole-dipole, electrostatic, van der Walls forces, hydrogen bonding, hydrophobic interaction, and the release of distortion energy of CD ring upon guest binding cooperatively govern the stability of an inclusion complex. The neutral -CDs are not charged (2.0 < pH < 11.0) and the major inclusion interactions are hydrophobic interactions between the guest and CD cavity, and hydrogen bonding of the guest to OH groups or other introduced groups on CD ring [7]. As a result of additional groups of HP--CD comparative parent -CD, the hydrophilic property of HP--CD was better than that -CD. Meanwhile, the chemical modification enlarged the cavity of the host molecule, thus as a rule its inclusion capability was enhanced. Table 3 showed that the K value of SAF-HP--CD was larger than that SAF--CD, Moreover, compared with HP-CD, the inclusion interactions of ES and TCTIF with -CD were not observed in these experiments. But the inclusion interactions of -CDs with dyes were complicated, besides the size of the internal cavity and the hydrophilic, hydrophobic characters of the host, the other effect factors (charge of host and guest, speciation of guest molecule) were contributing and could result from the difference of K value. 3.2.2. Effect of the guest molecule on inclusion interaction The structure of guest is very important for host-guest inclusion interaction. The shape and size of the guest must match with the cavity of -CDs, and its polarity should be smaller than water. For SAF and BRB, their hydrophobic properties were relatively stronger; they were easy to enter the hydrophobic cavity of CDs and to form the relatively stable inclusion com-
plexes. For MB, its hydrophobic was weaker than that of BRB, so the host-guest inclusion interaction of -CDs-MB was weaker (KBRB-CDs < KMB-CDs ), which could be proved by the chemical shifts’ degree of the protons (δ), the chemical shifts’ degree of the protons (δ) was BRB--CD > MB--CD, which indicated that BRB entered the cavity more deeply than MB, and the inclusion interaction of BRB with -CD was more strong than MB with -CD. ES and TCTIF were easy to dissolve in water for their good hydrophilic property. So their inclusion complexes with -CDs were relatively labile, and the K values were smaller. 3.2.3. Effect of pH on inclusion interaction The effect of pH on host-guest inclusion interaction mostly behaved that the conformation of guest were dissimilar at different pH values, namely the polarity of the guest changed. For example, SAF was a neutral molecule in neutral medium, but in acidic and basic media, it was a charged molecule. So SAF as molecule was easily included into HP--CD in neutral medium. Similarly, the inclusion complexes of ES and TCTIF with HP-CD were more stable in subacid medium than in acidic and basic media. The reason was probably that ES and TCTIF were neutral molecules in subacid medium and the hydrophobic interactions between them and the -CDs cavity were strong; while in acidic and basic media, the guest molecules were charged, so their hydrophilic properties were better than the neutral molecules. Table 3 was shown that the inclusion constants of dyes with -CD were more sensitive to the changes of pH values. In a word, the factors which influence inclusion interaction were complex, and the K value reflected the general outcome about all the factors. 3.3. The inclusion interaction between VB6 and HP-β-CD The VB6 itself could emit fluorescence. The wavelengths of excitation and emission were at 292 and 394 nm, respectively [17]. When VB6 was into the cavity of CDs, its fluorescence intensity did not changed obviously. So, the direct fluorescence method to determine the inclusion constants of VB6 -HP--CD could not be used, the competitive inclusion method should be chosen. Competitive inclusion method was a technique that based on two different guests completing the same CD’s cavity which caused a remarkable change of the observed signal. When VB6 was added into the solution which containing the CDs-probe, the fluorescence intensity of the fluorescence probe was reduced. That was because some VB6 molecules replaced portion of the fluorescence probe molecules from the CD’s cavity. The competitive inclusion process is as follows: CD-G + V = CD-V + G
(1)
where the symbols G, V, CD-G and CD-V represent fluorescence probe molecule, VB6 , and the inclusion complexes, respectively. With KCD-G , KCD-V to represent the inclusion constant of CD-G and CD-V, respectively, then KCD-V could be obtained from the
X. Zhu et al. / Talanta 72 (2007) 237–242
241
Fig. 3. Effect of VB6 on the fluorescence spectra of ES-HP--CD (pH 5.0). (1) 1.0 × 10−5 mol/L ES + 2.0 × 10−3 mol/L HP--CD; (2–3) 1 + VB6 (4.0 × 10−3 , 8.0 × 10−3 mol/L); (4) 1.0 × 10−5 mol/L ES.
Fig. 4. Effect of VB6 on the fluorescence spectra of TCTIF-HP--CD (pH 7.6). (1) 1.0 × 10−5 mol/L TCTIF + 2.0 × 10−3 mol/L HP--CD; (2–3) 1 + VB6 (4.0 × 10−3 , 1.2 × 10−2 mol/L); (4) 1.0 × 10−5 mol/L TCTIF.
following formula:
the inclusion constant of VB6 -HP--CD was determined to be 71(±4) L/mol. The inclusion constant of Dye-HP--CD (Table 3) was larger than that of VB6 -HP--CD, namely the dyes probes included more tightly with HP--CD. So enough VB6 would be added into the system as to make the VB6 molecules replace the probe molecules from the cavity of HP--CD.
