Send Orders for Reprints to reprints@benthamscience.net Current Analytical Chemistry, 2013, 9, 659-667 659 Development and Validation of a Simple Stability-indicating LC-method and UVA Photostability Study of Desonide Hair Lotion Fabrícia Dalla Santa1, Laura Escher Sperotto1, Muriele Picoli Braga2, Tássia Cristina Silveira Dalcin1, Cristiane Franco Codevilla3, Leonardo Zanchetti Meneghini 3, Eliane Maria Donato3, Clarice Madalena Bueno Rolim1, Ana Maria Bergold3 and Andréa Inês Horn Adams1* 1 Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Av. Roraima, 1000, 97105-900. Santa Maria - RS, Brasil; 2Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria. Santa Maria, Rio Grande do Sul, Brasil; 3Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752, 90610-000. Porto Alegre - RS, Brasil Abstract: The aim of this study was to develop and validate a LC stability-indicating method for the quantitation of desonide hair lotion currently marketed in Brazil, as well as delineate upon the photostability study of the same pharmaceutical product. The method used a RP-18 column (150 x 4.6 mm, 5 mm), with a methanol-acetonitrile-triethylamine solution as mobile phase (0.3 %, pH 4.0) (50:10:40, v/v/v) and UV detection at 244 nm. The method satisfied all the validation requirements, evidenced by good linearity (r > 0.9999, in the range from 5 to 100 µg mL-1), accuracy (recovery average 100.26%), precision (intraday RSD 0.45 and 1.76%; interday RSD 1.23%), robustness and specificity, indicated by good resolution between the analite peak from adjacent peaks (R>2.0) and suitable peak purity index in all the stress conditions. In order to evaluate the photostability of desonide hair lotion and desonide active pharmaceutical ingredient (desonide API), their portions were exposed to UVA light. The degradation of desonide hair lotion 0.1% in the conditions tested can be described by first-order kinetics, with the residual desonide content being around 39% after 15h exposure. The content of desonide API was 96.5%, after 72h exposure. The results indicate the instability of desonide in the evaluated drug product. Considering the characteristics of the developed method, it can be applied in the quality control routine of desonide in hair lotion and in stability studies. Keywords: Desonide, desonide assay, desonide hair lotion, desonide photostability, desonide stability, validation. INTRODUCTION Desonide (Fig. 1) is a synthetic and non-fluorinated mild strength corticosteroid, exhibiting anti-inflammatory and anti-pruritic properties [1, 2]. It is suitable for the treatment of inflammation and pruritus in corticosteroid-responsive dermatoses of mild to moderate severity, as well as in the long-term treatment of scaly, dry chronic dermatoses [3]. It has been used for over 30 years, and its safety and efficacy have been well demonstrated, as shown by a global pharmacovigilance program on desonide 0.05%, which highlighted only 62 incidences of treatment-related adverse events reported in a period of 9 years, despite the widespread use of this drug [4]. The safety of desonide foam 0.05% in shortterm treatment in children as young as 3 months was also indicated [5]. The available pharmaceutical forms of desonide include ointment, cream, lotion, gel and foam (all of them at 0.05%) and hair lotion (0.1%). Despite its usage since a long time, desonide has not achieved inclusion in any pharmacopoeia, nor has any *Address correspondence to this author at the Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Av. Roraima, 1000, 97105-900. Santa Maria - RS, Brasil; Tel/Fax: + 55 (55) 3220 8661; E-mail: andrea.ih.adams@gmail.com 1875-6727/13 $58.00+.00 analytical method been reported for quantitative analysis of the drug in dosage forms. There are only few analytical methods reported in the literature, designed to determine desonide in urine, focused on doping analysis [6-8]. Corticosteroids are known by their susceptibility to hydrolysis and photolysis [9]. The desonide photostability in organic solvents has also been reported [10]; however, there are no data about the drug stability in aqueous media or in pharmaceutical products. OH OH CH3 O CH3 O CH3 O H H CH3 H O Fig. (1). Chemical structure of desonide. Preliminary stability studies developed by our research group revealed that a desonide hair lotion currently marketed in Brazil has shown to undergo degradation upon exposure to light. According the International Conference on Harmonization guidelines [11], stress testing must be carried out to elu© 2013 Bentham Science Publishers 660 Current Analytical Chemistry, 2013, Vol. 9, No. 4 cidate the inherent stability characteristics of drugs in pharmaceutical preparations. Following its recommendation, the photostability testing should be an integral part of stress testing. Thus, a stability-indicating method is necessary for quantification of the drug in the presence of degradation products [12]. Considering the extensive use of desonide, the lack of pharmacopeial monographs and the preliminary results about its stability in pharmaceutical products, this work sought to develop and validate a simple stability-indicating method to assay desonide in hair lotion (0.1%) as well as to determine the photodegradation kinetics of this drug in the active pharmaceutical ingredient (desonide API). EXPERIMENTAL 2.1. Chemical and Reagents Desonide reference substance (98.55%, lot 0622-3) was obtained from Proactive Molecular Research (USA). Commercial pharmaceutical preparation (0.1% hair lotion, expiration date 2012/10, excipients isopropyl alcohol, methylparaben, propylparaben, propylene glycol and acetone) was purchased from local pharmacies. Methanol and acetonitrile (LC grade) were purchased from Tedia (Ohio, USA). Purified water was used to prepare the mobile phase, reagents and diluents solution (Millipore MilliQ® A 10 water system Billerica, USA). Analytical grade hydrochloric acid, sodium hydroxide and triethylamine were purchased from Merck (Darmstadt, Germany) and 30% hydrogen peroxide from Sinth (Brazil). 