Research article Received: 25 June 2014,
Revised: 7 November 2014,
Accepted: 16 November 2014
Published online in Wiley Online Library: 21 January 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2828
Micelle-enhanced spectrofluorimetric determination of amlexanox in bioadhesive buccal tablets: application to content uniformity testing M. I. Walash, F. Belal, M. M. Tolba and M. I. Halawa* ABSTRACT: A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of Amlexanox (AMX) in its bioadhesive buccal tablets. The proposed method is based on measuring the native fluorescence of the methanolic solution of AMX at 400 nm after excitation at 242 nm in 0.2 M borate buffer (pH 10) and 0.5% w/v sodium dodecyl sulfate (SDS) solution. The interaction of AMX with SDS was studied, and the enhanced fluorescence intensity was exploited to develop an assay method for the determination of AMX. The relative fluorescence intensity–concentration plot was rectilinear over the range 5.0–80.0 ng/mL, with a lower detection limit of 0.57 ng/mL and a lower quantification limit of 1.74 ng/mL. The proposed method was successfully applied to the analysis of AMX in its commercial tablets. Moreover, content uniformity testing was conducted by applying official USP guidelines. Statistical evaluation and comparison of the data obtained using the proposed and comparison methods revealed good accuracy and precision for the proposed method. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Amlexanox; spectrofluorimetric; bioadhesive buccal tablets; content uniformity testing
Introduction
Luminescence 2015; 30: 823–829
* Correspondence to: M. I. Halawa, Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516 Mansoura, Egypt. E-mail: [email protected] Department of Analytical Chemistry, University of Mansoura, Mansoura, Egypt
Copyright © 2015 John Wiley & Sons, Ltd.
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Amlexanox is a novel anti-inflammatory and anti-allergic agent. Clinically, it is the only drug approved by Food and Drug Administration (FDA) for the treatment of aphthous ulcers (1). AMX effectively treats aphthous ulcers by accelerating healing and complete pain resolution. It potently inhibits the release of histamine and leukotrienes from mast cells, basophils and neutrophils under in vitro settings, possibly by increasing the intracellular cyclic AMP content of inflammatory cells, a membrane-stabilizing effect or inhibition of calcium influx (1,2). Chemically, AMX is 2-amino-7-isopropyl-5-oxo-5H-[1]benzopyrano [2,3-b]pyridine-3-carboxylic acid (Figure 1). It is commercially available as a 5% oral paste, bioadhesive buccal tablets and as a biodegradable muco-adhesive disc (1). A review of the literature revealed that few HPLC methods have been developed for the analysis of AMX in bulk drugs (3) or human serum (4) or in its paste in the presence of related substances (5). Also, AMX was determined simultaneously with cromolyn and tranilast using the HPLC method (6). Nothing has been reported in the literature regarding the determination of AMX in its bioadhesive buccal tablets. In addition, no spectrofluorimetric method has been described for the determination of AMX in its tablets and this initiated the present study. Based on micellar enhancement of the native fluorescence intensity of AMX, a simple and highly sensitive spectrofluorimetric method has been developed for determination of AMX in its tablets. The use of surfactants in various areas of analytical chemistry has attracted much interest in the last three decades. It has been used in separation science as a modifier of mobile and stationary phases (7). Micellar catalysis has been used to enhance
several chromogenic derivatization reactions by the simultaneous alteration of various physical properties of the solution and physicochemical properties of the reagents, intermediates and products (8). The use of micellar media is the most commonly applied method for increasing the fluorescence intensity of target fluorophores and consequently, for lowing their limit of detection (LOD) (9). When a solute passes from an aqueous medium to a micellar medium, several properties (e.g. reactivity, solubility or spectroscopic characteristics) undergo important changes, increasing fluorescence intensity being the most outstanding and advantageous (10). In general, micellar media provide a very rigid microenvironment capable of restricting the freedom of fluorophores, and so diminish the probabilities of non-radiative processes and provide relatively high viscous microenvironments that can inhibit quenching by molecular oxygen. These factors might increase the fluorescence quantum yield and enhance the fluorescence signals of guest molecules (9). Sodium dodecyl sulfate (SDS), also known as sodium lauryl sulfate (C12H25SO4Na), is an anionic surfactant that has recently found many applications in different areas of analytical chemistry. The critical micelle concentration (CMC) of SDS in pure water at 25 °C is 8.2 × 10-3 M and the aggregation number at this concentration is usually considered to be 62 (11). SDS has been used
M. I. Walash et al. Procedures
Figure 1. Chemical formula of AMX.
as a fluorescence enhancer for the determination of some pharmaceutical compounds viz., verapamil hydrochloride (12), citalopram (13), labetalol (14), gatifloxacin (15), levofloxacin (16), histamine (17) and epinephrine (18). The method developed here is simple, timesaving and does not require elaborate treatment compared with the reported chromatographic method (5). In addition, content uniformity testing was conducted according to USP guidelines (19).
Experimental Instruments All fluorescence measurements were made using an RF-1501 Shimadzu spectrofluorometer, equipped with a 150 W xenon arc lamp (Kyoto, Japan). The excitation and emission wavelengths were 242 and 400 nm, respectively. The slit widths were 5 nm for both excitation and emission wavelengths, and the photomultiplier voltage was set to auto. A 1 cm quartz cuvette was used. A Consort NV P-901 pH meter (Belgium, Europe) was used for pH measurements.
Construction of the calibration graph. Aliquots of methanolic AMX standard solution were transferred to a series of 10 ml volumetric flasks to give final concentrations of 5.0–80.0 ng/mL. One milliliter of 0.2 M borate buffer (pH 10) was added to each flask followed by 0.6 ml of 0.5% w/v SDS solution. The volume was completed with methanol, the contents of the flasks were mixed well and the fluorescence intensity was measured at 400 nm after excitation at 242 nm. Relative fluorescence intensity (RFI) was plotted against final drug concentration (ng/mL) to give a calibration graph. Alternatively, the corresponding regression equation was derived.
