Research article Received: 06 June 2014,
Accepted: 24 July 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2758
Host–guest inclusion complex of mesalazine and β-cyclodextrin and spectrofluorometric determination of mesalazine Abdalla A. Elbashir,a* Fatima Altayib Alasha Abdallaa and Hassan Y. Aboul-Eneinb* ABSTRACT: The supramolecular interaction of mesalazine (MSZ) and β-cyclodextrin (β-CD) has been examined by ultraviolet– visible (UV–vis) light, infra-red (IR) light and fluorescence spectroscopy. The formation of an inclusion complex has been confirmed based on the changes of the spectral properties. MSZ–β-CD host–guest complex was formed in (1:1) stoichiometry and the inclusion constant (K = 1.359 × 102 L mol–1) was ascertained by typical double reciprocal plots. Furthermore, the thermodynamic parameters (ΔG°, ΔH° and ΔS°) of (MSZ–β-CD) were obtained. Based on the remarkable enhancement of the fluorescence intensity of MSZ produced through complex formation, a simple, accurate, rapid and highly sensitive spectrofluorometric method for the determination of MSZ in aqueous solution in the presence of β-CD was developed. The measurement of relative fluorescence intensity was carried with excitation at 330 nm and emission 493 nm. All variables affecting the reactions were studied and optimized. Beer’s law was obeyed in the concentration range 0.1–0.45 μg/mL. Absorbance was found to increase linearly with increasing concentration of MSZ, which is corroborated by the calculated correlation coefficient values of 0.99989. The molar absorptivity, Sandell’s sensitivity, detection and quantification limits were calculated. The validity of the described methods was assessed, and the method was successfully applied to the determination of MSZ in its pharmaceutical formulation. In addition, a solid inclusion complex was synthesized by co-precipitation method. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: mesalazine; inclusion complex; β-CD; supramolecular study; spectrofluorimetry; pharmaceutical analysis
Introduction Cyclodextrins are oligosaccharides obtained from the enzymatic hydrolysis of starch. One of these, β-cyclodextrin (β-CD), is one of the most abundant natural oligomers and corresponds to the association of seven glucose units with a cavity, which exhibits a hydrophobic character, whereas the exterior is strongly hydrophilic. Cyclodextrins are well known in supramolecular chemistry as the most efficient molecular hosts (1,2). The formation of an inclusion complex greatly affects the physical–chemical properties of the guest molecules, such as solubility, chemical reactivity and the spectroscopic and electrochemical properties. Most of these effects can be utilized in many fields including the pharmaceutical industry (3–5) to improve the solubility, stability and bioavailability of pharmaceuticals as carriers of active substances in biological systems and to retard the release of active substances from the pharmaceutical matrix and various branches of analytical chemistry (6). From an analytical view point the formation of inclusion complexes allows the improvement of fluorescence intensity (7,8) and the induction of chiral separation in capillary electrophoresis (CE) (9–12). Analysts have used this property of CDs, and many methods based on the fluorescence of inclusion complexes with CDs have been proposed for the determination of several pharmaceutical drugs, pesticides, and metal ions (12,13). In principle, inclusion complexes can be formed either in solution or in the crystalline state (14). The preparation of the CD solid inclusion complex and the study of its structure and nature are helpful to know the inclusion process. The results of this
Luminescence 2014
analysis can give more information about the interaction forces for inclusion complex formation, the detailed analysis of the solid inclusion complex provides a three-dimensional structure and lattice of the complex. The formation of host–guest complexes in the solid state effectively protects the compounds against some types of reactions (e.g. oxidation, hydrolysis) and decreases their sublimation and volatility (12,15,16). Mesalazine (MSZ) (Fig. 1) (5-amino-2-hydroxybenzoic acid) is an anti-inflammatory drug used to treat and also maintain the remission of mild to moderate ulcerative colitis or Crohn’s disease (17,18).
* Correspondence to: Hassan Y. Aboul-Enein. Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt. E-mail: [email protected] * Abdalla A. Elbashir. Chemistry Department, Faculty of Science, University of Khartoum, Khartoum 11115, Sudan. E-mail: [email protected] a
Chemistry Department, Faculty of Science, University of Khartoum, Khartoum 11115, Sudan
b
Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt Abbreviations: CE, capillary electrophoresis; FT-IR, Fourier transform infrared; GTS, glass thermoplastic system; LOQ, limit of quantification; MSZ, mesalazine; RSD, relative standard deviations.
Copyright © 2014 John Wiley & Sons, Ltd.
A. A. Elbashir et al. Stock and standard solutions An accurately weighed amount of MSZ (0.010 g) was dissolved in about 30 mL water then transferred into a 100 mL volumetric flask, filled to the mark with distilled water to obtain a stock solution of 100 μg/mL. The stock solution was further diluted with the same solvent to obtain working solution of 20 μg/mL. MSZ.
Figure 1. Chemical structure of mesalazine.
Mesalazine has been shown to be a potent scavenger of reactive oxygen species that plays a significant role in the pathogenesis of inflammatory bowel disease, inhibition of natural killer cell activity, inhibition of antibody synthesis, inhibition of cyclo-oxygenase and lipoxygenase pathways and impairment of neutrophil function (19). Several analytical methods have been reported for the determination of MSZ in pharmaceutical dosage forms and biological fluids including micellar electrokinetic chromatography (20), differential pulse voltammetry (21) high pressure liquid chromatography (HPLC) (22–27), liquid chromatography–tandem mass spectrometry (LC-MS/MS) (28) and spectrophotometric analysis (29–32). However, only a few spectrofluorometric methods (33,34) have been reported for the determination of MSZ in its pharmaceutical formulations. Spectrofluorometric methods are the most convenient technique because of their inherent simplicity, high sensitivity, low cost, and wide availability in quality control laboratories. In this paper, the host–guest inclusion interaction between β-CD and MSZ was investigated using fluorescence, ultra-violet–visible (UV–vis) light, and infra-red (IR) light spectroscopy. A series of conditions during the formation of the inclusion complex was studied. Based on the great enhancement of the fluorescence intensity of MSZ, a novel method spectrofluorometric method was developed to determine MSZ in pharmaceutical formulations. Moreover the solid complex of MSZ–β-CD was prepared by a co-precipitation method.
β-Cyclodextrin (β-CD). An accurately weighed amount of β-CD (1.1370 g) was dissolved in distilled water then transferred into a 100 mL volumetric flask, completed to the mark with distilled water to obtain a solution of 0.01 mol/L. The solution was freshly prepared.
A phosphate buffer solution of pH 7.0 was prepared by adding 24.40 mL of 0.1 mol/L HCl and 75.60 mL of 0.1 mol/L Na2HPO4 and adjusting to pH 7.0, Other buffer solutions were also prepared according to literature methods (35). Buffer solutions.
Spectrophotometric method Here, 10 μg/mL of MSZ solution was added into a 10 mL volumetric flask, then 1.5 mL of phosphate buffer solution (pH 7.0) was added into flask to control the pH value of the medium, followed by 3 mL of 0.01 mol/L β-CD solution. The reaction was allowed to proceed at room temperature for 10 min after which the reaction mixture was made up to the mark with distilled water. The absorbance spectra were measured at 330 nm. Spectrofluorometric method
Experimental Chemicals and reagents All chemicals were of analytical or HPLC grade. Distilled water was used in all experiments. Details of chemicals (suppliers) are given below. The MSZ standard was purchased from Sigma-Aldrich (St. Louis, MI, USA), the tablet formulation Pentasa [Pharbil Pharma Bielefeld, Germany (Marketing Authorization Holder: Ferring GmbH, Kiel, Germany)] was procured from a local pharmacy with a labeled amount 500 mg MSZ, β-cyclodextrin (β-CD) was obtained from Sigma-Aldrich (St. Louis, MI, USA).
Instruments and apparatus All spectrofluorimetric measurements were made with a spectrofluorimeter RF-1501 (Shimadzu, Japan) equipped with a 150 W xenon lamp, Slit widths for both monochromators were set at 10 nm. All spectrophotometric measurements were made with a double beam UV1800 UV–vis light spectrophotometer provided with a matched 1-cm quartz cell (Shimadzu, Japan). IR measurements were recorded by Fourier transform IR (FT-IR) spectroscopy using a spectrometer-8400S (Perkin-Elmer, Shimadzu, Japan). Samples were pressed into KBr pellets and recorded at frequencies from 4000 to 200 cm–1. A pH meter (model HI 255, Hanna Instruments, Mumbai, India) was used for pH measurements. A thermostatically controlled water bath (LAUDA, Ecoline model RE 220, Gaithersburg, MD, USA) was used.
wileyonlinelibrary.com/journal/luminescence
Sample solution. A sample of finely powdered tablet nominally equivalent to 0.0100 g MSZ was dissolved in about 30 mL distilled water in a 100 mL volumetric flask, swirled, sonicated for 15 min, then filled to the correct level with distilled water, the contents were mixed well and filtered. This prepared solution was diluted quantitatively to obtain a suitable concentration for the analysis.