KCD-V =
([CD]0 − [CD]) [CD]([V]0 − [CD]0 + [CD])
(2)
where [CD]0 and [V]0 represent the initial concentration of CD and VB6 respectively, [CD] was the concentration of the dissociative CD in the solution. [CD] is the only unknown in this formula. It can be obtained from the following formula: [CD] =
F − FG KCD-G (FCD-G − F )
(3)
where FG , FCD-G and F represent the fluorescence intensity of the dissociative G, the fluorescence intensity of the G after it included with the CD, and the fluorescence intensity of the G when G, VB6 and CD was coexisting in the solution, respectively. In this paper, ES and TCTIF were chosen as the fluorescence probes to observe the inclusion interaction betweenVB6 and HP--CD. It was found that the fluorescence intensities of ES and TCTIF were gradually decreased with an increase of VB6 concentration, respectively (Figs. 3 and 4). The inclusion interaction was taken in 5 min, and VB6 -ES(TCTIF)-CDs was stable in 30 min It indicated that the same cavity of HP--CD was competed by the two guests (ES and VB6 , TCTIF and VB6 , respectively) and portion of the probe molecules were replaced out of the cavity by VB6 . According to the formulas (2) and (3),
3.3.1. Applications It was observed from the above experiment that in these systems of ES-HP--CD or TCTIF-HP--CD, the VB6 concentration had a linear relationship with the change fluorescence intensity of the systems (F). The experimental conditions were optimized as follows: the concentration of ES and TCTIF was 1.0 × 10−5 mol/L, HP--CD concentration was 3.2 × 10−3 mol/L, and the pH value of the systems was 5.0 and 7.6, respectively. 3.3.2. Analytical parameters The analytical characteristics for the determination of VB6 were shown in Table 4. 3.3.3. Interferences A study was carried out on the effects of foreign interferences on the determination of 20 mg/25 mL of VB6 (in ES system). With a relative error of less than ±1%, the tolerance limits for the
Table 4 Analytical parameters for the determination of VB6 Analytical characteristic
ES-VB6
TCTIF-VB6
Linear regression equation Linear range (g/mL) Correlation coefficient Limit of detection (g/mL) RSD (%) (n = 5, c = 8.0 × 10−4 g/mL)
F = −3.75 + 104651.98c (g/mL) 4.0 × 10−4 –4.0 × 10−3 0.9987 1.0 × 10−4 0.11
F = 4.79 + 32390.79c (g/mL) 6.4 × 10−4 − 4.8 × 10−3 0.9992 1.5 × 10−4 0.27
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X. Zhu et al. / Talanta 72 (2007) 237–242
Table 5 Determination of VB6 in synthetic samples Synthetic sample
Sample ingredients (mg/mL)
Addition (mg/mL)
Amount found (mg/mL)a
RSD (%)
Recovery (%)
A (ES) A (TCTIF)
VB6 (1.6) + VC (0.6) + VB2 (4.0 × 10−3 ) + VB12 (1.6 × 10−4 )
1.60
1.63 ± 0.02 1.57 ± 0.02
1.1 1.3
100.6–103.1 96.7–99.4
B (ES) B (TCTIF)
VB6 (1.6) + VC (0.4) + VB2 (8.0 × 10−3 ) + VB12 (3.2 × 10−4 )
1.60
1.56 ± 0.02 1.54 ± 0.02
1.5 1.2
96.3–98.8 95.0–97.5
a
Average value of three determination, the confidence level was 95%.
Table 6 The results of determination of the sample Sample Tablet mg/piece (ES) Tablet mg/piece (TCTIF) Injection g/2 mL (ES) Injection g/2 mL (TCTIF) a
Marked content
Proposed methoda
10.0 0.10
10.6 10.7 0.096 0.095
± ± ± ±
0.1 0.1 0.001 0.001
RSD (%) n = 3
HPLC method
0.6 1.0 0.6 0.6
10.9 ± 0.1 0.11 ± 0.01
The confidence level was 95%.
foreign substance were as fallows (mg/25 mL): Serine, leucine, l-cysteine chloride (>300); Vitamin C (25); d-l-tryptophan (20); 3,5-iodogorgoic acid (15); Vitamin B2 (0.3); Vitamin B12 (0.02). It was shown that most of the amino acids did not influence the determination of VB6 . But VB12 could absorb the fluorescence of those systems [20], it would influence the analysis, and should be separated before determination. 3.3.4. Sample analysis The calibration graphs for VB6 were constructed from the results obtained under the optimal conditions, and VB6 in synthetic samples, tablets and injections were determined with standard curve method. The results were compared with the method of pharmacopoeia (HPLC) [21] (Tables 5 and 6). The ttest method was used to do a significant difference test for these data, and it was found that the data of both methods did not have significant difference. 4. Conclusion In this paper, the inclusion interactions of dyes with -CD and HP--CD were investigated, and the inclusion constants of dyes-CDs were determined by fluorescence technique. The related inclusion mechanism was discussed. The inclusion interaction of VB6 with HP--CD was studied by the competitive fluorescence inclusion method. A new method for the analysis of VB6 was developed on the basis of the linear relationship between the change fluorescence intensity of the systems (F) and the concentration of VB6 . The method had been successfully applied to the determination of VB6 in synthetic samples, tablets and injections with satisfactory results.
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