Twenty Aphthtab® tablets were crushed and a weight equivalent to 10.0 mg AMX was transferred quantitively into a 100 ml volumetric flask, ~ 80 ml of methanol was added and the flasks were sonicated for 30 min. Dissolution of the tablets was enhanced with the aid of a magnetic stirrer. The solutions were then diluted to volume with methanol, mixed and filtered. Aliquots covering a working concentration range of 5.0–80.0 ng/mL were transferred into 10 ml volumetric flasks. The procedure described under ‘Construction of the calibration graph’ was performed. The nominal contents of the tablets were calculated using the calibration graph or the corresponding regression equation. Analysis of AMX in aphthtab® tablets.
Content uniformity testing of aphthtab tablets. Ten different tablets were analyzed using the procedure applied to the analysis of the studied compound in tablets. The uniformity of the tablet contents was tested by applying the official USP guideline [see Chapter 905 (19)].
Reagents and materials
Results and discussion
All chemicals used were of analytical reagents (AR) grade, and the solvents were HPLC grade. AMX lot # 080112G (certified to have a purity of 98%) was purchased from AK Scientific, Inc. (Union City, CA, USA) and was used as received without further purification. Aphthtab® tablets labeled as containing 2 mg AMX per tablet (batch # 203369) were from EVA Pharma for Pharmaceuticals and Medical Appliances (Giza, Egypt). The following surfactants were used as 0.5% (w/v) aqueous solutions: SDS (95%) and cetyl trimethylammonium bromide (CTAB; 99%) (London, England); β-cyclodextrin (β-CD) and hydroxy propyl-β-cyclodextrin (HP-β-CD) (Darmstadt, Germany); carboxymethylcellulose (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt); Tween-80 (El-Nasr Pharmaceutical Chemicals Co.); methanol (Paris, France), n-propanol and acetonitrile (SigmaAldrich Chemie GmbH, Germany); dimethyl sulfoxide (RiedeldeHäen, Sleeze, Germany); glacial acetic acid, sodium acetate trihydrate, boric acid, sodium hydroxide and dimethyl formamide (El-Nasr Pharmaceutical Chemical Co.). Acetate buffer (0.2 M, pH 3.5–5.5) and borate buffer (0.2 M, pH 6.5–10.5) solutions were freshly prepared.
AMX is formulated in a very minute amount in tablets (2 mg/tablet); therefore, there is an urgent need to explore a sufficiently sensitive and specific method for its determination in tablets. Fluorimetric analysis, by virtue of its high inherent sensitivity, might successfully overcome this problem. A methanolic solution of AMX was found to exhibit three emission bands of different intensities at 400 nm, after excitation at 242, 281 or 350 nm. The highest sensitivity was achieved upon using 242 nm as the excitation wavelength, and so this value was selected. It is well known that, in many cases, the addition of a surfactant at a concentration above its CMC to a given fluorophore solution increases its molar absorbtivity and/or the fluorescence quantum yield (20,21). This has been used to improve the performance of spectrofluorimetric methods for various analytes. The fluorescence properties of AMX in various micellar media were studied; there was significant enhancement of the fluorescence intensity in the presence of SDS compared with a methanolic solution, therefore SDS was used as a fluorescence enhancer to develop a new sensitive spectrofluorimetric method for the determination of AMX.
Standard solutions
Fluorescence spectra of AMX in methanolic solution and in the SDS system
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Stock solution equivalent to μg/mL of AMX was prepared by dissolving 10.0 mg of the drug in 100 ml of methanol. This solution was further diluted using the same solvent as appropriate. Standard solutions were stable for 10 days when kept in the refrigerator.
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The fluorescence spectra of AMX in both the methanolic and SDS systems were studied. Figure 2 illustrates the fluorescence spectra of AMX in the two systems. The first system was methanolic borate buffer (pH 10) and the second was the same solution
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 823–829
Spectrofluorimetric determination of amlexanox
a
b Figure 2. (a) Fluorescence spectra of: (A, B) AMX (60 ng/mL) in methanolic borate buffer (pH 10). Where: A = excitation band; B1 = emission band at λex = 242 nm; B2 = emission band at λex = 281 nm; B3 = emission band at λex = 350 nm. (b) Fluorescence spectra of: (A, B) AMX (60 ng/mL) in methanolic borate buffer (pH 10) (SDS system); (A′, B′) blank. Where: A, A′ = excitation spectra; B, B′ = emission spectra.
but in the presence of SDS as a fluorescence enhancer. The percentage fluorescence enhancement in the presence of SDS was significant when compared with the native fluorescence intensity of the drug in a methanolic medium.
Optimization of the experimental conditions Effect of organized media. The effect of various organized media on the fluorescence intensity of AMX was studied by adding 1.0 ml of a 0.5% (w/v) aqueous solution of each to the drug solution. Different surfactants such as SDS (anionic surfactant), CTAB (cationic surfactant) and Tween-80 (nonionic surfactant) and also macromolecules like β-CD and HP-β-CD were studied. All the organized media studied caused a slight decrease in the RFI of the drug (Fig. 3). Only SDS gave a considerable increase in the RFI, so it was used throughout the present study.
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Figure 3. Effect of different organized media (1 ml of a 0.5% w/v solution of each) on the RFI of AMX (60 ng/mL).