Aliquots of MSZ solution were added to 10 mL volumetric flasks to give final concentrations of 0.1–0.45 μg/mL. Next, 1.5 mL buffer solution pH 7.0 was added followed by 2.0 mL β-CD solution (1 × 10–2 mol/L). The reaction was allowed to proceed at room temperature for 10 min after which time the reaction mixture was made up to the mark with distilled water and the fluorescence intensity of MSZ–β-CD was measured at λex/λem = 330 nm/493 nm. Preparation of solid complex of MSZ–β-CD Accurately weighed β-CD (0.3712 g) was placed into a 100 mL conical flask and 30 mL distilled water was added, stirred, then 0.05 g MSZ was put into a 100 mL beaker and 70 mL of distilled water was added and put over electromagnetic stirrer to stir until it was dissolved. Then slowly, the β-CD solution was poured into a stirred MSZ solution, and continuously stirred for 20 h at room temperature (36). The reaction mixture was freeze dried. At this time, the formation of white crystal was observed. This MSZ inclusion complex with β-CD was characterized by FT-IR spectroscopy. Stoichiometry of the reaction Here, 1.0 μg/mL of MSZ and 1.5 mL of phosphate buffer solution (pH 7.0) were added to a volumetric flask, then the various amounts of β-CD (0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, or 7.0 mL. of
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Spectrofluorometric determination of mesalazine 1 × 10–2 mol/L) were added sequentially. The reaction was allowed to proceed at room temperature for 10 min, after which time the reaction mixture was made up to the mark with distilled water.
Results and discussion Absorbance spectra The absorption spectra of MSZ in the absence and in the presence of 0.003 mol/L β-CD was first recorded Fig. 2. The results showed that the wavelength of maximum absorbance of MSZ (10 μg/mL) at pH 7.0 was 330 nm, when β-CD was added into the MSZ solution, the wavelength of maximum absorbance did not change but the absorbance was slightly increased and the shape of the absorption spectrum of MSZ remained constant in the broad spectral range 290–400 nm, this is due to the formation of the MSZ and β-CD complex. Emission spectra
Optimization of reaction variables
The spectral characteristics of MSZ were studied and the results showed that the wavelength of maximum emission of MSZ at pH 7.0 was 493 nm Fig. 3. When β-CD was added into the MSZ solution, the wavelength of maximum of emission did not change but the fluorescence intensity dramatically increased. This can be rationalized as MSZ entering the β-CD hydrophobic cavity and binding taking place through non-covalent bonding due to Van der Waals forces and hydrogen bonding. In the cavity, the degree of motional freedom of MSZ molecule was reduced, thus the cavity could shield the MSZ excitation signal from quenching in the aqueous solution. Therefore the fluorescence intensity increased when the MSZ–β-CD inclusion complex was produced. Infra-red spectra The solid complex formation was confirmed by FT-IR spectroscopy (Fig. 4), However some MSZ peaks were either absent (C–N stretching) or shifted (C–O stretching), which suggested a change in environment due to inclusion complex formation between MSZ and β-CD.
Figure 2. Absorbance spectra of β-CD (—), MSZ (–), inclusion complex (MSZ–β–3 CD) (…), MSZ (10 μg/mL), β-CD (3 × 10 mol/L) at room temperature, time 15 min, 2 mL buffer solution pH 7.0.
Luminescence 2014
Figure 3. Fluorescence excitation (left hand) and emission spectra (right hand) of –3 (1, green) β-CD 2 × 10 mol/L, (2, red) MSZ 1.0 μg/mL, and (3, black) inclusion complex MSZ–β-CD at room temperature, time 10 min, pH 7.0.
Effect of pH. The effect of pH was studied by forming the MSZ–β-CD complex in the presence of various buffers; fluorescence intensity was measured. The pKa values for MSZ have been reported as 6.0 for (–NH+3 ), 13.9 for (–OH), and 3 for (–COOH) (28) respectively. Figure 5 shows that the fluorescence intensity increased rapidly with increasing pH. Maximum absorbance was attained at a pH value of 7.0. At pH values greater than 7.0, a decrease in the fluorescence intensity of the product was observed, the formation constant (K) of the inclusion complex was determined by fluorescence measurement. Influence the volume of buffer solution. The effect of the volume of the phosphate buffer pH 7.0 added on the MSZ–βCD complex was also studied. It was found that increasing the buffer volume resulted in a subsequent increase in fluorescence intensity up to 1.5 mL, after which volume the effect of fluorescence intensity remained constant. A volume of 1.5 phosphate buffer pH 7.0 was recorded as optimum in this study as shown in Fig. 6.
Figure 4. IR spectra of MSZ and the MSZ–β-CD inclusion complex.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
A. A. Elbashir et al. 1000 900
Flourescence Intensity
800 700 600 500 400 300 200 100 0 0
10
20
30
40
50
60
70
Temperature( C)
Effect of temperature and time. The reaction time was determined by forming the MSZ–β-CD at room temperature and in a thermostatically controlled water-bath adjusted in the range 30–90 °C. The highest fluorescence intensity was obtained at room temperature (Fig. 7). Effect of time at room temperature was studied; the highest reading was obtained after 10 min (Fig. 8), therefore the fluorescence measured at room temperature after 10 min in this study was considered to be optimal. Effect of β-CD concentration. The effect of β-CD concentration on the fluorescence intensity of MSZ was examined. The concentration of MSZ was fixed and the concentration of β-CD varied from 0.001–0.007 mol/L. The reaction was found to be dependent on β-CD concentration (Fig. 9), and illustrated that with increase in β-CD concentration fluorescence intensity was enhanced. Therefore 2 mL of 1 × 10–2 mol/L β-CD was selected as optimal and adopted in all subsequent work.
Stoichiometry of the reaction The stoichiometry of the inclusion complex was studied under the established experimental conditions: assuming that the
Figure 7. Effect of temperature on the fluorescence intensity of MSZ–β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, time 10 min, buffer volume 1.5 ml, pH 7.0.
1000 900 800
Fluoresence Intensity
Figure 5. Effect of pH on the fluorescence intensity of the MSZ–β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, at room temperature, time 10 min, buffer volume 2.0 mL.
500 400 300
100 0 0
10
20
30
40
50
Time(minute) Figure 8. Effect of time on the fluorescence intensity of MSZ-β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, at room temperature, buffer volume 1.5 mL, pH 7.0.
720 700
Flourescence Intensity
612
Flourescence Intensity
600
200
614
610 608 606 604
680 660 640 620 600
602 600 0.0
700
580 0.5
1.0
1.5
2.0
2.5
0.001
Volume of buffer(ml)
0.003
0.004
0.005
0.006
0.007
Beta-CD in molar concentration
Figure 6. Effect of buffer volume on the fluorescence intensity of MSZ-β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, at room temperature, time 10 min, pH 7.0.
wileyonlinelibrary.com/journal/luminescence
0.002
Figure 9. Effect of β-CD concentration on the fluorescence intensity of MSZ–β-CD inclusion complex, [MSZ] = 1.0 μg/mL, at room temperature, time 10 min, buffer volume 1.5 mL, pH 7.0.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Spectrofluorometric determination of mesalazine Alternatively, assuming the stoichiometry of the inclusion complex was 1:2, the following expression was obtained:
0.025
1=F F0 ¼ 1=ðF∞ F0ÞK ½β CD0
1/(F-F0)
0.020
2
þ 1=F∞ F0
(2)
When making a plot of 1/F – F0 against 1/([β-CD]0)2, a non-linear relationship was obtained (Fig. 11), which indicated that the stoichiometry of the inclusion complex is not 1:2. These results confirmed that β-CD and MSZ formed a host–guest complex in 1:1 stoichiometry (37). The inclusion constant (K) was calculated to be 1.359 × 102 L/mol.
