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Simultaneous determination of econazole nitrate, main impurities and preservatives in cream formulation by high performance liquid chromatography
Talanta 77 (2008) 673–678
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Simultaneous determination of econazole nitrate, main impurities and preservatives in cream formulation by high performance liquid chromatography Angel Arturo Gaona-Galdos ∗ , Pedro López García, María Segunda Aurora-Prado, Maria Inês Rocha Miritello Santoro, Érika Rosa Maria Kedor-Hackmann Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580, 05508-900 São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history: Received 16 April 2008 Received in revised form 3 July 2008 Accepted 7 July 2008 Available online 15 July 2008 Keywords: Econazole nitrate Impurities Preservatives Gradient method
a b s t r a c t A reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of econazole nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-chlorobenzyl alcohol and alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol) in cream formulations, has been developed and validated. Separation was achieved on a column Bondclone® C18 (300 mm × 3.9 mm i.d., 10 m) using a gradient method with mobile phase composed of methanol and water. The flow rate was 1.4 mL min−1 , temperature of the column was 25 ◦ C and the detection was made at 220 nm. Miconazole nitrate was used as an internal standard. The total run time was less than 15 min. The analytical curves presented coefficient of correlation upper to 0.99 and detection and quantitation limits were calculated for all molecules. Excellent accuracy and precision were obtained for econazole nitrate. Recoveries varied from 97.9 to 102.3% and intra- and inter-day precisions, calculated as relative standard deviation (R.S.D.), were lower than 2.2%. Specificity, robustness and assay for econazole nitrate were also determined. The method allowed the quantitative determination of econazole nitrate, its impurities and preservatives and could be applied as a stability-indicating method for econazole nitrate in cream formulations. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Most fungal infections involve superficial invasion of skin or the mucous membranes of body orifices. Some species under certain conditions are capable of invading deeper body cavities and causing systemic mycoses. Such infections may become serious and occasionally life-threatening, and are frequently difficult to treat. The treatment of systemic mycoses is becoming very important in recent years as a result of the increased incidence of opportunistic yeast infections in immunocompromised patients. The widespread use of immunosuppressants following organ transplant operations and AIDS has been major contributors to this situation [1]. The azoles represent a class of versatile antifungal agents. In general, the azoles are effective against most fungi that cause superficial infections of the skin and mucous membranes. At high concentrations the azoles are fungicidal; at low concentrations they
∗ Corresponding author at: Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580 B-13, Butantã, 05508900 São Paulo, SP, Brazil. Tel.: +55 11 3091 3655; fax: +55 11 3815 4418. E-mail address: [email protected] (A.A. Gaona-Galdos). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.07.010
are fungistatic. The fungicidal effect is associated with damage to the fungi cell membrane, with the loss of essential cellular constituents. The fungistatic effects of the azoles have been correlated with the inhibition of membrane-bound enzymes by low concentration of the azoles [1]. Econazole nitrate (1-[2-(4-chlorophenyl)methoxy]-2-(2,4dichlorophenyl)ethyl)-1H-imidazole mononitrate (Fig. 1EN) is a potent broad-spectrum antifungal agent used topically in the treatment of skin infections. Further, results indicate that econazole could replace rifampicin/isoniazid as well as both rifampicin and isoniazid in chemotherapy of murine tuberculosis. Econazole alone or in combination with antitubercular drugs did not produce any hepatotoxicity in normal or Mycobacterium tuberculosis-infected mice [2,3]. Reported methods for the determination of econazole nitrate in pharmaceutical formulations include titrimetry [4], spectrophotometry [5,6], derivative spectrophotometry [7,8], high performance liquid chromatography (HPLC) [9–12], gas chromatography [13], high performance thin-layer chromatography [14] and capillary electrophoresis (CE) [15]. A variety of enantiomeric separations using HPLC and CE methods were also published [16–25]. Quantification of EN in biological samples has been developed using
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Fig. 1. Chemical structures of econazole nitrate (EN), alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (DCE), 4-chlorobenzyl alcohol (CBA), methylparaben (MP), propylparaben (PP) and miconazole nitrate (MN).
near infrared spectrometry [26,27] and liquid chromatography [28]. A stability-indicating assay method validated for econazole nitrate is described in the literature [29], showing specificity for its degradation products (4-chlorobenzyl alcohol and alpha-(2,4dichlorophenyl)-1H-imidazole-1-ethanol), as well as the inactive ingredients, but not for preservatives because they were not present in the formulation (methylparaben and propylparaben). This chromatographic separation was achieved with isocratic elution on a RP-18 column using methanol/aqueous ammonium carbonate solution/tetrahydrofurane as the mobile phase and miconazole nitrate as an internal standard. Nevertheless, this method allows only the quantitative determination of econazole nitrate. A pharmaceutical impurity is a component that is not the chemical entity defined as the drug substance or an excipient in the drug product. For this reason the safety of pharmaceuticals is dependent not only on the intrinsic toxicological properties of the active ingredient and excipients in the drug product, but also in part upon the impurities that it may contain [30]. Therefore, identification, quantification and control of impurities in the drug substance and drug product are important in the drug development. Preservatives would be effective at low concentrations against all possible microorganisms, nontoxic and compatible with other constituents used in the preparation. Esters of p-hydroxybenzoic acid (parabens) have antifungal properties. Their toxicity is generally low, owing to rapid in vivo hydrolysis to p-hydroxybenzoic acid, which is rapidly conjugated and excreted [1]. The present methodology in comparison with the methods described in the literature, shows a simple sample preparation and mobile phase composition and the last containing methanol–water without buffers. This proposed method allows the simultaneous determination of econazole nitrate, its impurities and preservatives in creams formulations with good resolution and peak symmetry.
2. Experimental 2.1. Instrumentation The gradient HPLC method was performed on a chromatographic system, consisted of a solvent delivery pump system model LC-10AD (Shimadzu® Corporation, Japan), an auto injector fitted with 20 L loop model SIL-10AD (Shimadzu® Corporation, Japan), an online degasification system model DGU-14A (Shimadzu® Corporation, Japan), a column thermostat oven model CTO-10AS (Shimadzu® Corporation, Japan) and an UV–VIS photodiode array detector model SPD-M10A (Shimadzu® Corporation, Japan). The output signal was monitored and integrated using CLASS VP® software v.5.91 (Shimadzu® Corporation, Japan). A reversed-phase C18 Column, Bondclone® (300 mm × 3.9 mm i.d., 10 m) PhenomenexTM California, USA, was used for separation. 2.2. Chemicals The reagents were of analytical grade. Methanol (HPLC grade), obtained from Merck (Darmstadt, Germany). Water was deionized and purified on a Milli-Q® water purification system (Millipore, Bedford, MA, USA) and used to prepare all solutions. 2.3. Chromatographic conditions The mobile phase was methanol–water. The analysis was carried out in a gradient elution mode with 57% methanol at 0 min gradually increased to 72% at 6.5 min, then increased to 98% at 10 min, from 10.01 to 15 min 98% using a flow rate of 1.4 mL min−1 at 25 ◦ C. Before delivering into the system the solvent was filtered through 0.45 m, HV membrane and degassed. The chromatograms were recorded at 220 nm.