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Effect of the volume of SDS. The influence of SDS on the RFI was studied using increasing volumes of 0.5% w/v SDS aqueous solution. It was found that increasing volumes of SDS solution resulted in a corresponding increase in RFI up to 0.4 ml, after
M. I. Walash et al. which no further increase in RFI was obtained. Therefore, 0.6 ml of a 0.5% w/v SDS solution was chosen as the optimum volume for AMX (Fig. 4). An SDS volume > 1 ml was not used because of a high blank reading. Effect of pH. The influence of pH on the micelle-enhanced fluorescence of AMX was studied using different types of buffers covering the whole pH range, for example, 0.2 M acetate buffer over the pH range 3.5–5.5 and 0.2 M borate buffer over the pH range 6.5–10.5. It was found that maximum and constant RFI was achieved over a pH range of 9.5–10.5; therefore, pH 10 was chosen as the optimum value for the study. Use of 0.2 M acetate buffer caused a gradual decrease in the RFI of AMX (Fig. 5). Effect of diluting solvent. The effect of dilution with different solvents such as distilled water, methanol, acetonitrile, n-propanol, dimethyl sulfoxide and dimethyl formamide was studied. It was found that methanol was the best solvent for dilution, as it gave the highest RFI and lowest blank reading Table 1. A distinct and sharp decrease in RFI was observed on using acetonitrile or n-propanol. Addition of these solvents may result in a reduction in the size of the micelles and a progressive breakdown of the surfactant aggregate at very high concentrations (22). Both dimethyl sulfoxide and dimethyl formamide gave high blank readings and greatly quenched the fluorescence intensities of AMX because they exhibited an intersystem-crossing process (similar to the heavy atom effect) (23).
Table 1. Effect of diluting solvents on the fluorescence intensity (FI) of AMX (60 ng/mL) Solvent
FI
Water Methanol n-propanol Acetonitrile Dimethyl sulfoxide Dimethyl formamide
420 660 365 340 135 50
Effect of time. The effect of time on the RFI of the drug was also studied. It was found that the fluorescence intensity developed immediately and remained stable for more than 2 h. Effect of temperature. Another factor that may affect the fluorescence intensity is temperature. The effect of temperature was studied in the range 40–100 °C in a thermostatically controlled water bath. It was found that increasing the temperature resulted in a decrease in the RFI. This can be explained by higher internal conversion as the temperature increases, facilitating nonradiative deactivation of the excited singlet state and an increase in the loss of energy via collision with solvent molecules (24). Therefore, all experiments were carried out at room temperature. Method validation The validity of proposed method was evaluated regarding linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, robustness and specificity. Linearity and range. The calibration graph for the determination of AMX using the proposed method was constructed by plotting the RFI against the concentration of the drug (ng/mL). The calibration graph showed a linear dependence of RFI on drug concentration over a range of 5.0–80.0 ng/mL for AMX. Linear regression analysis of the data gave the following equation: RFI ¼ 44:73 þ 9:82C
Figure 4. Effect of the volume of SDS (0.5% w/v) on the RFI of AMX (60 ng/mL).
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Figure 5. Effect of pH on the RFI of AMX (60 ng/mL).
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Where RFI is the relative fluorescence intensity and C is the concentration of the drug in ng/mL for AMX. Statistical analysis (25) of the data gave high values for the correlation coefficients (r) of the regression equations, small for the standard deviation of residuals (Sy/x), of intercept (Sa) and of slope (Sb), and small value for the percentage relative standard deviation (%RSD) and the percentage relative error Table 2. These data proved the linearity of the calibration graph of the studied drug. LOQ and LOD. LOQ and LOD were calculated according to the ICH Q2 (R1) recommendations (26). LOQ values were determined by establishing the measurable concentrations below which the calibration graph is nonlinear. LOD values were determined by evaluating the lowest analyte concentrations that can be readily detected. The results are also summarized in Table 2. The values of LOQ and LOD were calculated according to the following equations (26):
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Spectrofluorimetric determination of amlexanox significant difference between the performance of the methods regarding accuracy and precision Tables 3 and 4. The comparison HPLC method involved the use of a mobile phase consisting of methanol : 0.01 M phosphate buffer (70:30, v/v; pH 4.8) and an Octadecylsilane (ODS) column with UV detection at 350 nm (5).
Table 2. Analytical performance data for the spectrofluorimetric determination of AMX Parameter
AMX
Wavelength [λex/λem] (nm) Concentration (ng/mL) Intercept (a) Slope (b) Correlation coefficient (r) SD of residuals (Sy/x) SD of intercept (Sa) SD of slope (Sb) % RSD LOD (ng/mL) LOQ (ng/mL)
242/400 5.0–80.0 44.73 9.82 0.9999 2.64 1.70 0.04 0.80 0.57 1.74
Precision. Intraday precision was evaluated by determining three concentrations of AMX in its pure form on three successive occasions over the course of one day. Interday precision was also evaluated by replicate analysis of three concentrations over a period of three successive days. The results of intraday and interday precision are summarized in Table 4. Robustness of the method. The robustness of the adopted method was demonstrated by the constancy of the RFI with minor changes in the experimental conditions, such as a change in the pH (10 ± 0.2) and SDS volume (0.6 ± 0.2 ml). These minor changes that may occur during the experimental operation did not affect the RFI.