0.015
0.010
0.005 200
400
600
800
Inclusion complex thermodynamics
1000
1 / [Beta-CD]0 mole/L Figure 10. Plot of 1/(F – F0) vs. 1/[β-CD] of the MSZ–β-CD complex; MSZ = 1.0 μg/mL, at room temperature, time 10 min, buffer volume 1.5 mL, pH 7.0.
0.025
1/(F-F0)
0.020
0.015
0.010
0.005 0
200000
400000
600000
800000 1000000
2
1 / [Beta-CD] 0 2
Figure 11. Plot of 1/(F – F0) vs. 1/[β-CD] of the MSZ-β-CD complex; MSZ = 1 μg/mL, at room temperature, time 10 min, buffer volume 1.5 mL, pH 7.0.
composition of the complex was 1:1, and using typical double reciprocal or Benesi–Hildebrand plots: 1=F F0 ¼ 1=ðF∞–F0ÞK½β CD0 þ 1=F∞ F0
(1)
Where [β-CD]0 denotes the β-CD concentration; F0 the fluorescence intensity of MSZ in the absence of β-CD; F∞ the fluorescence intensity when all MSZ molecules are essentially complexed with β-CD; and F the observed fluorescence intensity at each β-CD concentration tested. When a plot of 1/F – F0 versus 1/[β-CD]0 was constructed (Fig. 10), a straight line was obtained ,which is indicative of a 1:1 stoichiometry for the MSZ–β-CD complex. Table 1. Thermodynamic parameter of the reaction Thermodynamic parameters ΔH° ΔS° ΔG°
Luminescence 2014
Value kJ/mole –544.295 –1.414 –122.961
The thermodynamic parameters (ΔH°, ΔS° and ΔG°) for the formation of inclusion complex were determined from temperature dependence of apparent association constants, by using the classical van ’t Hoff equation (ln K = –ΔH°/RT + ΔS°/R), and plotting ln K versus 1/T (38,39). The corresponding enthalpy and entropy can be obtained from the slope and intercept, respectively, which indicated the marked tendency of MSZ to complex with β-CD. ΔG° was obtained according to the equation: ΔG° = ΔH° TΔS°. The results are shown in Table 1. We noted that the association constant for MSZ–β-CD increased as the temperature rose. Thermodynamic parameters were calculated based on the temperature dependence of the association constant for MSZ–β-CD binding. Thermodynamic parameters, such as the enthalpy changes (ΔH) and entropy changes (ΔS) of the binding reaction, are important to confirm the force of interactions of MSZ with β-CD. Four driving forces for the inclusion of CDs with substrates were proposed, including hydrogen bonding between the hydroxyl groups of CDs and the MSZ molecules, van der Waals interactions between host and guest molecules, hydrophobic interaction, and the release of ‘high-energy water’ molecules from the cavities of β-CDs to the bulk water. Hydrophobic interaction essentially involved favourable positive entropy together with a slightly positive enthalpy change, whereas the other forces involved negative ΔH and ΔS (40). Upon complex formation, the negative ΔG° value strongly suggested that the inclusion process proceeded spontaneously, as both negative enthalpic changes and negative entropic values were obtained, which mean that the inclusion complexation is exothermic and enthalpy controlled, but not entropy driven. This is the common situation concerning the formation of inclusion complexes between cyclodextrins
Table 2. Summary of quantitative parameters and statistical data using the proposed procedure
Parameters
Result
Linear range (μg/mL) Intercept Slope Standard deviation Correlation coefficient (r) Limit of detection, LOD (μg/mL) Limit of quantitation, LOQ (μg/mL) Molar absorptivity (L mol–1 cm–1) Sandell’s sensitivity
Copyright © 2014 John Wiley & Sons, Ltd.
0.1–0.5 12.79 719.0 1.922 0.9998 0.009 0.015 1.294 × 108 1.183 × 10–6
wileyonlinelibrary.com/journal/luminescence
A. A. Elbashir et al. and various guest molecules. It is widely accepted that the negative entropy change is due to the steric barrier caused by their molecular geometrical shape and the limit of the β-CD cavity to the freedom of shift and rotation of guest molecules.
Table 6. Application of the proposed spectrofluorometric method
Validation of the proposed method
Pentasa
Linearity and sensitivity. In the proposed method, a linear plot (n = 6) with good correlation coefficients (0.99989) was obtained in the concentration range 0.1–0.5 μg/mL. The molar absorptivity (ε) was 1.294 × 108 (L /mol/ cm). The Sandell’s sensitivity value was 1.183 × 10–6. The detection limit (LOD) is defined as the minimum level at which the analyte can be reliably detected, it was calculated using the following equation:
* Values are the mean of three determinations.
LOD ¼ 3:3 s=k
(3)
Where s is the standard deviation of replicate determination
Brand name Labeled claim and dosage form mg/tablet
0.2 0.3 0.4
Recovery (% ± RSD)*
Relative error %
98.86 ± 0.19 101.41 ± 1.03 99.42 ± 1.27
–1.360 1.418 –0.598
Table 4. Recovery of the proposed method
0.1 0.1 0.1
Amount of pure drug added (μg/mL) 0.1 0.2 0.3
Recovery (% ± SD)*
Amount recovered (μg/mL) 0.194 0.308 0.398
96.85 ± 1.81 102.62 ± 0.19 99.60 ± 0.61
* Values are the mean of three determinations.
Table 5. Robustness of the proposed spectrofluorometric method Method conditions Standard condition pH 6.8 7.2 Temperature (°C) 20 30 Time(minutes) 8 12 β-CD concentration (mol/L) 1.8 × 10–3 2.2 × 10–3
Recovery ± RSD* 98.864 ± 0.194 99.464 ± 1.085 99.154 ± 0.888 100.105 ± 0.498 97.654 ± 0.228 100.715 ± 0.457 104.682 ± 0.202 100.575 ± 0.883 100.055 ± 1.066
* Values are the mean of three determinations.
wileyonlinelibrary.com/journal/luminescence
487.36
97.47 ± 0.634
500
LOQ ¼ 10 s=k
* Values are the mean of three determinations.
Initial amount (μg/mL)
Recovery (% ± SD)*
values under the same conditions for sample analysis in the absence of the analyte, and k is the sensitivity, namely the slope of the calibration graph. In accordance with the formula, the detection limit was found to be 0.009 μg/mL. The limit of quantification (LOQ) is defined as the lowest concentration that can be measured with acceptable accuracy and precision:
Table 3. Precision of the proposed method Concentration (μg/mL)
Amount found
(4)
According to this equation, the limit of quantization was found to be 0.015 μg/mL. These parameters for the proposed method are summarized in Table 2. Accuracy and precision. The accuracy and precision of the proposed method was determined at three concentration levels of MSZ (within the linear range) by analyzing three replicates of pure drug of each concentration. The percentage relative error (as accuracy) and percentage relative standard deviations (RSD) (as precision) for the results did not exceed 2% for the proposed method as shown in Tables 3 and 4, indicating good reproducibility and repeatability. This good level of precision and accuracy was suitable for quality control analysis of MSZ in their pharmaceutical formulation. Robustness. Robustness was examined by evaluating the influence of small variation in the method variables on its analytical performance. In these experiments, one parameter was changed whereas the others were kept unchanged, and the recovery percentage was calculated each time. It was found that small variation in the method variable did not significantly affect the procedure .This provided an indication of the reliability of the proposed method during routine work; recovery values were shown in Table 5. Applications of the methods. The proposed method was applied to the pharmaceutical formulation containing MSZ. The results are shown in Table 6, and indicate the high accuracy of the proposed method for the determination of the studied drug. The proposed method has the advantage of being virtually free from interferences by excipients.