Table 1 Development of solvent gradient systems System 1 T (min)
Methanol
Flow rate (mL min−1 )
System 2 %
0 45
55 100
1.0
T (min) 0 35
1.2
System 3
System 4
System 5
System 6
%
T (min)
%
T (min)
%
T (min)
%
T (min)
%
55 100
0 13 15 23
55 72 90 95
0 13 15 23
55 72 90 95
0 10 14 19
57 72 97 97
0 6.5 10 15
57 72 98 98
1.2
1.4
1.4
1.4
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Fig. 2. Gradient developing systems for methylparaben (MP), 4-chlorobenzyl alcohol (CBA), propylparaben (PP), alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (DCE), econazole nitrate (EN) and miconazole nitrate (MN). (Systems as in Table 1.)
2.4. Standards Econazole nitrate (100.55% purity) (Fig. 1EN) and miconazole nitrate (100.32% purity) (Fig. 1MN) were kindly donated by Formil Química Ltda. (São Paulo, Brazil). Impurities of econazole nitrate: alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (98% purity) (Fig. 1DCE) and 4-chlorobenzyl alcohol (99% purity) (Fig. 1CBA) ¯ Organics (Geel, Belgium). Preservawere purchased from Acros tives: methylparaben (Fig. 1MP) and propylparaben (Fig. 1PP) were donated by Laboratório Stiefel Ltda. (São Paulo, Brazil). 2.5. Sample The cream sample was supplied by Laboratório Stiefel Ltda. (São Paulo, Brazil) containing 1% (w/w) of econazole nitrate.
For the preparation of sample solution an amount equivalent to about 0.25 g of cream was accurately weighed into a 25-mL beaker and dissolved in 10 mL of a mixture of methanol–water (50:50, v/v) in a water-bath at 60 ◦ C for 5 min. After this period, the solution was quantitatively transferred to a 25-mL volumetric flask. A 2.5mL aliquot of the miconazole nitrate solution (1000 g mL−1 , stock solution) was transferred into a 25-mL volumetric flask. Then the solution was homogenized, cooled to room temperature and the volume was completed to 25 mL with the mixture methanol–water. The sample solution was then filtered using blue strip filter paper (Schleicher & Schull, Germany). Before injection on chromatograph, the solution was filtered through a 0.22 m filter (Millex PTFE, Millipore® ). Final concentrations of econazole nitrate and miconazole nitrate solutions were 100 g mL−1 . 2.7. Method validation
2.6. Preparation of standard and sample solutions (1000 g mL−1 ),
Standard stock solutions of econazole nitrate miconazole nitrate (1000 g mL−1 ), alpha-(2,4-dichlorophenyl)1H-imidazole-1-ethanol (20 and 40 g mL−1 ), 4-chlorobenzyl alcohol (20 and 40 g mL−1 ), methylparaben (500 g mL−1 ) and propylparaben (200 g mL−1 ) were prepared in methanol. The solutions were stored under refrigeration. Working standard solutions were prepared fresh daily by diluting appropriately the stock solutions with the same solvent.
The method was validated according to the United States Pharmacopeia requirements [31]. The following validation charac-
Table 2 Chromatographic parameters Compounds
MP CBA PP DCE EN MN
Parameters tR (min)
K
˛
Rs
4.03 5.00 7.11 7.85 13.25 13.83
1.47 2.06 3.40 3.81 7.12 7.48
MP/CBA = 1.40 CBA/PP = 1.65 PP/DCE = 1.12 DCE/EN = 1.87 EN/MN = 1.05
2.31 4.92 1.47 12.46 2.25
MP: methylparaben; CBA: 4-chlorobenzyl alcohol; PP: propylparaben; DCE: alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol; EN: econazole nitrate; MN: miconazole nitrate; tR : retention time; ˛: separation factor; K: retention factor; Rs : resolution.
Fig. 3. Chromatogram of standard solutions. Peaks: methylparaben (MP), 4-chlorobenzyl alcohol (CBA), propylparaben (PP), alpha-(2,4-dichlorophenyl)-1Himidazole-1-ethanol (DCE), econazole nitrate (EN) and miconazole nitrate (MN). Conditions: Bondclone® C18 Column, 300 mm × 3.9 mm i.d., 10 m; mobile phase: gradient elution starting with 57%, v/v methanol–water; temperature: 25 ± 1 ◦ C; flow rate: 1.4 mL min−1 ; UV detection at 220 nm.
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Table 3 Linearity, detection and quantitation limits determined for the proposed RP-HPLC method for all compounds Statistical parameter −1
Concentration range (g mL ) Intercept Slope Correlation coefficient (r2 ) Residual S.D. of the regression line () DL (g mL−1 ) QL (g mL−1 ) F
EN
MP
PP
CBA
DCE
70–120 0.0834 0.0101 0.9977 0.0144 4.71 14.28 857.57
30–55 0.0192 0.0066 0.9977 0.0047 2.35 7.13 861.19
8–18 0.0031 0.0050 0.9991 0.0009 0.57 1.72 2356.78
0.2–6.2 −0.0015 0.0144 0.9991 0.0015 0.35 1.06 2220.68
0.2–6.2 −0.0011 0.0086 0.9991 0.0009 0.35 1.05 2278.52
EN: econazole nitrate; MP: methylparaben; PP: propylparaben; CBA: 4-chlorobenzyl alcohol; DCE: alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol; DL: detection limit; QL: quantitation limit; F-test tabulated(0.05,1.4) = 7.71.