LOQ ¼ 10Sa =b LOD ¼ 3:3Sa =b Where Sa is the standard deviation of the intercept of the regression line and b is the slope of the calibration graph. Accuracy. To prove the accuracy of the proposed method, the results of the drug assay were compared with those obtained using a comparison HPLC method (5). Statistical analysis (25) of the results obtained from the proposed and comparison methods using Student’s t-test and variance ratio F-test showed no
Specificity. The specificity of the method was investigated by observing any interference encountered from common tablet excipients. It was shown that these compounds did not interfere with the results of the proposed method (Table 3). Pharmaceutical applications Application of the proposed method to the analysis of AMX in bioadhesive buccal tablets. The proposed method was successfully applied to the assay of AMX in its conventional tablets. Percent recoveries of different concentrations were based
Table 3. Assay results for the determination of AMX in raw material and its bioadhesive buccal tablets using the proposed and comparison methods Compound
Comparison method[5]
Proposed method Amount taken (ng/mL)
AMX
Mean ± SD % RSD t F Aphthtab tabletsb (AMX 2 mg/ tablet) Mean ± SD t F
5.0 10.0 20.0 30.0 40.0 50.0 60.0 80.0
20.00 40.00 60.00
Amount found (ng/mL) 4.915 10.006 19.883 29.963 40.247 50.430 59.594 79.958
19.776 39.792 60.120
% Found
Amount taken (μg/mL)
98.31 100.07 99.42 99.88 100.62 100.86 99.32 99.95 99.80 ± 0.80 0.80 0.89 (2.26) 1.17 (4.74) 98.88 99.48 100.20 99.52 ± 0.66 1.05 (2.78) 4.53 (19.0)
6.0 8.0 10.0
Amount found (μg/mL) 5.9670 8.0200 10.118
% Found 99.45 100.25 101.18
100.29 ± 0.87 0.86
6.0 8.0 10.0
5.949 8.156 10.030
99.15 101.95 100.30 100.47 ± 1.41
a
Average of three separate determinations. Labeled to contain 2 mg AMX/tablet (batch # 203369), product of EVA Pharma for Pharmaceuticals and Medical Appliances, Giza, Egypt. c Values in parentheses are the tabulated t and F values at P = 0.05 (25). b
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M. I. Walash et al. Table 4. Precision data for the determination of AMX using the proposed method Parameter
AMX (ng/mL) 10.0
Intra-day
% Found
Inter-day
Mean (SD) % RSD % Found
Mean (SD) % RSD
100.35 101.20 99.45 100.33 (0.88) 0.87 100.48 98.78 101.82 100.36 (1.53) 1.52
Table 5. Results of content uniformity testing of AMX tablets using the proposed method Parameter Data
Mean (SD) % RSD % Error Acceptance value (AV) (19) Max. allowed AV (L1) (19)
Tablet no.a 1 2 3 4 5 6 7 8 9 10
Percentage of the label claim 98.85 100.72 101.45 101.35 99.64 100.18 99.28 97.92 102.55 101.98 100.39 (1.48) 1.47 0.47
3.55 15.0
a
Aphthtab tablets: labeled to contain 2 mg AMX per tablet (batch # 203369), product of EVA Pharma for Pharmaceuticals and Medical Appliances, Giza, Egypt.
on the average of three replicate determinations. The results were in good agreement with those obtained using the comparison HPLC method (5). Content uniformity testing for tablets. Because of the high sensitivity of the proposed method and its ability to rapidly measure the fluorescence intensity of a single tablet extract with sufficient accuracy, it is ideally suited for content uniformity testing, which is a time-consuming process when using conventional assay techniques. The steps of the test were adopted according to the USP (19) procedure. The acceptance value (AV) was calculated and found to be smaller than the maximum allowed AV (L1). The results demonstrated excellent drug uniformity, as shown in Table 5.
Conclusion
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A simple and sensitive spectrofluorimetric method was developed for the determination of AMX through enhancement of
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20.0 99.58 101.45 101.10 100.71 (0.99) 0.99 99.30 101.55 99.76 100.20 (1.19) 1.19
40.0 99.82 100.45 99.30 99.86 (0.58) 0.58 98.60 99.95 101.20 99.92 (1.30) 1.30
its native fluorescence upon using SDS. The proposed method is rapid, time-saving and does not require elaborate treatment compared with the reported chromatographic methods. By virtue of its simplicity and sensitivity, the proposed method can be applied to the analysis of Amlexanox for the first time in its bioadhesive buccal tablets, and moreover, in quality control laboratories. The proposed method is suitable for use in content uniformity testing. Acknowledgements The authors extend their appreciation to Analytical Chemistry Department Mansoura University for providing instruments and chemicals. Also Ak Scientific company and EVA Pharma company for kindly providing pure powder and tablets of amlexanox.
References 1. Sweetman SC (Ed). editor#. Martindale, the complete drug reference. 37th ed. Pharmaceutical Press: London, 2009 1115. 2. Makino H, Saijo T, Ashida Y, Kuriki H, Maki Y . Mechanism of action of an antiallergic agent, Amlexanox (AA-673), in inhibiting histamine release from mast cells. Int Arch Aller A Imm 1987;82:66–71. 3. Sundar BS, Nazeerunnisa M. A validated stability indicating LC method for Amlexanox in bulk drugs. Am J Anal Chem 2011;2:533–8. 4. McComish S, Mize A, Harris J, Premkumar N, Colon LE . A highpressure liquid chromatographic method for measuring mitotane: [1,1-(o,p-dichlorodiphenyl)-2,2-dichloroethane] and its metabolite 1,1-(o,p′-dichlorodi-phenyl)-2,2-dichloroethene in plasma. Ther Drug Monit 1995;17:526–31. 5. Sun L, Ding J, Zhang D. RP-HPLC determination of related substances of Amlexanox and its content in paste. Yaowu Fenxi Zazhi 2009;29:1220–2. 6. Shishibori T . Three distinct anti-allergic drugs, Amlexanox, cromolyn and tranilast, bind to S100A12 and S100A13 of the S100 protein family. Biochem J 1999;338:583–9. 7. Hinze WL, Pramauro E . Crit Rev Anal Chem 1993;24:133. 8. Esteve-Romero JS, Simó-Alfonso EF, García-Alvarez-Coque MC, Ramis-Ramos G. Trends Anal Chem 1995;14:29. 9. Yang HM, Wang YS, Li JH, Li GR, Wang Y, Tan X, et al. Anal Chim Acta 2009;636:51. 10. Aidin BM, Acar M, Arik M, Onganer Y . Dyes Pigment 2009;81:156. 11. Turo NJ, Yekta A. J Am Chem Soc 1978;100:5951. 12. Walash MI, Belal F, El-Enany N, Abdelal A. J Am Chem Soc 2006;89:1565. 13. El-Sherbiny DT. J AOAC Int 2006;89:1288. 14. El-Enany N. J AOAC Int 2007;90:948. 15. Ocaña JA, Barragán FJ, Callejón M. J Pharm Biomed Anal 2005;37:327. 16. González JA, Callejón Mochón M, de la Rosa FJ . Talanta 2000;52:1149.