Conclusion The supramolecular interaction of MSZ with β-CD was been investigated by spectrofluorimetry. The results showed that β-CD reacted with MSZ to form a 1:1 (host: guest) complex with inclusion constant K = 1.359 × 102 L/mol. Based upon the enhancement effect observed, a simple, sensitive, accurate spectrofluorometric method for the determination of MSZ is proposed. Under optimized experimental conditions, the method was applied to the determination of MSZ in its pharmaceutical formulations.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Spectrofluorometric determination of mesalazine
References 1. Brewster ME, Vandecruys R, Verreck G, Noppe M, Peeters J. A novel cyclodextrin-containing glass thermoplastic system (GTS) for formulating poorly water soluble drug candidates: preclinical and clinical results. J Inclusion Phenom 2002;44:35–8. 2. Qi AD, Li L, Liu Y. Molecular binding ability and selectivity of natural α-, β-, γ-cyclodextrins and oligo(ethylenediamino) modified β-cyclodextrins with Chinese traditional medicines. J Inclusion Phenom 2003;45:69–72. 3. Diez MA, Pena IMA, Garera MCM, Gil DB, Canada FC. Fluorimetric determination of sulphaguanidine and sulphamethoxazole by host–guest complexation β-cyclodextrin and partial least squares calibration. J Fluoresc 2007;17:309–18. 4. Elbashir AA, Dsugi NFA, Aboul-Enein HY. Supramolecular study on the interaction between ofloxacin and methyl β-cyclodextrin by fluorescence spectroscopy and its analytical application. J Fluoresc 2014;24:355–61. 5. Chao J-B, Tong H-B, Huang S-P, Liu D-S. Preparation and study on the solid inclusion complex of sparfloxacin with β-cyclodextrin. Spectrochim Acta A Mol Biomol Spectrosc 2005;60:161–6. 6. Linare M, Bertorello MM, Longhi M. Preparation and characterization of solid complexes of napthoquinone and hydroxypropyl-βcyclodextrin. Molecules 2000;5:342–4. 7. Xiea H, Wangb HY, Mac Li Y, Xiaob Y, Hanb J. Spectrophotometric study of the inclusion complex between β-cyclodextrin and dibenzoyl peroxide and its analytical application. Spectrochim Acta A Mol Biomol Spectrosc 2005;62:197–202. 8. Chao J-B, Tong H-B, Liu D-S, Huang S-P. Preparation and characterization of inclusion complexes of pefloxacin mesylate with three kinds of cyclodextrins. Spectrochim Acta A Mol Biomol Spectrosc 2006;64:166–70. 9. Elbashir AA, Suliman FEO, Saad B, Aboul-Enein HY. Capillary electrophoretic separation and computational modeling of inclusion complexes of β-cyclodextrin and 18-crown-6 ether with primaquine and quinocide. Biomed Chromatogr 2010;24:393–8. 10. Elbashir AA, Suliman FO. Computational modeling of capillary electrophoretic behavior of primary amines using dual system of 18-crown-6 and β-cyclodextrin. J Chromatogr A 2011;1218:5344–51. 11. Elbashir AA, Saad B, Ali ASM, Saleh MI, Aboul-Enein HY. Recent developments of enantioseparations for fluoroquinolone drugs using liquid chromatography and capillary. Curr Pharm Anal 2010;6:246–55. 12. Elbashir AA, Dsugi NFA, Mohmed TOM, Aboul-Enein HY. Spectrofluorometric analytical applications of cyclodextrins. Luminescence 2014;29:1–7. 13. Monson PL, Mueller RA, Breese GR. Principles of pharmacology. New York: Chapman & Hall, 1995:l064. 14. Bender ML, Komiyama M. Cyclodextrin chemistry. Berlin, Heidelberg, New York: Springer-Verlag, 1978:10. 15. Tong LH. Cyclodextrin chemistry foundation and application [Huanhujing Huaxue Jichu Yu Yingyong]. Beijing: Science Press, 2001;191 pp. 16. Li S, Purdy WC. Cyclodextrin and their application in analytical chemistry. Chem Rev 1992;92:1458–70. 17. Hanauer S, Schwartz J, Robinson M, Roufail W, Arora S, Cello J, Safdi M. Mesalamine capsules for treatment of active ulcerative colitis: results of a controlled trial. Am J Gastroenterol1993;88:1188–97. 18. Thomson AB. New developments in the use of 5-aminosalicylic acid in patients with inflammatory bowel disease. Aliment Pharmacol Ther 1991;5:449–70. 19. Geier DL, Miner PB. New therapeutic agents in the treatment of inflammatory bowel disease. Am J Med 1992;93:208–98. 20. Gotti R, Pomponio R, Bertucci C, Cavrini V. Determination of 5-aminosalicylic acid related impurities by micellar electrokinetic chromatography with an ion-pair reagent. J Chromatogr A 2001;916:175–83. 21. Biljana N, Branimir I. Determination of 5-aminosalicylic acid in pharmaceutical formulation by differential pulse voltammetry, J Pharm Biomed Anal 2003;31:169–74.
Luminescence 2014
22. Palumbo G, Carlucci G, Mazzeo P. Simultaneous determination of 5-aminosalicylic acid, acetyl-5-aminosalicylic acid and 2,5dihydroxybenzoic acid in endoscopic intestinal biopsy samples in humans by high performance liquid chromatography with electrochemical detection. J Pharm Biomed Anal Oxford 1995;14:175–80. 23. Janice AR, José RJ, Rúbia C, Sandra RG, Maria D., Fátima B, Maria José VF. Validation of HPLC, DPPH and nitrosation methods for mesalamine determination in pharmaceutical dosage forms. Rev Brasil Cienc Farm 2007;43:97–103. 24. Haney PW, Dash AK. Simple liquid chromatographic method for the analysis of 5- aminosalicyclic acid and its degradation product. J Chromatogr A 1997;765:233–9. 25. Palumbo G, Bacchi S, Primavera L, Palumbo P, Carlucci G. A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma. Biomed Chromatogr 2005;19:350–4. 26. Bystrowska B, Nowak J, Brandys J. Validation of a LC method for the determination of 5-aminosalicylic acid and its metabolite in plasma and urine. J Pharm Biomed Anal 2000;22:341–51. 27. Nobilis M, Vybíralová Z, Sládková K, Lísa M, Holcapek M, Kvetina J. High-performance liquid-chromatographic determination of 5-aminosalicylic acid and its metabolites in blood plasma. J Chromatogr A 2006;1119:299–308. 28. Pastorini E, Locatelli M, Simoni P, Roda G, Roda V, Roda A. Development and validation of a HPLC–ESI–MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5aminosalicylic acid in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2008;872:99–106. 29. Patel KM, Patel CN, Panigrahi B, Parikh AS, Patel HN. Development and validation of spectrophotometric methods for the estimation of mesalazine in tablet dosage forms. J Young Pharmacists 2010;2:284–8. 30. Madhavi V, Panchakshari V, Prathyusha TN, Sekaran CB. Spectrophotometric determination of mesalazine in bulk and tablet dosage forms based on diazocoupling reaction with resorcinol. Int J Pharm Sci Rev Res 2011;11:105–9. 31. Narala S, Saraswathi K. Spectrophotometric methods for quantitative determination of mesalamine in bulk and tablet dosage forms. Int J Res Pharm Biomed Sci 2010;1:10–3. 32. Reddy MP, Prabhavathi K, Reddy NR, Reddy PR. Two simple spectrophotometric methods for the estimation of mesalamine in bulk sample and its pharmaceutical dosage forms. Global J Pharmacol 2011;5:101–5. 33. Zadeh HA, Kohansal S. Determination of mesalamine by spectrofluorometry in human serum after solid-phase extraction with Ni-Al layered double hydroxide as nanosorbent. J Brazil Chem Soc 2012;23:473–81. 34. Cui FL, Fan J, Li W, Fan YC, Hu ZD. Fluorescence spectroscopy studies on 5-aminosalicylic acid and zinc 5-aminosalylicylate interaction with human serum albumin. J Pharm Biomed Anal 2004;34:189–97. 35. Analytical Chemistry Studio of Zhongnan Mineral College. Handbook of chemical analysis. Beijing: Science Press, 1984:383 pp. 36. Chao J-B, Tong H-B, Huang Sh-P, Liu D-Sh. Preparation and study on the solid inclusion complex of sparfloxacin with β-cyclodextrin. Spectrochimica Acta Part A 2005;60:161–6. 37. Catena GC, Bright FV. Thermodynamic study on the effects of beta-cyclodextrin inclusion with anilinonaphthalene sulfonates. Anal Chem 1989;61:905–9. 38. Rekharsky MV, Inoue Y. Complexation thermodynamics of cyclodextrins. Chem Rev 1998;98:1875–918. 39. Inoue Y, Hakushi T, Liu Y, Tong LH, Shen BJ, Jin DS. Thermodynamics of molecular recognition by cyclodextrins. 1. Calorimetric titration of inclusion complexation of naphthalenesulfonates with. alpha-, beta-, and gamma-cyclodextrins: enthalpy–entropy compensation. J Am Chem Soc 1993;115:475–81. 40. Zhang Q, Jiang Z, Guo Y, Li R. Complexation study of brilliant cresyl blue with β-cyclodextrin and its derivatives by UV-vis and fluorospectrometry. Spectrochim Acta Part A 2008;69:65–70.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
Accepted: 24 July 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2758