teristics were addressed to: linearity, detection and quantitation limits, precision, accuracy, robustness, system suitability, selectivity, assay and specificity. 2.7.1. Linearity, detection and quantitation limits, precision and accuracy Appropriate aliquots of stock solutions were transferred into 10 mL volumetric flasks and diluted to volume with methanol. Concentration range from 70 to 120 g mL−1 for econazole nitrate, 30–55 g mL−1 for methylparaben, 8–18 g mL−1 for propylparaben, and 0.2–6.2 g mL−1 for degradation products (4-chlorobenzyl alcohol and alpha-(2,4-dichlorophenyl)-1Himidazole-1-ethanol) were obtained. Then, the solutions were filtered using a 0.22-m filter (Millex PTFE, Millipore® ) and injected on the HPLC instrument. Each solution was injected in triplicate. Peak area ratios (compound/miconazole nitrate) were plotted versus the respective compound concentrations. Detection (DL) and quantitation limits (QL) were calculated from the residual standard deviation of the regression line () of the analytical curve and its slope (S) in accordance with the equations DL = 3.3 (/S) and QL = 10 (/S) [32]. In order to measure repeatability of the system (while keeping the operating conditions identical), 20 consecutive injections were made using a standard solution containing 80 g mL−1 of econazole nitrate and 100 g mL−1 of miconazole nitrate (IS). The results were expressed as the percentage relative standard deviation (%R.S.D.) for peak area ratio (PAR) of econazole nitrate/miconazole nitrate and retention time of econazole nitrate. For the determination of repeatability sample solutions were prepared at 100 g mL−1 of econazole nitrate and 100 g mL−1 of miconazole nitrate. Ten determinations were performed to establish the intra-day precision. The intra-day precision was evaluated by injecting sample solutions prepared at lower, middle and higher concentrations of the analytical curve (80–120 g mL−1 econazole nitrate) containing 100 g mL−1 of miconazole nitrate, in 1 day. The inter-day precision was evaluated by injecting the same solutions on three consecutive days. Three determinations for each concentration were performed. Precision was expressed as the %R.S.D. for peak area ratio of econazole nitrate/miconazole nitrate. The accuracy was calculated as the percentage recovery of a known amount of standard added to the sample. The accuracy of method was evaluated in triplicate using three concentration levels 80, 100 and 120 g mL−1 . Econazole nitrate standard solution was added to commercial sample solution and analyzed by the proposed method. 2.7.2. Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters, and provides an indication of its
reliability during normal usage [33]. In order to the robustness study of the proposed method, deliberate modifications in temperature–wavelength values were made. Thus, three temperatures values were selected, one below and one above of the chosen temperature. The same was made with wavelength. 2.7.3. Specificity The specificity of the method for econazole nitrate was tested by analyzing a mixture of the inactive ingredients (placebo), the commercial samples of econazole nitrate and a mixture of standard solutions. 2.7.4. Selectivity The selectivity of the method was established through study of retention time, separation factor, retention factor, resolution of all peaks and the absorption spectra of the eluted peaks. 3. Results and discussion 3.1. Method development In order to develop a simple HPLC method for quantitative determination of EN, its impurities and preservatives in cream formulations, different solvent systems were evaluated (acetonitrile, methanol and isopropanol), each one in different proportions. Rate gradients and flow rates also were tested to achieve efficient separation with a satisfactory resolution in a short time of analysis. Both solvents acetonitrile and isopropanol do not provided satisfactory
Fig. 4. Chromatograms of placebo (A), commercial sample containing econazole nitrate (B) and standard solutions(C). Conditions: Bondclone® C18 Column, 300 mm × 3.9 mm i.d., 10 m; mobile phase: gradient elution starting with 57%, v/v methanol–water; temperature: 25 ± 1 ◦ C; flow rate: 1.4 mL min−1 ; UV detection at 220 nm.
A.A. Gaona-Galdos et al. / Talanta 77 (2008) 673–678
chromatographic profiles (overlaps in degradation products peaks and inadequate drift). Several gradient systems were tested with the mobile phase, methanol–water (Table 1). The optimization of the methodology had targets as an adequate analysis time, avoid a pronounced rise of drift and an adequate resolution factor of all compounds separated. To reduce time analysis it was determined the elapsed time between the increase in the composition of mobile phase and its passage through the detector. In order, to avoid the drift elevation, it was also determined the adequate time in which the percentage of methanol increases from 72 to 98% in 3.5 min, since a smaller time produced an abrupt rise in the baseline. On the other hand, a greater time does not cause significant difference. To achieve an adequate resolution between all eluted peaks, it was calculated the resolution factor, which was higher than 1.45 for all the substances. Time analysis was significantly reduced in the system 6 in comparison with the others tested systems. Fig. 2 shows the retention times for the 6 compounds in each one of the systems. The gradient developed system 6 was the one who provides the best results in time, resolution and symmetry of the peaks, as depicted in Fig. 3. Other parameters are shown in Table 2. 3.2. Method validation Analytical curves were obtained by plotting peak area ratios (compound/IS) against the concentrations of respective substances. In all cases, straight regression lines with correlation coefficients (r) above 0.997 were obtained. F-test was applied for all calibration curves and the data provide conclusive evidence of a linear effect between concentration and instrumental response [34]. Data are summarized in Table 3. The DL and QL were calculated using analytical curves results (Table 3). DL and QL values were obtained by injecting 20 L of standard solutions in the chromatographic system.
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Table 4 Intra- and inter-day precision of the proposed RP-HPLC method for econazole nitrate quantitative determination 80 g mL−1
100 g mL−1
120 g mL−1
Intra-day (n = 3) %R.S.D.
2.20
0.81
1.56
Inter-day (n = 9) %R.S.D.
0.52
1.27
0.93
Injection precision was determined after injecting 20 times in the chromatographic system an econazole nitrate standard solution containing 80 g mL−1 . The results obtained for PAR were, %R.S.D. 0.25 and for retention time, %R.S.D. 0.04. The values obtained demonstrate that the system is reliable for analysis. The precision of the method was evaluated by repeatability and intermediate precision determinations. For repeatability, 10 sample solutions at 100 g mL−1 were analyzed in the same day and the R.S.D. obtained was 1.74%. The intermediate precision was achieved by analyzing three different concentrations on three consecutive days. The one-way ANOVA was used to estimate the total variability within and between days. The results, which, are shown in Table 4 present good agreement. The accuracy of the method was evaluated at three concentration levels. Triplicate determinations were made at each concentration level. The accuracy is expressed as percentage of standard recovered from sample matrix as R.S.D. The results are shown in Table 5. For robustness determination, changes in the temperature and wavelength were evaluated. About 1.8% of difference was observed in the more critical result when the analytical parameters were modified and compared with the original conditions. The specificity of the method was demonstrated by the absence of interference among econazole nitrate, methylparaben, propy-
Fig. 5. Chromatogram of separation of standard solutions and spectra for all compounds. Peaks: methylparaben (MP), 4-chlorobenzyl alcohol (CBA), propylparaben (PP), alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (DCE), econazole nitrate (EN) and miconazole nitrate (MN). Conditions: Bondclone® C18 Column, 300 mm × 3.9 mm i.d., 10 m; mobile phase: gradient elution starting with 57%, v/v methanol–water; temperature: 25 ± 1 ◦ C; flow rate: 1.4 mL min−1 ; UV detection at 220 nm.