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Spectrofluorimetric determination of amlexanox 17. Adamou R, Coly A, Douabalé SE, Saleck ML, Gaye-Seye MD, Tine AJ . Fluorescence 2005;15:679. 18. Adeniyi WK, Wright AR. Spectrochim Acta 2009;74:1001. 19. The United States Pharmacopoeia 30, The National Formulary 25. US Pharmacopeial Convention, 2007. Electronic version. 20. Hinze WL, Singh HN, Baba Y, Harvey NG. Micellar enhanced analytical fluorimetry. Trends Anal Chem 1984;3:193–9. 21. McIntire GL. Micelles in analytical chemistry. Crit Rev Anal Chem 1990;21:257–78. 22. Leung R, Shah DO. Dynamic properties of micellar solutions: I. Effects of short chain alcohols and polymers on micellar stability. J Colloid Interface Sci 1986;113:484–99.
23. Skoog DA, Holler FJ, Crouch SR. Principles of instrumental analysis. 6th ed. Belmont, NV: Thomson, 2007; 406. 24. Skoog DA, West DM, Holler FJ, Crouch SR. editor#. Fundamentals of analytical chemistry. 8th ed. Saunders College Publishing: Philadelphia, PA, 2004 1003–6. 25. Miller JN, Miller JC. Statistics and chemometrics for analytical chemistry. 5th ed. Harlow, UK: Pearson Education, 2005; 39–73, 107–49, 256. 26. ICH Expert Working Group. ICH harmonized tripartite guidelines. Validation of analytical procedures: text and methodology, Q2(R1), 2005. http://www.ich.org/LOB/media/ MEDIA417.pdf (accessed 15 February 2008).
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Copyright © 2015 John Wiley & Sons, Ltd.
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Revised: 7 November 2014,
Accepted: 16 November 2014
Published online in Wiley Online Library: 21 January 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2828
Micelle-enhanced spectrofluorimetric determination of amlexanox in bioadhesive buccal tablets: application to content uniformity testing M. I. Walash, F. Belal, M. M. Tolba and M. I. Halawa* ABSTRACT: A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of Amlexanox (AMX) in its bioadhesive buccal tablets. The proposed method is based on measuring the native fluorescence of the methanolic solution of AMX at 400 nm after excitation at 242 nm in 0.2 M borate buffer (pH 10) and 0.5% w/v sodium dodecyl sulfate (SDS) solution. The interaction of AMX with SDS was studied, and the enhanced fluorescence intensity was exploited to develop an assay method for the determination of AMX. The relative fluorescence intensity–concentration plot was rectilinear over the range 5.0–80.0 ng/mL, with a lower detection limit of 0.57 ng/mL and a lower quantification limit of 1.74 ng/mL. The proposed method was successfully applied to the analysis of AMX in its commercial tablets. Moreover, content uniformity testing was conducted by applying official USP guidelines. Statistical evaluation and comparison of the data obtained using the proposed and comparison methods revealed good accuracy and precision for the proposed method. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Amlexanox; spectrofluorimetric; bioadhesive buccal tablets; content uniformity testing
Introduction
Luminescence 2015; 30: 823–829
* Correspondence to: M. I. Halawa, Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516 Mansoura, Egypt. E-mail: [email protected] Department of Analytical Chemistry, University of Mansoura, Mansoura, Egypt
Copyright © 2015 John Wiley & Sons, Ltd.
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Amlexanox is a novel anti-inflammatory and anti-allergic agent. Clinically, it is the only drug approved by Food and Drug Administration (FDA) for the treatment of aphthous ulcers (1). AMX effectively treats aphthous ulcers by accelerating healing and complete pain resolution. It potently inhibits the release of histamine and leukotrienes from mast cells, basophils and neutrophils under in vitro settings, possibly by increasing the intracellular cyclic AMP content of inflammatory cells, a membrane-stabilizing effect or inhibition of calcium influx (1,2). Chemically, AMX is 2-amino-7-isopropyl-5-oxo-5H-[1]benzopyrano [2,3-b]pyridine-3-carboxylic acid (Figure 1). It is commercially available as a 5% oral paste, bioadhesive buccal tablets and as a biodegradable muco-adhesive disc (1). A review of the literature revealed that few HPLC methods have been developed for the analysis of AMX in bulk drugs (3) or human serum (4) or in its paste in the presence of related substances (5). Also, AMX was determined simultaneously with cromolyn and tranilast using the HPLC method (6). Nothing has been reported in the literature regarding the determination of AMX in its bioadhesive buccal tablets. In addition, no spectrofluorimetric method has been described for the determination of AMX in its tablets and this initiated the present study. Based on micellar enhancement of the native fluorescence intensity of AMX, a simple and highly sensitive spectrofluorimetric method has been developed for determination of AMX in its tablets. The use of surfactants in various areas of analytical chemistry has attracted much interest in the last three decades. It has been used in separation science as a modifier of mobile and stationary phases (7). Micellar catalysis has been used to enhance
several chromogenic derivatization reactions by the simultaneous alteration of various physical properties of the solution and physicochemical properties of the reagents, intermediates and products (8). The use of micellar media is the most commonly applied method for increasing the fluorescence intensity of target fluorophores and consequently, for lowing their limit of detection (LOD) (9). When a solute passes from an aqueous medium to a micellar medium, several properties (e.g. reactivity, solubility or spectroscopic characteristics) undergo important changes, increasing fluorescence intensity being the most outstanding and advantageous (10). In general, micellar media provide a very rigid microenvironment capable of restricting the freedom of fluorophores, and so diminish the probabilities of non-radiative processes and provide relatively high viscous microenvironments that can inhibit quenching by molecular oxygen. These factors might increase the fluorescence quantum yield and enhance the fluorescence signals of guest molecules (9). Sodium dodecyl sulfate (SDS), also known as sodium lauryl sulfate (C12H25SO4Na), is an anionic surfactant that has recently found many applications in different areas of analytical chemistry. The critical micelle concentration (CMC) of SDS in pure water at 25 °C is 8.2 × 10-3 M and the aggregation number at this concentration is usually considered to be 62 (11). SDS has been used
M. I. Walash et al. Procedures
Figure 1. Chemical formula of AMX.