Host–guest inclusion complex of mesalazine and β-cyclodextrin and spectrofluorometric determination of mesalazine Abdalla A. Elbashir,a* Fatima Altayib Alasha Abdallaa and Hassan Y. Aboul-Eneinb* ABSTRACT: The supramolecular interaction of mesalazine (MSZ) and β-cyclodextrin (β-CD) has been examined by ultraviolet– visible (UV–vis) light, infra-red (IR) light and fluorescence spectroscopy. The formation of an inclusion complex has been confirmed based on the changes of the spectral properties. MSZ–β-CD host–guest complex was formed in (1:1) stoichiometry and the inclusion constant (K = 1.359 × 102 L mol–1) was ascertained by typical double reciprocal plots. Furthermore, the thermodynamic parameters (ΔG°, ΔH° and ΔS°) of (MSZ–β-CD) were obtained. Based on the remarkable enhancement of the fluorescence intensity of MSZ produced through complex formation, a simple, accurate, rapid and highly sensitive spectrofluorometric method for the determination of MSZ in aqueous solution in the presence of β-CD was developed. The measurement of relative fluorescence intensity was carried with excitation at 330 nm and emission 493 nm. All variables affecting the reactions were studied and optimized. Beer’s law was obeyed in the concentration range 0.1–0.45 μg/mL. Absorbance was found to increase linearly with increasing concentration of MSZ, which is corroborated by the calculated correlation coefficient values of 0.99989. The molar absorptivity, Sandell’s sensitivity, detection and quantification limits were calculated. The validity of the described methods was assessed, and the method was successfully applied to the determination of MSZ in its pharmaceutical formulation. In addition, a solid inclusion complex was synthesized by co-precipitation method. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: mesalazine; inclusion complex; β-CD; supramolecular study; spectrofluorimetry; pharmaceutical analysis
Introduction Cyclodextrins are oligosaccharides obtained from the enzymatic hydrolysis of starch. One of these, β-cyclodextrin (β-CD), is one of the most abundant natural oligomers and corresponds to the association of seven glucose units with a cavity, which exhibits a hydrophobic character, whereas the exterior is strongly hydrophilic. Cyclodextrins are well known in supramolecular chemistry as the most efficient molecular hosts (1,2). The formation of an inclusion complex greatly affects the physical–chemical properties of the guest molecules, such as solubility, chemical reactivity and the spectroscopic and electrochemical properties. Most of these effects can be utilized in many fields including the pharmaceutical industry (3–5) to improve the solubility, stability and bioavailability of pharmaceuticals as carriers of active substances in biological systems and to retard the release of active substances from the pharmaceutical matrix and various branches of analytical chemistry (6). From an analytical view point the formation of inclusion complexes allows the improvement of fluorescence intensity (7,8) and the induction of chiral separation in capillary electrophoresis (CE) (9–12). Analysts have used this property of CDs, and many methods based on the fluorescence of inclusion complexes with CDs have been proposed for the determination of several pharmaceutical drugs, pesticides, and metal ions (12,13). In principle, inclusion complexes can be formed either in solution or in the crystalline state (14). The preparation of the CD solid inclusion complex and the study of its structure and nature are helpful to know the inclusion process. The results of this
Luminescence 2014
analysis can give more information about the interaction forces for inclusion complex formation, the detailed analysis of the solid inclusion complex provides a three-dimensional structure and lattice of the complex. The formation of host–guest complexes in the solid state effectively protects the compounds against some types of reactions (e.g. oxidation, hydrolysis) and decreases their sublimation and volatility (12,15,16). Mesalazine (MSZ) (Fig. 1) (5-amino-2-hydroxybenzoic acid) is an anti-inflammatory drug used to treat and also maintain the remission of mild to moderate ulcerative colitis or Crohn’s disease (17,18).
* Correspondence to: Hassan Y. Aboul-Enein. Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt. E-mail: [email protected] * Abdalla A. Elbashir. Chemistry Department, Faculty of Science, University of Khartoum, Khartoum 11115, Sudan. E-mail: [email protected] a
Chemistry Department, Faculty of Science, University of Khartoum, Khartoum 11115, Sudan
b
Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt Abbreviations: CE, capillary electrophoresis; FT-IR, Fourier transform infrared; GTS, glass thermoplastic system; LOQ, limit of quantification; MSZ, mesalazine; RSD, relative standard deviations.
Copyright © 2014 John Wiley & Sons, Ltd.
A. A. Elbashir et al. Stock and standard solutions An accurately weighed amount of MSZ (0.010 g) was dissolved in about 30 mL water then transferred into a 100 mL volumetric flask, filled to the mark with distilled water to obtain a stock solution of 100 μg/mL. The stock solution was further diluted with the same solvent to obtain working solution of 20 μg/mL. MSZ.
Figure 1. Chemical structure of mesalazine.
Mesalazine has been shown to be a potent scavenger of reactive oxygen species that plays a significant role in the pathogenesis of inflammatory bowel disease, inhibition of natural killer cell activity, inhibition of antibody synthesis, inhibition of cyclo-oxygenase and lipoxygenase pathways and impairment of neutrophil function (19). Several analytical methods have been reported for the determination of MSZ in pharmaceutical dosage forms and biological fluids including micellar electrokinetic chromatography (20), differential pulse voltammetry (21) high pressure liquid chromatography (HPLC) (22–27), liquid chromatography–tandem mass spectrometry (LC-MS/MS) (28) and spectrophotometric analysis (29–32). However, only a few spectrofluorometric methods (33,34) have been reported for the determination of MSZ in its pharmaceutical formulations. Spectrofluorometric methods are the most convenient technique because of their inherent simplicity, high sensitivity, low cost, and wide availability in quality control laboratories. In this paper, the host–guest inclusion interaction between β-CD and MSZ was investigated using fluorescence, ultra-violet–visible (UV–vis) light, and infra-red (IR) light spectroscopy. A series of conditions during the formation of the inclusion complex was studied. Based on the great enhancement of the fluorescence intensity of MSZ, a novel method spectrofluorometric method was developed to determine MSZ in pharmaceutical formulations. Moreover the solid complex of MSZ–β-CD was prepared by a co-precipitation method.
β-Cyclodextrin (β-CD). An accurately weighed amount of β-CD (1.1370 g) was dissolved in distilled water then transferred into a 100 mL volumetric flask, completed to the mark with distilled water to obtain a solution of 0.01 mol/L. The solution was freshly prepared.
A phosphate buffer solution of pH 7.0 was prepared by adding 24.40 mL of 0.1 mol/L HCl and 75.60 mL of 0.1 mol/L Na2HPO4 and adjusting to pH 7.0, Other buffer solutions were also prepared according to literature methods (35). Buffer solutions.
Spectrophotometric method Here, 10 μg/mL of MSZ solution was added into a 10 mL volumetric flask, then 1.5 mL of phosphate buffer solution (pH 7.0) was added into flask to control the pH value of the medium, followed by 3 mL of 0.01 mol/L β-CD solution. The reaction was allowed to proceed at room temperature for 10 min after which the reaction mixture was made up to the mark with distilled water. The absorbance spectra were measured at 330 nm. Spectrofluorometric method
Experimental Chemicals and reagents All chemicals were of analytical or HPLC grade. Distilled water was used in all experiments. Details of chemicals (suppliers) are given below. The MSZ standard was purchased from Sigma-Aldrich (St. Louis, MI, USA), the tablet formulation Pentasa [Pharbil Pharma Bielefeld, Germany (Marketing Authorization Holder: Ferring GmbH, Kiel, Germany)] was procured from a local pharmacy with a labeled amount 500 mg MSZ, β-cyclodextrin (β-CD) was obtained from Sigma-Aldrich (St. Louis, MI, USA).
Instruments and apparatus All spectrofluorimetric measurements were made with a spectrofluorimeter RF-1501 (Shimadzu, Japan) equipped with a 150 W xenon lamp, Slit widths for both monochromators were set at 10 nm. All spectrophotometric measurements were made with a double beam UV1800 UV–vis light spectrophotometer provided with a matched 1-cm quartz cell (Shimadzu, Japan). IR measurements were recorded by Fourier transform IR (FT-IR) spectroscopy using a spectrometer-8400S (Perkin-Elmer, Shimadzu, Japan). Samples were pressed into KBr pellets and recorded at frequencies from 4000 to 200 cm–1. A pH meter (model HI 255, Hanna Instruments, Mumbai, India) was used for pH measurements. A thermostatically controlled water bath (LAUDA, Ecoline model RE 220, Gaithersburg, MD, USA) was used.
wileyonlinelibrary.com/journal/luminescence
Sample solution. A sample of finely powdered tablet nominally equivalent to 0.0100 g MSZ was dissolved in about 30 mL distilled water in a 100 mL volumetric flask, swirled, sonicated for 15 min, then filled to the correct level with distilled water, the contents were mixed well and filtered. This prepared solution was diluted quantitatively to obtain a suitable concentration for the analysis.