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Table 5 Recovery of a standard solution of econazole nitrate added to sample and determined using the proposed HPLC method
Acknowledgements
Standard added to commercial samplea (g mL−1 )
Standard found (g mL−1 )
Recovery (%)b
The authors thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico of Brazil (CNPq, 130652/2007-5; CNPq, 142664/2005-7) for fellowship.
40.00 50.00 60.00
39.15 51.13 60.68
97.88 102.26 101.13
References
a b
Commercial sample (econazole nitrate cream). Average of three determinations.
lparaben, alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol, 4chlorobenzyl alcohol, miconazole nitrate and excipients in the samples, using the criteria defined in the USP 30 for assays [31]. A mixture of the inactive ingredients (placebo) (Fig. 4A), the commercial sample of econazole nitrate (Fig. 4B) and a standard mixture solution (Fig. 4C), were analyzed by the proposed methodology. As it can be observed, neither the cream excipients nor preservatives and the impurity interfere in the analysis of EN. Two preservative substances present in the formulation and impurities of econazole nitrate were used to evaluate the selectivity of the method. Results are showed in Table 2. The absorption spectra of the eluted peaks were achieved using a photodiode array detector and then compared with those of the reference standards. The results showed equivalent spectrophotometric profiles (Fig. 5). For the assay, a sample was analyzed in triplicate and the average obtained was 105.50%. The British Pharmacopoeia [35] established a range between 90 and 110%. The tested sample using the proposed method, presented satisfactory results. System suitability test is an important part of liquid chromatographic method. It is used to verify if the chromatographic system is adequate and reliable. Data from 5 injections of a solution containing 80 g mL−1 of econazole nitrate standard solutions were analyzed. The R.S.D. was 0.09%. This result agrees with those specified in the United States Pharmacopeia [31]. 4. Conclusion The validated method is rapid and efficient, and allows the separation of econazole nitrate in the presence of its degradation products, impurities and excipients, without using buffers or pH modifier in the mobile phase. Since it was possible to identify and quantify econazole nitrate impurities as (4-chlorobenzyl alcohol and alpha-(2,4dichlorophenyl)-1H-imidazole-1-ethanol), the proposed method can be used as a stability-indicating method for this drug in pharmaceutical formulations.
[1] A.R. Martin, in: J.N. Delgado, W.A. Remers (Eds.), Wilson and Davold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry, Lippincot Williams & Wilkins, England, 1998, pp. 173–221. [2] A. Zahoor, S. Sharma, G.K. Khuller, FEMS Microbiol. Lett. 258 (2006) 200. [3] A. Zahoor, S. Sharma, G.K. Khuller, FEMS Microbiol. Lett. 261 (2006) 181. [4] M. Massaccesi, Analyst 111 (1986) 987. [5] S.R. El-Shabouri, K.M. Emara, P.Y. Khashaba, A.M. Mohamed, Anal. Lett. 31 (1998) 1367. [6] P.Y. Khashaba, S.R. El-Shabouri, K.M. Emara, A.M. Mohamed, J. Pharm. Biomed. Anal. 22 (2000) 363. [7] D. Bonazzi, V. Cavrini, R. Gatti, E. Boselli, M. Caboni, J. Pharm. Biomed. Anal. 18 (1998) 235. [8] V. Cavrini, A.M. Di Pietra, R. Gatti, J. Pharm. Biomed. Anal. 7 (1989) 1535. [9] X.-L. Yang, Z.-F. Xi, J.-F. Sheng, Chin. J. Antib. 29 (2004) 403. [10] L. Gagliardi, D. De Orsi, P. Chimenti, R. Porra’, D. Tonelli, Anal. Sci. 19 (2003) 1195. [11] E. Kublin, T. Kaniewska, J. Pharm. Belg. 53 (1998) 208. [12] A.M. Di Pietra, V. Andrisano, R. Gotti, V. Cavrini, J. Pharm. Biomed. Anal. 14 (1996) 1191. [13] E. Kublin, T. Kaniewska, Chem. Anal. (Warsaw, Pol.) 41 (1996) 19. [14] G. Popovic, M. Cakar, K. Vucicevic, S. Vladimirov, D. Agbaba, J. Planar Chromatogr. Mod. TLC 17 (2004) 109. [15] A. Arranz, C. Echevarria, J.M. Moreda, A. Cid, J.F. Arranz, J. Chromatogr. A 871 (2000) 399. [16] L. Toribio, M.J. Del Nozal, J.L. Bernal, C. Alonso, J.J. Jimenez, J. Chromatogr. A 1144 (2007) 255. [17] X. Lin, C. Zhu, C. Hao, Electrophoresis 26 (2005) 3890. [18] I. Ali, H.Y. Aboul-Enein, Biomed. Chromatogr. 17 (2003) 113. [19] H.Y. Aboul-Enein, I. Ali, J. Pharm. Biomed. Anal. 27 (2002) 441. [20] H.Y. Aboul-Enein, I. Ali, Anal. Bioanal. Chem. 370 (2001) 951. [21] H.Y. Aboul-Enein, I. Ali, Chromatographia 54 (2001) 200. [22] A. Van Eeckhaut, S. Boonkerd, M.R. Detaevernier, Y. Michotte, J. Chromatogr. A 903 (2000) 245. [23] J.Y. Liu, Y.Y. Dong, T.S. Wang, H.W. Liu, A.J. Huang, Y.L. Sun, Z.P. Sun, Chin. Chem. Lett. 10 (1999) 39. [24] E. Peyrin, Y.C. Guillaume, Chromatographia 49 (1999) 691. [25] N. Morin, Y.C. Guillaume, J.C. Rouland, Chromatographia 48 (1998) 388. [26] J.P. Medendorp, K.S. Paudel, R.A. Lodder, A.L. Stinchcomb, Pharm. Res. 24 (2007) 186. [27] J. Medendorp, J. Yedluri, D.C. Hammell, T. Ji, R.A. Lodder, A.L. Stinchcomb, Pharm. Res. 23 (2006) 835. [28] P. Chiap, Ph. Hubert, J. Crommen, J. Chromatogr. A 948 (2002) 151. [29] R. Christinat, H.W. Zulliger, Arzneim. Forsch. 34 (1984) 551. [30] A.K. Basak, A.S. Raw, L.X. Yu, Adv. Drug Delivery Rev. 59 (2007) 1. [31] United States Pharmacopeia, 31st ed. The United States Pharmacopeial Convention, Rockville, 2008, pp. 683–687. [32] E. McEvoy, S. Donegan, J. Power, K. Altria, J. Pharm. Biomed. Anal. 44 (2007) 137. [33] International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, Topic Q2 (R1): Validation of Analytical Procedures: Text and Methodology, Geneva, 2005. [34] J.O. Rawlings, S.G. Pantula, D.A. Dickey, Applied Regression Analysis, 2nd ed., Springer, 1998, pp. 16–18. [35] British Pharmacopoeia. http://www.pharmacopeia.co.uk, 2008 (accessed 3/7/2008).