as a fluorescence enhancer for the determination of some pharmaceutical compounds viz., verapamil hydrochloride (12), citalopram (13), labetalol (14), gatifloxacin (15), levofloxacin (16), histamine (17) and epinephrine (18). The method developed here is simple, timesaving and does not require elaborate treatment compared with the reported chromatographic method (5). In addition, content uniformity testing was conducted according to USP guidelines (19).
Experimental Instruments All fluorescence measurements were made using an RF-1501 Shimadzu spectrofluorometer, equipped with a 150 W xenon arc lamp (Kyoto, Japan). The excitation and emission wavelengths were 242 and 400 nm, respectively. The slit widths were 5 nm for both excitation and emission wavelengths, and the photomultiplier voltage was set to auto. A 1 cm quartz cuvette was used. A Consort NV P-901 pH meter (Belgium, Europe) was used for pH measurements.
Construction of the calibration graph. Aliquots of methanolic AMX standard solution were transferred to a series of 10 ml volumetric flasks to give final concentrations of 5.0–80.0 ng/mL. One milliliter of 0.2 M borate buffer (pH 10) was added to each flask followed by 0.6 ml of 0.5% w/v SDS solution. The volume was completed with methanol, the contents of the flasks were mixed well and the fluorescence intensity was measured at 400 nm after excitation at 242 nm. Relative fluorescence intensity (RFI) was plotted against final drug concentration (ng/mL) to give a calibration graph. Alternatively, the corresponding regression equation was derived.
Twenty Aphthtab® tablets were crushed and a weight equivalent to 10.0 mg AMX was transferred quantitively into a 100 ml volumetric flask, ~ 80 ml of methanol was added and the flasks were sonicated for 30 min. Dissolution of the tablets was enhanced with the aid of a magnetic stirrer. The solutions were then diluted to volume with methanol, mixed and filtered. Aliquots covering a working concentration range of 5.0–80.0 ng/mL were transferred into 10 ml volumetric flasks. The procedure described under ‘Construction of the calibration graph’ was performed. The nominal contents of the tablets were calculated using the calibration graph or the corresponding regression equation. Analysis of AMX in aphthtab® tablets.
Content uniformity testing of aphthtab tablets. Ten different tablets were analyzed using the procedure applied to the analysis of the studied compound in tablets. The uniformity of the tablet contents was tested by applying the official USP guideline [see Chapter 905 (19)].
Reagents and materials
Results and discussion
All chemicals used were of analytical reagents (AR) grade, and the solvents were HPLC grade. AMX lot # 080112G (certified to have a purity of 98%) was purchased from AK Scientific, Inc. (Union City, CA, USA) and was used as received without further purification. Aphthtab® tablets labeled as containing 2 mg AMX per tablet (batch # 203369) were from EVA Pharma for Pharmaceuticals and Medical Appliances (Giza, Egypt). The following surfactants were used as 0.5% (w/v) aqueous solutions: SDS (95%) and cetyl trimethylammonium bromide (CTAB; 99%) (London, England); β-cyclodextrin (β-CD) and hydroxy propyl-β-cyclodextrin (HP-β-CD) (Darmstadt, Germany); carboxymethylcellulose (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt); Tween-80 (El-Nasr Pharmaceutical Chemicals Co.); methanol (Paris, France), n-propanol and acetonitrile (SigmaAldrich Chemie GmbH, Germany); dimethyl sulfoxide (RiedeldeHäen, Sleeze, Germany); glacial acetic acid, sodium acetate trihydrate, boric acid, sodium hydroxide and dimethyl formamide (El-Nasr Pharmaceutical Chemical Co.). Acetate buffer (0.2 M, pH 3.5–5.5) and borate buffer (0.2 M, pH 6.5–10.5) solutions were freshly prepared.
AMX is formulated in a very minute amount in tablets (2 mg/tablet); therefore, there is an urgent need to explore a sufficiently sensitive and specific method for its determination in tablets. Fluorimetric analysis, by virtue of its high inherent sensitivity, might successfully overcome this problem. A methanolic solution of AMX was found to exhibit three emission bands of different intensities at 400 nm, after excitation at 242, 281 or 350 nm. The highest sensitivity was achieved upon using 242 nm as the excitation wavelength, and so this value was selected. It is well known that, in many cases, the addition of a surfactant at a concentration above its CMC to a given fluorophore solution increases its molar absorbtivity and/or the fluorescence quantum yield (20,21). This has been used to improve the performance of spectrofluorimetric methods for various analytes. The fluorescence properties of AMX in various micellar media were studied; there was significant enhancement of the fluorescence intensity in the presence of SDS compared with a methanolic solution, therefore SDS was used as a fluorescence enhancer to develop a new sensitive spectrofluorimetric method for the determination of AMX.
Standard solutions
Fluorescence spectra of AMX in methanolic solution and in the SDS system
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Stock solution equivalent to μg/mL of AMX was prepared by dissolving 10.0 mg of the drug in 100 ml of methanol. This solution was further diluted using the same solvent as appropriate. Standard solutions were stable for 10 days when kept in the refrigerator.