Aliquots of MSZ solution were added to 10 mL volumetric flasks to give final concentrations of 0.1–0.45 μg/mL. Next, 1.5 mL buffer solution pH 7.0 was added followed by 2.0 mL β-CD solution (1 × 10–2 mol/L). The reaction was allowed to proceed at room temperature for 10 min after which time the reaction mixture was made up to the mark with distilled water and the fluorescence intensity of MSZ–β-CD was measured at λex/λem = 330 nm/493 nm. Preparation of solid complex of MSZ–β-CD Accurately weighed β-CD (0.3712 g) was placed into a 100 mL conical flask and 30 mL distilled water was added, stirred, then 0.05 g MSZ was put into a 100 mL beaker and 70 mL of distilled water was added and put over electromagnetic stirrer to stir until it was dissolved. Then slowly, the β-CD solution was poured into a stirred MSZ solution, and continuously stirred for 20 h at room temperature (36). The reaction mixture was freeze dried. At this time, the formation of white crystal was observed. This MSZ inclusion complex with β-CD was characterized by FT-IR spectroscopy. Stoichiometry of the reaction Here, 1.0 μg/mL of MSZ and 1.5 mL of phosphate buffer solution (pH 7.0) were added to a volumetric flask, then the various amounts of β-CD (0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, or 7.0 mL. of
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Spectrofluorometric determination of mesalazine 1 × 10–2 mol/L) were added sequentially. The reaction was allowed to proceed at room temperature for 10 min, after which time the reaction mixture was made up to the mark with distilled water.
Results and discussion Absorbance spectra The absorption spectra of MSZ in the absence and in the presence of 0.003 mol/L β-CD was first recorded Fig. 2. The results showed that the wavelength of maximum absorbance of MSZ (10 μg/mL) at pH 7.0 was 330 nm, when β-CD was added into the MSZ solution, the wavelength of maximum absorbance did not change but the absorbance was slightly increased and the shape of the absorption spectrum of MSZ remained constant in the broad spectral range 290–400 nm, this is due to the formation of the MSZ and β-CD complex. Emission spectra
Optimization of reaction variables
The spectral characteristics of MSZ were studied and the results showed that the wavelength of maximum emission of MSZ at pH 7.0 was 493 nm Fig. 3. When β-CD was added into the MSZ solution, the wavelength of maximum of emission did not change but the fluorescence intensity dramatically increased. This can be rationalized as MSZ entering the β-CD hydrophobic cavity and binding taking place through non-covalent bonding due to Van der Waals forces and hydrogen bonding. In the cavity, the degree of motional freedom of MSZ molecule was reduced, thus the cavity could shield the MSZ excitation signal from quenching in the aqueous solution. Therefore the fluorescence intensity increased when the MSZ–β-CD inclusion complex was produced. Infra-red spectra The solid complex formation was confirmed by FT-IR spectroscopy (Fig. 4), However some MSZ peaks were either absent (C–N stretching) or shifted (C–O stretching), which suggested a change in environment due to inclusion complex formation between MSZ and β-CD.
Figure 2. Absorbance spectra of β-CD (—), MSZ (–), inclusion complex (MSZ–β–3 CD) (…), MSZ (10 μg/mL), β-CD (3 × 10 mol/L) at room temperature, time 15 min, 2 mL buffer solution pH 7.0.
Luminescence 2014
Figure 3. Fluorescence excitation (left hand) and emission spectra (right hand) of –3 (1, green) β-CD 2 × 10 mol/L, (2, red) MSZ 1.0 μg/mL, and (3, black) inclusion complex MSZ–β-CD at room temperature, time 10 min, pH 7.0.
Effect of pH. The effect of pH was studied by forming the MSZ–β-CD complex in the presence of various buffers; fluorescence intensity was measured. The pKa values for MSZ have been reported as 6.0 for (–NH+3 ), 13.9 for (–OH), and 3 for (–COOH) (28) respectively. Figure 5 shows that the fluorescence intensity increased rapidly with increasing pH. Maximum absorbance was attained at a pH value of 7.0. At pH values greater than 7.0, a decrease in the fluorescence intensity of the product was observed, the formation constant (K) of the inclusion complex was determined by fluorescence measurement. Influence the volume of buffer solution. The effect of the volume of the phosphate buffer pH 7.0 added on the MSZ–βCD complex was also studied. It was found that increasing the buffer volume resulted in a subsequent increase in fluorescence intensity up to 1.5 mL, after which volume the effect of fluorescence intensity remained constant. A volume of 1.5 phosphate buffer pH 7.0 was recorded as optimum in this study as shown in Fig. 6.
Figure 4. IR spectra of MSZ and the MSZ–β-CD inclusion complex.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
A. A. Elbashir et al. 1000 900
Flourescence Intensity
800 700 600 500 400 300 200 100 0 0
10
20
30
40
50
60
70
Temperature( C)
Effect of temperature and time. The reaction time was determined by forming the MSZ–β-CD at room temperature and in a thermostatically controlled water-bath adjusted in the range 30–90 °C. The highest fluorescence intensity was obtained at room temperature (Fig. 7). Effect of time at room temperature was studied; the highest reading was obtained after 10 min (Fig. 8), therefore the fluorescence measured at room temperature after 10 min in this study was considered to be optimal. Effect of β-CD concentration. The effect of β-CD concentration on the fluorescence intensity of MSZ was examined. The concentration of MSZ was fixed and the concentration of β-CD varied from 0.001–0.007 mol/L. The reaction was found to be dependent on β-CD concentration (Fig. 9), and illustrated that with increase in β-CD concentration fluorescence intensity was enhanced. Therefore 2 mL of 1 × 10–2 mol/L β-CD was selected as optimal and adopted in all subsequent work.
Stoichiometry of the reaction The stoichiometry of the inclusion complex was studied under the established experimental conditions: assuming that the
Figure 7. Effect of temperature on the fluorescence intensity of MSZ–β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, time 10 min, buffer volume 1.5 ml, pH 7.0.
1000 900 800
Fluoresence Intensity
Figure 5. Effect of pH on the fluorescence intensity of the MSZ–β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, at room temperature, time 10 min, buffer volume 2.0 mL.
500 400 300
100 0 0
10
20
30
40
50
Time(minute) Figure 8. Effect of time on the fluorescence intensity of MSZ-β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, at room temperature, buffer volume 1.5 mL, pH 7.0.
720 700
Flourescence Intensity
612
Flourescence Intensity
600
200
614
610 608 606 604
680 660 640 620 600
602 600 0.0
700
580 0.5
1.0
1.5
2.0
2.5
0.001
Volume of buffer(ml)
0.003
0.004
0.005
0.006
0.007
Beta-CD in molar concentration
Figure 6. Effect of buffer volume on the fluorescence intensity of MSZ-β-CD inclusion complex, [MSZ] = 1.0 μg/mL, [β-CD] = 0.002 mol/L, at room temperature, time 10 min, pH 7.0.
wileyonlinelibrary.com/journal/luminescence
0.002
Figure 9. Effect of β-CD concentration on the fluorescence intensity of MSZ–β-CD inclusion complex, [MSZ] = 1.0 μg/mL, at room temperature, time 10 min, buffer volume 1.5 mL, pH 7.0.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Spectrofluorometric determination of mesalazine Alternatively, assuming the stoichiometry of the inclusion complex was 1:2, the following expression was obtained:
0.025
1=F F0 ¼ 1=ðF∞ F0ÞK ½β CD0
1/(F-F0)
0.020
2
þ 1=F∞ F0
(2)
When making a plot of 1/F – F0 against 1/([β-CD]0)2, a non-linear relationship was obtained (Fig. 11), which indicated that the stoichiometry of the inclusion complex is not 1:2. These results confirmed that β-CD and MSZ formed a host–guest complex in 1:1 stoichiometry (37). The inclusion constant (K) was calculated to be 1.359 × 102 L/mol.