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Simultaneous determination of econazole nitrate, main impurities and preservatives in cream formulation by high performance liquid chromatography Angel Arturo Gaona-Galdos ∗ , Pedro López García, María Segunda Aurora-Prado, Maria Inês Rocha Miritello Santoro, Érika Rosa Maria Kedor-Hackmann Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580, 05508-900 São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history: Received 16 April 2008 Received in revised form 3 July 2008 Accepted 7 July 2008 Available online 15 July 2008 Keywords: Econazole nitrate Impurities Preservatives Gradient method
a b s t r a c t A reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of econazole nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-chlorobenzyl alcohol and alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol) in cream formulations, has been developed and validated. Separation was achieved on a column Bondclone® C18 (300 mm × 3.9 mm i.d., 10 m) using a gradient method with mobile phase composed of methanol and water. The flow rate was 1.4 mL min−1 , temperature of the column was 25 ◦ C and the detection was made at 220 nm. Miconazole nitrate was used as an internal standard. The total run time was less than 15 min. The analytical curves presented coefficient of correlation upper to 0.99 and detection and quantitation limits were calculated for all molecules. Excellent accuracy and precision were obtained for econazole nitrate. Recoveries varied from 97.9 to 102.3% and intra- and inter-day precisions, calculated as relative standard deviation (R.S.D.), were lower than 2.2%. Specificity, robustness and assay for econazole nitrate were also determined. The method allowed the quantitative determination of econazole nitrate, its impurities and preservatives and could be applied as a stability-indicating method for econazole nitrate in cream formulations. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Most fungal infections involve superficial invasion of skin or the mucous membranes of body orifices. Some species under certain conditions are capable of invading deeper body cavities and causing systemic mycoses. Such infections may become serious and occasionally life-threatening, and are frequently difficult to treat. The treatment of systemic mycoses is becoming very important in recent years as a result of the increased incidence of opportunistic yeast infections in immunocompromised patients. The widespread use of immunosuppressants following organ transplant operations and AIDS has been major contributors to this situation [1]. The azoles represent a class of versatile antifungal agents. In general, the azoles are effective against most fungi that cause superficial infections of the skin and mucous membranes. At high concentrations the azoles are fungicidal; at low concentrations they
∗ Corresponding author at: Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580 B-13, Butantã, 05508900 São Paulo, SP, Brazil. Tel.: +55 11 3091 3655; fax: +55 11 3815 4418. E-mail address: [email protected] (A.A. Gaona-Galdos). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.07.010
are fungistatic. The fungicidal effect is associated with damage to the fungi cell membrane, with the loss of essential cellular constituents. The fungistatic effects of the azoles have been correlated with the inhibition of membrane-bound enzymes by low concentration of the azoles [1]. Econazole nitrate (1-[2-(4-chlorophenyl)methoxy]-2-(2,4dichlorophenyl)ethyl)-1H-imidazole mononitrate (Fig. 1EN) is a potent broad-spectrum antifungal agent used topically in the treatment of skin infections. Further, results indicate that econazole could replace rifampicin/isoniazid as well as both rifampicin and isoniazid in chemotherapy of murine tuberculosis. Econazole alone or in combination with antitubercular drugs did not produce any hepatotoxicity in normal or Mycobacterium tuberculosis-infected mice [2,3]. Reported methods for the determination of econazole nitrate in pharmaceutical formulations include titrimetry [4], spectrophotometry [5,6], derivative spectrophotometry [7,8], high performance liquid chromatography (HPLC) [9–12], gas chromatography [13], high performance thin-layer chromatography [14] and capillary electrophoresis (CE) [15]. A variety of enantiomeric separations using HPLC and CE methods were also published [16–25]. Quantification of EN in biological samples has been developed using
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Fig. 1. Chemical structures of econazole nitrate (EN), alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (DCE), 4-chlorobenzyl alcohol (CBA), methylparaben (MP), propylparaben (PP) and miconazole nitrate (MN).
near infrared spectrometry [26,27] and liquid chromatography [28]. A stability-indicating assay method validated for econazole nitrate is described in the literature [29], showing specificity for its degradation products (4-chlorobenzyl alcohol and alpha-(2,4dichlorophenyl)-1H-imidazole-1-ethanol), as well as the inactive ingredients, but not for preservatives because they were not present in the formulation (methylparaben and propylparaben). This chromatographic separation was achieved with isocratic elution on a RP-18 column using methanol/aqueous ammonium carbonate solution/tetrahydrofurane as the mobile phase and miconazole nitrate as an internal standard. Nevertheless, this method allows only the quantitative determination of econazole nitrate. A pharmaceutical impurity is a component that is not the chemical entity defined as the drug substance or an excipient in the drug product. For this reason the safety of pharmaceuticals is dependent not only on the intrinsic toxicological properties of the active ingredient and excipients in the drug product, but also in part upon the impurities that it may contain [30]. Therefore, identification, quantification and control of impurities in the drug substance and drug product are important in the drug development. Preservatives would be effective at low concentrations against all possible microorganisms, nontoxic and compatible with other constituents used in the preparation. Esters of p-hydroxybenzoic acid (parabens) have antifungal properties. Their toxicity is generally low, owing to rapid in vivo hydrolysis to p-hydroxybenzoic acid, which is rapidly conjugated and excreted [1]. The present methodology in comparison with the methods described in the literature, shows a simple sample preparation and mobile phase composition and the last containing methanol–water without buffers. This proposed method allows the simultaneous determination of econazole nitrate, its impurities and preservatives in creams formulations with good resolution and peak symmetry.
2. Experimental 2.1. Instrumentation The gradient HPLC method was performed on a chromatographic system, consisted of a solvent delivery pump system model LC-10AD (Shimadzu® Corporation, Japan), an auto injector fitted with 20 L loop model SIL-10AD (Shimadzu® Corporation, Japan), an online degasification system model DGU-14A (Shimadzu® Corporation, Japan), a column thermostat oven model CTO-10AS (Shimadzu® Corporation, Japan) and an UV–VIS photodiode array detector model SPD-M10A (Shimadzu® Corporation, Japan). The output signal was monitored and integrated using CLASS VP® software v.5.91 (Shimadzu® Corporation, Japan). A reversed-phase C18 Column, Bondclone® (300 mm × 3.9 mm i.d., 10 m) PhenomenexTM California, USA, was used for separation. 2.2. Chemicals The reagents were of analytical grade. Methanol (HPLC grade), obtained from Merck (Darmstadt, Germany). Water was deionized and purified on a Milli-Q® water purification system (Millipore, Bedford, MA, USA) and used to prepare all solutions. 2.3. Chromatographic conditions The mobile phase was methanol–water. The analysis was carried out in a gradient elution mode with 57% methanol at 0 min gradually increased to 72% at 6.5 min, then increased to 98% at 10 min, from 10.01 to 15 min 98% using a flow rate of 1.4 mL min−1 at 25 ◦ C. Before delivering into the system the solvent was filtered through 0.45 m, HV membrane and degassed. The chromatograms were recorded at 220 nm.