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The fluorescence spectra of AMX in both the methanolic and SDS systems were studied. Figure 2 illustrates the fluorescence spectra of AMX in the two systems. The first system was methanolic borate buffer (pH 10) and the second was the same solution
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Spectrofluorimetric determination of amlexanox
a
b Figure 2. (a) Fluorescence spectra of: (A, B) AMX (60 ng/mL) in methanolic borate buffer (pH 10). Where: A = excitation band; B1 = emission band at λex = 242 nm; B2 = emission band at λex = 281 nm; B3 = emission band at λex = 350 nm. (b) Fluorescence spectra of: (A, B) AMX (60 ng/mL) in methanolic borate buffer (pH 10) (SDS system); (A′, B′) blank. Where: A, A′ = excitation spectra; B, B′ = emission spectra.
but in the presence of SDS as a fluorescence enhancer. The percentage fluorescence enhancement in the presence of SDS was significant when compared with the native fluorescence intensity of the drug in a methanolic medium.
Optimization of the experimental conditions Effect of organized media. The effect of various organized media on the fluorescence intensity of AMX was studied by adding 1.0 ml of a 0.5% (w/v) aqueous solution of each to the drug solution. Different surfactants such as SDS (anionic surfactant), CTAB (cationic surfactant) and Tween-80 (nonionic surfactant) and also macromolecules like β-CD and HP-β-CD were studied. All the organized media studied caused a slight decrease in the RFI of the drug (Fig. 3). Only SDS gave a considerable increase in the RFI, so it was used throughout the present study.
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Figure 3. Effect of different organized media (1 ml of a 0.5% w/v solution of each) on the RFI of AMX (60 ng/mL).
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Effect of the volume of SDS. The influence of SDS on the RFI was studied using increasing volumes of 0.5% w/v SDS aqueous solution. It was found that increasing volumes of SDS solution resulted in a corresponding increase in RFI up to 0.4 ml, after
M. I. Walash et al. which no further increase in RFI was obtained. Therefore, 0.6 ml of a 0.5% w/v SDS solution was chosen as the optimum volume for AMX (Fig. 4). An SDS volume > 1 ml was not used because of a high blank reading. Effect of pH. The influence of pH on the micelle-enhanced fluorescence of AMX was studied using different types of buffers covering the whole pH range, for example, 0.2 M acetate buffer over the pH range 3.5–5.5 and 0.2 M borate buffer over the pH range 6.5–10.5. It was found that maximum and constant RFI was achieved over a pH range of 9.5–10.5; therefore, pH 10 was chosen as the optimum value for the study. Use of 0.2 M acetate buffer caused a gradual decrease in the RFI of AMX (Fig. 5). Effect of diluting solvent. The effect of dilution with different solvents such as distilled water, methanol, acetonitrile, n-propanol, dimethyl sulfoxide and dimethyl formamide was studied. It was found that methanol was the best solvent for dilution, as it gave the highest RFI and lowest blank reading Table 1. A distinct and sharp decrease in RFI was observed on using acetonitrile or n-propanol. Addition of these solvents may result in a reduction in the size of the micelles and a progressive breakdown of the surfactant aggregate at very high concentrations (22). Both dimethyl sulfoxide and dimethyl formamide gave high blank readings and greatly quenched the fluorescence intensities of AMX because they exhibited an intersystem-crossing process (similar to the heavy atom effect) (23).
Table 1. Effect of diluting solvents on the fluorescence intensity (FI) of AMX (60 ng/mL) Solvent
FI
Water Methanol n-propanol Acetonitrile Dimethyl sulfoxide Dimethyl formamide
420 660 365 340 135 50
Effect of time. The effect of time on the RFI of the drug was also studied. It was found that the fluorescence intensity developed immediately and remained stable for more than 2 h. Effect of temperature. Another factor that may affect the fluorescence intensity is temperature. The effect of temperature was studied in the range 40–100 °C in a thermostatically controlled water bath. It was found that increasing the temperature resulted in a decrease in the RFI. This can be explained by higher internal conversion as the temperature increases, facilitating nonradiative deactivation of the excited singlet state and an increase in the loss of energy via collision with solvent molecules (24). Therefore, all experiments were carried out at room temperature. Method validation The validity of proposed method was evaluated regarding linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, robustness and specificity. Linearity and range. The calibration graph for the determination of AMX using the proposed method was constructed by plotting the RFI against the concentration of the drug (ng/mL). The calibration graph showed a linear dependence of RFI on drug concentration over a range of 5.0–80.0 ng/mL for AMX. Linear regression analysis of the data gave the following equation: RFI ¼ 44:73 þ 9:82C
Figure 4. Effect of the volume of SDS (0.5% w/v) on the RFI of AMX (60 ng/mL).
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Figure 5. Effect of pH on the RFI of AMX (60 ng/mL).
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Where RFI is the relative fluorescence intensity and C is the concentration of the drug in ng/mL for AMX. Statistical analysis (25) of the data gave high values for the correlation coefficients (r) of the regression equations, small for the standard deviation of residuals (Sy/x), of intercept (Sa) and of slope (Sb), and small value for the percentage relative standard deviation (%RSD) and the percentage relative error Table 2. These data proved the linearity of the calibration graph of the studied drug. LOQ and LOD. LOQ and LOD were calculated according to the ICH Q2 (R1) recommendations (26). LOQ values were determined by establishing the measurable concentrations below which the calibration graph is nonlinear. LOD values were determined by evaluating the lowest analyte concentrations that can be readily detected. The results are also summarized in Table 2. The values of LOQ and LOD were calculated according to the following equations (26):
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Spectrofluorimetric determination of amlexanox significant difference between the performance of the methods regarding accuracy and precision Tables 3 and 4. The comparison HPLC method involved the use of a mobile phase consisting of methanol : 0.01 M phosphate buffer (70:30, v/v; pH 4.8) and an Octadecylsilane (ODS) column with UV detection at 350 nm (5).