0.015
0.010
0.005 200
400
600
800
Inclusion complex thermodynamics
1000
1 / [Beta-CD]0 mole/L Figure 10. Plot of 1/(F – F0) vs. 1/[β-CD] of the MSZ–β-CD complex; MSZ = 1.0 μg/mL, at room temperature, time 10 min, buffer volume 1.5 mL, pH 7.0.
0.025
1/(F-F0)
0.020
0.015
0.010
0.005 0
200000
400000
600000
800000 1000000
2
1 / [Beta-CD] 0 2
Figure 11. Plot of 1/(F – F0) vs. 1/[β-CD] of the MSZ-β-CD complex; MSZ = 1 μg/mL, at room temperature, time 10 min, buffer volume 1.5 mL, pH 7.0.
composition of the complex was 1:1, and using typical double reciprocal or Benesi–Hildebrand plots: 1=F F0 ¼ 1=ðF∞–F0ÞK½β CD0 þ 1=F∞ F0
(1)
Where [β-CD]0 denotes the β-CD concentration; F0 the fluorescence intensity of MSZ in the absence of β-CD; F∞ the fluorescence intensity when all MSZ molecules are essentially complexed with β-CD; and F the observed fluorescence intensity at each β-CD concentration tested. When a plot of 1/F – F0 versus 1/[β-CD]0 was constructed (Fig. 10), a straight line was obtained ,which is indicative of a 1:1 stoichiometry for the MSZ–β-CD complex. Table 1. Thermodynamic parameter of the reaction Thermodynamic parameters ΔH° ΔS° ΔG°
Luminescence 2014
Value kJ/mole –544.295 –1.414 –122.961
The thermodynamic parameters (ΔH°, ΔS° and ΔG°) for the formation of inclusion complex were determined from temperature dependence of apparent association constants, by using the classical van ’t Hoff equation (ln K = –ΔH°/RT + ΔS°/R), and plotting ln K versus 1/T (38,39). The corresponding enthalpy and entropy can be obtained from the slope and intercept, respectively, which indicated the marked tendency of MSZ to complex with β-CD. ΔG° was obtained according to the equation: ΔG° = ΔH° TΔS°. The results are shown in Table 1. We noted that the association constant for MSZ–β-CD increased as the temperature rose. Thermodynamic parameters were calculated based on the temperature dependence of the association constant for MSZ–β-CD binding. Thermodynamic parameters, such as the enthalpy changes (ΔH) and entropy changes (ΔS) of the binding reaction, are important to confirm the force of interactions of MSZ with β-CD. Four driving forces for the inclusion of CDs with substrates were proposed, including hydrogen bonding between the hydroxyl groups of CDs and the MSZ molecules, van der Waals interactions between host and guest molecules, hydrophobic interaction, and the release of ‘high-energy water’ molecules from the cavities of β-CDs to the bulk water. Hydrophobic interaction essentially involved favourable positive entropy together with a slightly positive enthalpy change, whereas the other forces involved negative ΔH and ΔS (40). Upon complex formation, the negative ΔG° value strongly suggested that the inclusion process proceeded spontaneously, as both negative enthalpic changes and negative entropic values were obtained, which mean that the inclusion complexation is exothermic and enthalpy controlled, but not entropy driven. This is the common situation concerning the formation of inclusion complexes between cyclodextrins
Table 2. Summary of quantitative parameters and statistical data using the proposed procedure
Parameters
Result
Linear range (μg/mL) Intercept Slope Standard deviation Correlation coefficient (r) Limit of detection, LOD (μg/mL) Limit of quantitation, LOQ (μg/mL) Molar absorptivity (L mol–1 cm–1) Sandell’s sensitivity
Copyright © 2014 John Wiley & Sons, Ltd.
0.1–0.5 12.79 719.0 1.922 0.9998 0.009 0.015 1.294 × 108 1.183 × 10–6
wileyonlinelibrary.com/journal/luminescence
A. A. Elbashir et al. and various guest molecules. It is widely accepted that the negative entropy change is due to the steric barrier caused by their molecular geometrical shape and the limit of the β-CD cavity to the freedom of shift and rotation of guest molecules.
Table 6. Application of the proposed spectrofluorometric method
Validation of the proposed method
Pentasa
Linearity and sensitivity. In the proposed method, a linear plot (n = 6) with good correlation coefficients (0.99989) was obtained in the concentration range 0.1–0.5 μg/mL. The molar absorptivity (ε) was 1.294 × 108 (L /mol/ cm). The Sandell’s sensitivity value was 1.183 × 10–6. The detection limit (LOD) is defined as the minimum level at which the analyte can be reliably detected, it was calculated using the following equation:
* Values are the mean of three determinations.
LOD ¼ 3:3 s=k
(3)
Where s is the standard deviation of replicate determination
Brand name Labeled claim and dosage form mg/tablet
0.2 0.3 0.4
Recovery (% ± RSD)*
Relative error %
98.86 ± 0.19 101.41 ± 1.03 99.42 ± 1.27
–1.360 1.418 –0.598
Table 4. Recovery of the proposed method
0.1 0.1 0.1
Amount of pure drug added (μg/mL) 0.1 0.2 0.3
Recovery (% ± SD)*
Amount recovered (μg/mL) 0.194 0.308 0.398
96.85 ± 1.81 102.62 ± 0.19 99.60 ± 0.61
* Values are the mean of three determinations.
Table 5. Robustness of the proposed spectrofluorometric method Method conditions Standard condition pH 6.8 7.2 Temperature (°C) 20 30 Time(minutes) 8 12 β-CD concentration (mol/L) 1.8 × 10–3 2.2 × 10–3
Recovery ± RSD* 98.864 ± 0.194 99.464 ± 1.085 99.154 ± 0.888 100.105 ± 0.498 97.654 ± 0.228 100.715 ± 0.457 104.682 ± 0.202 100.575 ± 0.883 100.055 ± 1.066
* Values are the mean of three determinations.
wileyonlinelibrary.com/journal/luminescence
487.36
97.47 ± 0.634
500
LOQ ¼ 10 s=k
* Values are the mean of three determinations.
Initial amount (μg/mL)
Recovery (% ± SD)*
values under the same conditions for sample analysis in the absence of the analyte, and k is the sensitivity, namely the slope of the calibration graph. In accordance with the formula, the detection limit was found to be 0.009 μg/mL. The limit of quantification (LOQ) is defined as the lowest concentration that can be measured with acceptable accuracy and precision:
Table 3. Precision of the proposed method Concentration (μg/mL)
Amount found
(4)
According to this equation, the limit of quantization was found to be 0.015 μg/mL. These parameters for the proposed method are summarized in Table 2. Accuracy and precision. The accuracy and precision of the proposed method was determined at three concentration levels of MSZ (within the linear range) by analyzing three replicates of pure drug of each concentration. The percentage relative error (as accuracy) and percentage relative standard deviations (RSD) (as precision) for the results did not exceed 2% for the proposed method as shown in Tables 3 and 4, indicating good reproducibility and repeatability. This good level of precision and accuracy was suitable for quality control analysis of MSZ in their pharmaceutical formulation. Robustness. Robustness was examined by evaluating the influence of small variation in the method variables on its analytical performance. In these experiments, one parameter was changed whereas the others were kept unchanged, and the recovery percentage was calculated each time. It was found that small variation in the method variable did not significantly affect the procedure .This provided an indication of the reliability of the proposed method during routine work; recovery values were shown in Table 5. Applications of the methods. The proposed method was applied to the pharmaceutical formulation containing MSZ. The results are shown in Table 6, and indicate the high accuracy of the proposed method for the determination of the studied drug. The proposed method has the advantage of being virtually free from interferences by excipients.