Table 1 Development of solvent gradient systems System 1 T (min)
Methanol
Flow rate (mL min−1 )
System 2 %
0 45
55 100
1.0
T (min) 0 35
1.2
System 3
System 4
System 5
System 6
%
T (min)
%
T (min)
%
T (min)
%
T (min)
%
55 100
0 13 15 23
55 72 90 95
0 13 15 23
55 72 90 95
0 10 14 19
57 72 97 97
0 6.5 10 15
57 72 98 98
1.2
1.4
1.4
1.4
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Fig. 2. Gradient developing systems for methylparaben (MP), 4-chlorobenzyl alcohol (CBA), propylparaben (PP), alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (DCE), econazole nitrate (EN) and miconazole nitrate (MN). (Systems as in Table 1.)
2.4. Standards Econazole nitrate (100.55% purity) (Fig. 1EN) and miconazole nitrate (100.32% purity) (Fig. 1MN) were kindly donated by Formil Química Ltda. (São Paulo, Brazil). Impurities of econazole nitrate: alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (98% purity) (Fig. 1DCE) and 4-chlorobenzyl alcohol (99% purity) (Fig. 1CBA) ¯ Organics (Geel, Belgium). Preservawere purchased from Acros tives: methylparaben (Fig. 1MP) and propylparaben (Fig. 1PP) were donated by Laboratório Stiefel Ltda. (São Paulo, Brazil). 2.5. Sample The cream sample was supplied by Laboratório Stiefel Ltda. (São Paulo, Brazil) containing 1% (w/w) of econazole nitrate.
For the preparation of sample solution an amount equivalent to about 0.25 g of cream was accurately weighed into a 25-mL beaker and dissolved in 10 mL of a mixture of methanol–water (50:50, v/v) in a water-bath at 60 ◦ C for 5 min. After this period, the solution was quantitatively transferred to a 25-mL volumetric flask. A 2.5mL aliquot of the miconazole nitrate solution (1000 g mL−1 , stock solution) was transferred into a 25-mL volumetric flask. Then the solution was homogenized, cooled to room temperature and the volume was completed to 25 mL with the mixture methanol–water. The sample solution was then filtered using blue strip filter paper (Schleicher & Schull, Germany). Before injection on chromatograph, the solution was filtered through a 0.22 m filter (Millex PTFE, Millipore® ). Final concentrations of econazole nitrate and miconazole nitrate solutions were 100 g mL−1 . 2.7. Method validation
2.6. Preparation of standard and sample solutions (1000 g mL−1 ),
Standard stock solutions of econazole nitrate miconazole nitrate (1000 g mL−1 ), alpha-(2,4-dichlorophenyl)1H-imidazole-1-ethanol (20 and 40 g mL−1 ), 4-chlorobenzyl alcohol (20 and 40 g mL−1 ), methylparaben (500 g mL−1 ) and propylparaben (200 g mL−1 ) were prepared in methanol. The solutions were stored under refrigeration. Working standard solutions were prepared fresh daily by diluting appropriately the stock solutions with the same solvent.
The method was validated according to the United States Pharmacopeia requirements [31]. The following validation charac-
Table 2 Chromatographic parameters Compounds
MP CBA PP DCE EN MN
Parameters tR (min)
K
˛
Rs
4.03 5.00 7.11 7.85 13.25 13.83
1.47 2.06 3.40 3.81 7.12 7.48
MP/CBA = 1.40 CBA/PP = 1.65 PP/DCE = 1.12 DCE/EN = 1.87 EN/MN = 1.05
2.31 4.92 1.47 12.46 2.25
MP: methylparaben; CBA: 4-chlorobenzyl alcohol; PP: propylparaben; DCE: alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol; EN: econazole nitrate; MN: miconazole nitrate; tR : retention time; ˛: separation factor; K: retention factor; Rs : resolution.
Fig. 3. Chromatogram of standard solutions. Peaks: methylparaben (MP), 4-chlorobenzyl alcohol (CBA), propylparaben (PP), alpha-(2,4-dichlorophenyl)-1Himidazole-1-ethanol (DCE), econazole nitrate (EN) and miconazole nitrate (MN). Conditions: Bondclone® C18 Column, 300 mm × 3.9 mm i.d., 10 m; mobile phase: gradient elution starting with 57%, v/v methanol–water; temperature: 25 ± 1 ◦ C; flow rate: 1.4 mL min−1 ; UV detection at 220 nm.
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Table 3 Linearity, detection and quantitation limits determined for the proposed RP-HPLC method for all compounds Statistical parameter −1
Concentration range (g mL ) Intercept Slope Correlation coefficient (r2 ) Residual S.D. of the regression line () DL (g mL−1 ) QL (g mL−1 ) F
EN
MP
PP
CBA
DCE
70–120 0.0834 0.0101 0.9977 0.0144 4.71 14.28 857.57
30–55 0.0192 0.0066 0.9977 0.0047 2.35 7.13 861.19
8–18 0.0031 0.0050 0.9991 0.0009 0.57 1.72 2356.78
0.2–6.2 −0.0015 0.0144 0.9991 0.0015 0.35 1.06 2220.68
0.2–6.2 −0.0011 0.0086 0.9991 0.0009 0.35 1.05 2278.52
EN: econazole nitrate; MP: methylparaben; PP: propylparaben; CBA: 4-chlorobenzyl alcohol; DCE: alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol; DL: detection limit; QL: quantitation limit; F-test tabulated(0.05,1.4) = 7.71.