Table 2. Analytical performance data for the spectrofluorimetric determination of AMX Parameter
AMX
Wavelength [λex/λem] (nm) Concentration (ng/mL) Intercept (a) Slope (b) Correlation coefficient (r) SD of residuals (Sy/x) SD of intercept (Sa) SD of slope (Sb) % RSD LOD (ng/mL) LOQ (ng/mL)
242/400 5.0–80.0 44.73 9.82 0.9999 2.64 1.70 0.04 0.80 0.57 1.74
Precision. Intraday precision was evaluated by determining three concentrations of AMX in its pure form on three successive occasions over the course of one day. Interday precision was also evaluated by replicate analysis of three concentrations over a period of three successive days. The results of intraday and interday precision are summarized in Table 4. Robustness of the method. The robustness of the adopted method was demonstrated by the constancy of the RFI with minor changes in the experimental conditions, such as a change in the pH (10 ± 0.2) and SDS volume (0.6 ± 0.2 ml). These minor changes that may occur during the experimental operation did not affect the RFI.
LOQ ¼ 10Sa =b LOD ¼ 3:3Sa =b Where Sa is the standard deviation of the intercept of the regression line and b is the slope of the calibration graph. Accuracy. To prove the accuracy of the proposed method, the results of the drug assay were compared with those obtained using a comparison HPLC method (5). Statistical analysis (25) of the results obtained from the proposed and comparison methods using Student’s t-test and variance ratio F-test showed no
Specificity. The specificity of the method was investigated by observing any interference encountered from common tablet excipients. It was shown that these compounds did not interfere with the results of the proposed method (Table 3). Pharmaceutical applications Application of the proposed method to the analysis of AMX in bioadhesive buccal tablets. The proposed method was successfully applied to the assay of AMX in its conventional tablets. Percent recoveries of different concentrations were based
Table 3. Assay results for the determination of AMX in raw material and its bioadhesive buccal tablets using the proposed and comparison methods Compound
Comparison method[5]
Proposed method Amount taken (ng/mL)
AMX
Mean ± SD % RSD t F Aphthtab tabletsb (AMX 2 mg/ tablet) Mean ± SD t F
5.0 10.0 20.0 30.0 40.0 50.0 60.0 80.0
20.00 40.00 60.00
Amount found (ng/mL) 4.915 10.006 19.883 29.963 40.247 50.430 59.594 79.958
19.776 39.792 60.120
% Found
Amount taken (μg/mL)
98.31 100.07 99.42 99.88 100.62 100.86 99.32 99.95 99.80 ± 0.80 0.80 0.89 (2.26) 1.17 (4.74) 98.88 99.48 100.20 99.52 ± 0.66 1.05 (2.78) 4.53 (19.0)
6.0 8.0 10.0
Amount found (μg/mL) 5.9670 8.0200 10.118
% Found 99.45 100.25 101.18
100.29 ± 0.87 0.86
6.0 8.0 10.0
5.949 8.156 10.030
99.15 101.95 100.30 100.47 ± 1.41
a
Average of three separate determinations. Labeled to contain 2 mg AMX/tablet (batch # 203369), product of EVA Pharma for Pharmaceuticals and Medical Appliances, Giza, Egypt. c Values in parentheses are the tabulated t and F values at P = 0.05 (25). b
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M. I. Walash et al. Table 4. Precision data for the determination of AMX using the proposed method Parameter
AMX (ng/mL) 10.0
Intra-day
% Found
Inter-day
Mean (SD) % RSD % Found
Mean (SD) % RSD
100.35 101.20 99.45 100.33 (0.88) 0.87 100.48 98.78 101.82 100.36 (1.53) 1.52
Table 5. Results of content uniformity testing of AMX tablets using the proposed method Parameter Data
Mean (SD) % RSD % Error Acceptance value (AV) (19) Max. allowed AV (L1) (19)
Tablet no.a 1 2 3 4 5 6 7 8 9 10
Percentage of the label claim 98.85 100.72 101.45 101.35 99.64 100.18 99.28 97.92 102.55 101.98 100.39 (1.48) 1.47 0.47
3.55 15.0
a
Aphthtab tablets: labeled to contain 2 mg AMX per tablet (batch # 203369), product of EVA Pharma for Pharmaceuticals and Medical Appliances, Giza, Egypt.
on the average of three replicate determinations. The results were in good agreement with those obtained using the comparison HPLC method (5). Content uniformity testing for tablets. Because of the high sensitivity of the proposed method and its ability to rapidly measure the fluorescence intensity of a single tablet extract with sufficient accuracy, it is ideally suited for content uniformity testing, which is a time-consuming process when using conventional assay techniques. The steps of the test were adopted according to the USP (19) procedure. The acceptance value (AV) was calculated and found to be smaller than the maximum allowed AV (L1). The results demonstrated excellent drug uniformity, as shown in Table 5.
Conclusion
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A simple and sensitive spectrofluorimetric method was developed for the determination of AMX through enhancement of
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20.0 99.58 101.45 101.10 100.71 (0.99) 0.99 99.30 101.55 99.76 100.20 (1.19) 1.19
40.0 99.82 100.45 99.30 99.86 (0.58) 0.58 98.60 99.95 101.20 99.92 (1.30) 1.30
its native fluorescence upon using SDS. The proposed method is rapid, time-saving and does not require elaborate treatment compared with the reported chromatographic methods. By virtue of its simplicity and sensitivity, the proposed method can be applied to the analysis of Amlexanox for the first time in its bioadhesive buccal tablets, and moreover, in quality control laboratories. The proposed method is suitable for use in content uniformity testing. Acknowledgements The authors extend their appreciation to Analytical Chemistry Department Mansoura University for providing instruments and chemicals. Also Ak Scientific company and EVA Pharma company for kindly providing pure powder and tablets of amlexanox.
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