Conclusion The supramolecular interaction of MSZ with β-CD was been investigated by spectrofluorimetry. The results showed that β-CD reacted with MSZ to form a 1:1 (host: guest) complex with inclusion constant K = 1.359 × 102 L/mol. Based upon the enhancement effect observed, a simple, sensitive, accurate spectrofluorometric method for the determination of MSZ is proposed. Under optimized experimental conditions, the method was applied to the determination of MSZ in its pharmaceutical formulations.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014
Spectrofluorometric determination of mesalazine
References 1. Brewster ME, Vandecruys R, Verreck G, Noppe M, Peeters J. A novel cyclodextrin-containing glass thermoplastic system (GTS) for formulating poorly water soluble drug candidates: preclinical and clinical results. J Inclusion Phenom 2002;44:35–8. 2. Qi AD, Li L, Liu Y. Molecular binding ability and selectivity of natural α-, β-, γ-cyclodextrins and oligo(ethylenediamino) modified β-cyclodextrins with Chinese traditional medicines. J Inclusion Phenom 2003;45:69–72. 3. Diez MA, Pena IMA, Garera MCM, Gil DB, Canada FC. Fluorimetric determination of sulphaguanidine and sulphamethoxazole by host–guest complexation β-cyclodextrin and partial least squares calibration. J Fluoresc 2007;17:309–18. 4. Elbashir AA, Dsugi NFA, Aboul-Enein HY. Supramolecular study on the interaction between ofloxacin and methyl β-cyclodextrin by fluorescence spectroscopy and its analytical application. J Fluoresc 2014;24:355–61. 5. Chao J-B, Tong H-B, Huang S-P, Liu D-S. Preparation and study on the solid inclusion complex of sparfloxacin with β-cyclodextrin. Spectrochim Acta A Mol Biomol Spectrosc 2005;60:161–6. 6. Linare M, Bertorello MM, Longhi M. Preparation and characterization of solid complexes of napthoquinone and hydroxypropyl-βcyclodextrin. Molecules 2000;5:342–4. 7. Xiea H, Wangb HY, Mac Li Y, Xiaob Y, Hanb J. Spectrophotometric study of the inclusion complex between β-cyclodextrin and dibenzoyl peroxide and its analytical application. Spectrochim Acta A Mol Biomol Spectrosc 2005;62:197–202. 8. Chao J-B, Tong H-B, Liu D-S, Huang S-P. Preparation and characterization of inclusion complexes of pefloxacin mesylate with three kinds of cyclodextrins. Spectrochim Acta A Mol Biomol Spectrosc 2006;64:166–70. 9. Elbashir AA, Suliman FEO, Saad B, Aboul-Enein HY. Capillary electrophoretic separation and computational modeling of inclusion complexes of β-cyclodextrin and 18-crown-6 ether with primaquine and quinocide. Biomed Chromatogr 2010;24:393–8. 10. Elbashir AA, Suliman FO. Computational modeling of capillary electrophoretic behavior of primary amines using dual system of 18-crown-6 and β-cyclodextrin. J Chromatogr A 2011;1218:5344–51. 11. Elbashir AA, Saad B, Ali ASM, Saleh MI, Aboul-Enein HY. Recent developments of enantioseparations for fluoroquinolone drugs using liquid chromatography and capillary. Curr Pharm Anal 2010;6:246–55. 12. Elbashir AA, Dsugi NFA, Mohmed TOM, Aboul-Enein HY. Spectrofluorometric analytical applications of cyclodextrins. Luminescence 2014;29:1–7. 13. Monson PL, Mueller RA, Breese GR. Principles of pharmacology. New York: Chapman & Hall, 1995:l064. 14. Bender ML, Komiyama M. Cyclodextrin chemistry. Berlin, Heidelberg, New York: Springer-Verlag, 1978:10. 15. Tong LH. Cyclodextrin chemistry foundation and application [Huanhujing Huaxue Jichu Yu Yingyong]. Beijing: Science Press, 2001;191 pp. 16. Li S, Purdy WC. Cyclodextrin and their application in analytical chemistry. Chem Rev 1992;92:1458–70. 17. Hanauer S, Schwartz J, Robinson M, Roufail W, Arora S, Cello J, Safdi M. Mesalamine capsules for treatment of active ulcerative colitis: results of a controlled trial. Am J Gastroenterol1993;88:1188–97. 18. Thomson AB. New developments in the use of 5-aminosalicylic acid in patients with inflammatory bowel disease. Aliment Pharmacol Ther 1991;5:449–70. 19. Geier DL, Miner PB. New therapeutic agents in the treatment of inflammatory bowel disease. Am J Med 1992;93:208–98. 20. Gotti R, Pomponio R, Bertucci C, Cavrini V. Determination of 5-aminosalicylic acid related impurities by micellar electrokinetic chromatography with an ion-pair reagent. J Chromatogr A 2001;916:175–83. 21. Biljana N, Branimir I. Determination of 5-aminosalicylic acid in pharmaceutical formulation by differential pulse voltammetry, J Pharm Biomed Anal 2003;31:169–74.
Luminescence 2014
22. Palumbo G, Carlucci G, Mazzeo P. Simultaneous determination of 5-aminosalicylic acid, acetyl-5-aminosalicylic acid and 2,5dihydroxybenzoic acid in endoscopic intestinal biopsy samples in humans by high performance liquid chromatography with electrochemical detection. J Pharm Biomed Anal Oxford 1995;14:175–80. 23. Janice AR, José RJ, Rúbia C, Sandra RG, Maria D., Fátima B, Maria José VF. Validation of HPLC, DPPH and nitrosation methods for mesalamine determination in pharmaceutical dosage forms. Rev Brasil Cienc Farm 2007;43:97–103. 24. Haney PW, Dash AK. Simple liquid chromatographic method for the analysis of 5- aminosalicyclic acid and its degradation product. J Chromatogr A 1997;765:233–9. 25. Palumbo G, Bacchi S, Primavera L, Palumbo P, Carlucci G. A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma. Biomed Chromatogr 2005;19:350–4. 26. Bystrowska B, Nowak J, Brandys J. Validation of a LC method for the determination of 5-aminosalicylic acid and its metabolite in plasma and urine. J Pharm Biomed Anal 2000;22:341–51. 27. Nobilis M, Vybíralová Z, Sládková K, Lísa M, Holcapek M, Kvetina J. High-performance liquid-chromatographic determination of 5-aminosalicylic acid and its metabolites in blood plasma. J Chromatogr A 2006;1119:299–308. 28. Pastorini E, Locatelli M, Simoni P, Roda G, Roda V, Roda A. Development and validation of a HPLC–ESI–MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5aminosalicylic acid in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2008;872:99–106. 29. Patel KM, Patel CN, Panigrahi B, Parikh AS, Patel HN. Development and validation of spectrophotometric methods for the estimation of mesalazine in tablet dosage forms. J Young Pharmacists 2010;2:284–8. 30. Madhavi V, Panchakshari V, Prathyusha TN, Sekaran CB. Spectrophotometric determination of mesalazine in bulk and tablet dosage forms based on diazocoupling reaction with resorcinol. Int J Pharm Sci Rev Res 2011;11:105–9. 31. Narala S, Saraswathi K. Spectrophotometric methods for quantitative determination of mesalamine in bulk and tablet dosage forms. Int J Res Pharm Biomed Sci 2010;1:10–3. 32. Reddy MP, Prabhavathi K, Reddy NR, Reddy PR. Two simple spectrophotometric methods for the estimation of mesalamine in bulk sample and its pharmaceutical dosage forms. Global J Pharmacol 2011;5:101–5. 33. Zadeh HA, Kohansal S. Determination of mesalamine by spectrofluorometry in human serum after solid-phase extraction with Ni-Al layered double hydroxide as nanosorbent. J Brazil Chem Soc 2012;23:473–81. 34. Cui FL, Fan J, Li W, Fan YC, Hu ZD. Fluorescence spectroscopy studies on 5-aminosalicylic acid and zinc 5-aminosalylicylate interaction with human serum albumin. J Pharm Biomed Anal 2004;34:189–97. 35. Analytical Chemistry Studio of Zhongnan Mineral College. Handbook of chemical analysis. Beijing: Science Press, 1984:383 pp. 36. Chao J-B, Tong H-B, Huang Sh-P, Liu D-Sh. Preparation and study on the solid inclusion complex of sparfloxacin with β-cyclodextrin. Spectrochimica Acta Part A 2005;60:161–6. 37. Catena GC, Bright FV. Thermodynamic study on the effects of beta-cyclodextrin inclusion with anilinonaphthalene sulfonates. Anal Chem 1989;61:905–9. 38. Rekharsky MV, Inoue Y. Complexation thermodynamics of cyclodextrins. Chem Rev 1998;98:1875–918. 39. Inoue Y, Hakushi T, Liu Y, Tong LH, Shen BJ, Jin DS. Thermodynamics of molecular recognition by cyclodextrins. 1. Calorimetric titration of inclusion complexation of naphthalenesulfonates with. alpha-, beta-, and gamma-cyclodextrins: enthalpy–entropy compensation. J Am Chem Soc 1993;115:475–81. 40. Zhang Q, Jiang Z, Guo Y, Li R. Complexation study of brilliant cresyl blue with β-cyclodextrin and its derivatives by UV-vis and fluorospectrometry. Spectrochim Acta Part A 2008;69:65–70.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