teristics were addressed to: linearity, detection and quantitation limits, precision, accuracy, robustness, system suitability, selectivity, assay and specificity. 2.7.1. Linearity, detection and quantitation limits, precision and accuracy Appropriate aliquots of stock solutions were transferred into 10 mL volumetric flasks and diluted to volume with methanol. Concentration range from 70 to 120 g mL−1 for econazole nitrate, 30–55 g mL−1 for methylparaben, 8–18 g mL−1 for propylparaben, and 0.2–6.2 g mL−1 for degradation products (4-chlorobenzyl alcohol and alpha-(2,4-dichlorophenyl)-1Himidazole-1-ethanol) were obtained. Then, the solutions were filtered using a 0.22-m filter (Millex PTFE, Millipore® ) and injected on the HPLC instrument. Each solution was injected in triplicate. Peak area ratios (compound/miconazole nitrate) were plotted versus the respective compound concentrations. Detection (DL) and quantitation limits (QL) were calculated from the residual standard deviation of the regression line () of the analytical curve and its slope (S) in accordance with the equations DL = 3.3 (/S) and QL = 10 (/S) [32]. In order to measure repeatability of the system (while keeping the operating conditions identical), 20 consecutive injections were made using a standard solution containing 80 g mL−1 of econazole nitrate and 100 g mL−1 of miconazole nitrate (IS). The results were expressed as the percentage relative standard deviation (%R.S.D.) for peak area ratio (PAR) of econazole nitrate/miconazole nitrate and retention time of econazole nitrate. For the determination of repeatability sample solutions were prepared at 100 g mL−1 of econazole nitrate and 100 g mL−1 of miconazole nitrate. Ten determinations were performed to establish the intra-day precision. The intra-day precision was evaluated by injecting sample solutions prepared at lower, middle and higher concentrations of the analytical curve (80–120 g mL−1 econazole nitrate) containing 100 g mL−1 of miconazole nitrate, in 1 day. The inter-day precision was evaluated by injecting the same solutions on three consecutive days. Three determinations for each concentration were performed. Precision was expressed as the %R.S.D. for peak area ratio of econazole nitrate/miconazole nitrate. The accuracy was calculated as the percentage recovery of a known amount of standard added to the sample. The accuracy of method was evaluated in triplicate using three concentration levels 80, 100 and 120 g mL−1 . Econazole nitrate standard solution was added to commercial sample solution and analyzed by the proposed method. 2.7.2. Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters, and provides an indication of its
reliability during normal usage [33]. In order to the robustness study of the proposed method, deliberate modifications in temperature–wavelength values were made. Thus, three temperatures values were selected, one below and one above of the chosen temperature. The same was made with wavelength. 2.7.3. Specificity The specificity of the method for econazole nitrate was tested by analyzing a mixture of the inactive ingredients (placebo), the commercial samples of econazole nitrate and a mixture of standard solutions. 2.7.4. Selectivity The selectivity of the method was established through study of retention time, separation factor, retention factor, resolution of all peaks and the absorption spectra of the eluted peaks. 3. Results and discussion 3.1. Method development In order to develop a simple HPLC method for quantitative determination of EN, its impurities and preservatives in cream formulations, different solvent systems were evaluated (acetonitrile, methanol and isopropanol), each one in different proportions. Rate gradients and flow rates also were tested to achieve efficient separation with a satisfactory resolution in a short time of analysis. Both solvents acetonitrile and isopropanol do not provided satisfactory
Fig. 4. Chromatograms of placebo (A), commercial sample containing econazole nitrate (B) and standard solutions(C). Conditions: Bondclone® C18 Column, 300 mm × 3.9 mm i.d., 10 m; mobile phase: gradient elution starting with 57%, v/v methanol–water; temperature: 25 ± 1 ◦ C; flow rate: 1.4 mL min−1 ; UV detection at 220 nm.
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chromatographic profiles (overlaps in degradation products peaks and inadequate drift). Several gradient systems were tested with the mobile phase, methanol–water (Table 1). The optimization of the methodology had targets as an adequate analysis time, avoid a pronounced rise of drift and an adequate resolution factor of all compounds separated. To reduce time analysis it was determined the elapsed time between the increase in the composition of mobile phase and its passage through the detector. In order, to avoid the drift elevation, it was also determined the adequate time in which the percentage of methanol increases from 72 to 98% in 3.5 min, since a smaller time produced an abrupt rise in the baseline. On the other hand, a greater time does not cause significant difference. To achieve an adequate resolution between all eluted peaks, it was calculated the resolution factor, which was higher than 1.45 for all the substances. Time analysis was significantly reduced in the system 6 in comparison with the others tested systems. Fig. 2 shows the retention times for the 6 compounds in each one of the systems. The gradient developed system 6 was the one who provides the best results in time, resolution and symmetry of the peaks, as depicted in Fig. 3. Other parameters are shown in Table 2. 3.2. Method validation Analytical curves were obtained by plotting peak area ratios (compound/IS) against the concentrations of respective substances. In all cases, straight regression lines with correlation coefficients (r) above 0.997 were obtained. F-test was applied for all calibration curves and the data provide conclusive evidence of a linear effect between concentration and instrumental response [34]. Data are summarized in Table 3. The DL and QL were calculated using analytical curves results (Table 3). DL and QL values were obtained by injecting 20 L of standard solutions in the chromatographic system.
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Table 4 Intra- and inter-day precision of the proposed RP-HPLC method for econazole nitrate quantitative determination 80 g mL−1
100 g mL−1
120 g mL−1
Intra-day (n = 3) %R.S.D.
2.20
0.81
1.56
Inter-day (n = 9) %R.S.D.
0.52
1.27
0.93
Injection precision was determined after injecting 20 times in the chromatographic system an econazole nitrate standard solution containing 80 g mL−1 . The results obtained for PAR were, %R.S.D. 0.25 and for retention time, %R.S.D. 0.04. The values obtained demonstrate that the system is reliable for analysis. The precision of the method was evaluated by repeatability and intermediate precision determinations. For repeatability, 10 sample solutions at 100 g mL−1 were analyzed in the same day and the R.S.D. obtained was 1.74%. The intermediate precision was achieved by analyzing three different concentrations on three consecutive days. The one-way ANOVA was used to estimate the total variability within and between days. The results, which, are shown in Table 4 present good agreement. The accuracy of the method was evaluated at three concentration levels. Triplicate determinations were made at each concentration level. The accuracy is expressed as percentage of standard recovered from sample matrix as R.S.D. The results are shown in Table 5. For robustness determination, changes in the temperature and wavelength were evaluated. About 1.8% of difference was observed in the more critical result when the analytical parameters were modified and compared with the original conditions. The specificity of the method was demonstrated by the absence of interference among econazole nitrate, methylparaben, propy-
Fig. 5. Chromatogram of separation of standard solutions and spectra for all compounds. Peaks: methylparaben (MP), 4-chlorobenzyl alcohol (CBA), propylparaben (PP), alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (DCE), econazole nitrate (EN) and miconazole nitrate (MN). Conditions: Bondclone® C18 Column, 300 mm × 3.9 mm i.d., 10 m; mobile phase: gradient elution starting with 57%, v/v methanol–water; temperature: 25 ± 1 ◦ C; flow rate: 1.4 mL min−1 ; UV detection at 220 nm.
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Table 5 Recovery of a standard solution of econazole nitrate added to sample and determined using the proposed HPLC method
Acknowledgements
Standard added to commercial samplea (g mL−1 )
Standard found (g mL−1 )
Recovery (%)b
The authors thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico of Brazil (CNPq, 130652/2007-5; CNPq, 142664/2005-7) for fellowship.
40.00 50.00 60.00
39.15 51.13 60.68
97.88 102.26 101.13
References
a b
Commercial sample (econazole nitrate cream). Average of three determinations.
lparaben, alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol, 4chlorobenzyl alcohol, miconazole nitrate and excipients in the samples, using the criteria defined in the USP 30 for assays [31]. A mixture of the inactive ingredients (placebo) (Fig. 4A), the commercial sample of econazole nitrate (Fig. 4B) and a standard mixture solution (Fig. 4C), were analyzed by the proposed methodology. As it can be observed, neither the cream excipients nor preservatives and the impurity interfere in the analysis of EN. Two preservative substances present in the formulation and impurities of econazole nitrate were used to evaluate the selectivity of the method. Results are showed in Table 2. The absorption spectra of the eluted peaks were achieved using a photodiode array detector and then compared with those of the reference standards. The results showed equivalent spectrophotometric profiles (Fig. 5). For the assay, a sample was analyzed in triplicate and the average obtained was 105.50%. The British Pharmacopoeia [35] established a range between 90 and 110%. The tested sample using the proposed method, presented satisfactory results. System suitability test is an important part of liquid chromatographic method. It is used to verify if the chromatographic system is adequate and reliable. Data from 5 injections of a solution containing 80 g mL−1 of econazole nitrate standard solutions were analyzed. The R.S.D. was 0.09%. This result agrees with those specified in the United States Pharmacopeia [31]. 4. Conclusion The validated method is rapid and efficient, and allows the separation of econazole nitrate in the presence of its degradation products, impurities and excipients, without using buffers or pH modifier in the mobile phase. Since it was possible to identify and quantify econazole nitrate impurities as (4-chlorobenzyl alcohol and alpha-(2,4dichlorophenyl)-1H-imidazole-1-ethanol), the proposed method can be used as a stability-indicating method for this drug in pharmaceutical formulations.
[1] A.R. Martin, in: J.N. Delgado, W.A. Remers (Eds.), Wilson and Davold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry, Lippincot Williams & Wilkins, England, 1998, pp. 173–221. [2] A. Zahoor, S. Sharma, G.K. Khuller, FEMS Microbiol. Lett. 258 (2006) 200. [3] A. Zahoor, S. Sharma, G.K. Khuller, FEMS Microbiol. Lett. 261 (2006) 181. [4] M. Massaccesi, Analyst 111 (1986) 987. [5] S.R. El-Shabouri, K.M. Emara, P.Y. Khashaba, A.M. Mohamed, Anal. Lett. 31 (1998) 1367. [6] P.Y. Khashaba, S.R. El-Shabouri, K.M. Emara, A.M. Mohamed, J. Pharm. Biomed. Anal. 22 (2000) 363. [7] D. Bonazzi, V. Cavrini, R. Gatti, E. Boselli, M. Caboni, J. Pharm. Biomed. Anal. 18 (1998) 235. [8] V. Cavrini, A.M. Di Pietra, R. Gatti, J. Pharm. Biomed. Anal. 7 (1989) 1535. [9] X.-L. Yang, Z.-F. Xi, J.-F. Sheng, Chin. J. Antib. 29 (2004) 403. [10] L. Gagliardi, D. De Orsi, P. Chimenti, R. Porra’, D. Tonelli, Anal. Sci. 19 (2003) 1195. [11] E. Kublin, T. Kaniewska, J. Pharm. Belg. 53 (1998) 208. [12] A.M. Di Pietra, V. Andrisano, R. Gotti, V. Cavrini, J. Pharm. Biomed. Anal. 14 (1996) 1191. [13] E. Kublin, T. Kaniewska, Chem. Anal. (Warsaw, Pol.) 41 (1996) 19. [14] G. Popovic, M. Cakar, K. Vucicevic, S. Vladimirov, D. Agbaba, J. Planar Chromatogr. Mod. TLC 17 (2004) 109. [15] A. Arranz, C. Echevarria, J.M. Moreda, A. Cid, J.F. Arranz, J. Chromatogr. A 871 (2000) 399. [16] L. Toribio, M.J. Del Nozal, J.L. Bernal, C. Alonso, J.J. Jimenez, J. Chromatogr. A 1144 (2007) 255. [17] X. Lin, C. Zhu, C. Hao, Electrophoresis 26 (2005) 3890. [18] I. Ali, H.Y. Aboul-Enein, Biomed. Chromatogr. 17 (2003) 113. [19] H.Y. Aboul-Enein, I. Ali, J. Pharm. Biomed. Anal. 27 (2002) 441. [20] H.Y. Aboul-Enein, I. Ali, Anal. Bioanal. Chem. 370 (2001) 951. [21] H.Y. Aboul-Enein, I. Ali, Chromatographia 54 (2001) 200. [22] A. Van Eeckhaut, S. Boonkerd, M.R. Detaevernier, Y. Michotte, J. Chromatogr. A 903 (2000) 245. [23] J.Y. Liu, Y.Y. Dong, T.S. Wang, H.W. Liu, A.J. Huang, Y.L. Sun, Z.P. Sun, Chin. Chem. Lett. 10 (1999) 39. [24] E. Peyrin, Y.C. Guillaume, Chromatographia 49 (1999) 691. [25] N. Morin, Y.C. Guillaume, J.C. Rouland, Chromatographia 48 (1998) 388. [26] J.P. Medendorp, K.S. Paudel, R.A. Lodder, A.L. Stinchcomb, Pharm. Res. 24 (2007) 186. [27] J. Medendorp, J. Yedluri, D.C. Hammell, T. Ji, R.A. Lodder, A.L. Stinchcomb, Pharm. Res. 23 (2006) 835. [28] P. Chiap, Ph. Hubert, J. Crommen, J. Chromatogr. A 948 (2002) 151. [29] R. Christinat, H.W. Zulliger, Arzneim. Forsch. 34 (1984) 551. [30] A.K. Basak, A.S. Raw, L.X. Yu, Adv. Drug Delivery Rev. 59 (2007) 1. [31] United States Pharmacopeia, 31st ed. The United States Pharmacopeial Convention, Rockville, 2008, pp. 683–687. [32] E. McEvoy, S. Donegan, J. Power, K. Altria, J. Pharm. Biomed. Anal. 44 (2007) 137. [33] International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, Topic Q2 (R1): Validation of Analytical Procedures: Text and Methodology, Geneva, 2005. [34] J.O. Rawlings, S.G. Pantula, D.A. Dickey, Applied Regression Analysis, 2nd ed., Springer, 1998, pp. 16–18. [35] British Pharmacopoeia. http://www.pharmacopeia.co.uk, 2008 (accessed 3/7/2008).