2.2. Instrumentation and Analytical Conditions Quantitation analyses were conducted in a Shimadzu LC system (Kyoto, Japan) provided with a LC-10AVP pump, a Rheodyne manual injector (20µL loop), a SPD- 10 Avp UV/VIS detector, and a SCL-10AVP controller with CLASSVP software. Another chromatographic system was employed to evaluate the specificity of the analytical method, which consisted of: Shimadzu LC system (Kyoto, Japan), LC-20 AT pump, DGU-20A5 degasser, SIL-20A autosampler (20µL loop), SPD-M20A photodiode array detector (PDA) and CBM-20A controller, with CLASS-VP software. A Phenomenex Luna® RP-18 column (150 x 4.6 mm, 5 µm, 100 Å) was used to perform chromatographic separation. Desonide detection was carried out at 244 nm. Mobile phase consisted of a methanol: acetonitrile: triethylamine solution 0.3%, pH 4.0 (50:10:40), at a flow rate of 1.0 mL minute-1 and isocratic elution. The pH of the mobile phase was adjusted with orthophosporic acid before the addition to the organic solvents. It was prepared daily, filtered through a 0.45 µm Nylon membrane filter (Millipore) and degassed by sonication and vacuum before use. Methanol:water solution (50:50) was used as diluent. 2.3. Preparation of Reference Substance and Sample Solutions Methanol was used to prepare the reference substance stock solutions (500 µg mL-1), considering the desonide solubility in this solvent. Working solutions were obtained Dalla Santa et al. from the dilution of stock solution to appropriate concentrations in the diluent. In order to prepare sample solutions, a portion of hair lotion (2.5 g, equivalent to 2.5 mg of desonide) was accurately weighed and then diluted to 10 mL using the diluent, in order to achieve a 250 µg mL-1 solution. An aliquot from this solution was diluted to 50 µg mL-1, with the diluent solution. 2.4. Validation of the LC-UV Method The method developed was planned to be applied to quantify desonide in hair lotion and to be used in stability studies, being validated by current ICH guidelines [12], under the following parameters: specificity, linearity, accuracy, precision and robustness. 2.4.1. Specificity Specificity and stability-indicating properties of the proposed method were verified through chromatographic runs of placebo solution and of solutions submitted to stress testing. During stress testing, desonide solutions were subjected to different pH values, oxidant agent and radiation. The ability of the method to quantify desonide among its degradation products and excipients was verified by the peak purity index, using the PDA detector, considering acceptable peak purity index above 0.9999 [13]. The preparation of the stressed desonide samples and placebo solution is described below (n=3/condition). 2.4.1.1. Acid Hydrolysis Five milliliters of methanolic sample solutions containing 500 µg mL-1 of desonide were kept with 5 mL of HCl 0.1 M, at 50 ºC, for 2 h. Alternatively, 5 mL of HCl 1M, at 50ºC, for 0.5 h were used to promote degradation. After treatment, solutions were neutralized with NaOH 0.1 or 1M, respectively, and diluted with diluent solution to theoretical concentration of 100 µg mL-1. 2.4.1.2. Basic Hydrolysis Five milliliters of methanolic sample solutions containing 500 µg mL-1 of desonide were kept with 5 mL of NaOH 0.1 M, at 50ºC, for 2 h. Alternatively, 5 mL of NaOH 1 M, at 50 ºC, for 0.5 h were used to promote degradation. After the specified time, solutions were neutralized with HCl 0.1 or 1M, respectively, and diluted to theoretical concentration of 100 µg mL-1, with diluent solution. 2.4.1.3. Oxidation To 5 mL of methanolic desonide solution (500 µg mL-1), 5 mL of 30% H2O2 were added. The solutions obtained were kept under heating (50 ºC), for 2 h. After treatment, solutions were diluted with diluent solution to a theoretical concentration of 100 µg mL-1 and analyzed shortly after. 2.4.1.4. Photodegradation One milliliter of desonide methanolic solutions (500 µg mL-1) was exposed to UVA radiation (352 nm) for 2 h, into a covered transparent container, in a light chamber. Samples subjected to the same conditions, but protected from light, were used as control. After the degradation treatment, the Assay and Photostability Study of Desonide Hair Lotion Current Analytical Chemistry, 2013, Vol. 9, No. 4 661 total volume of containers was diluted with diluent solution of 100 µg mL-1 and analyzed shortly after. tected from light were placed and exposed concurrently in the light chamber. 2.4.1.5. Excipients 2.5.1. Photostability of Desonide Hair Lotion 0.1% The relative amount of excipients contained in 2.5 g of formulation were accurately measured and dissolved in a volumetric flask, with methanol. Excipients included isopropyl alcohol, methylparaben, propylparaben, propylene glycol and acetone. An aliquot was removed and diluted in the same way as to prepare sample solutions. An accurate amount (1 g) of hair lotion was transferred to a covered transparent container and exposed to UVA radiation (352 nm) for 15 hours. At 1, 2, 4, 6, 8 and 15 h (n=3/time), containers were removed and samples diluted with diluent to theoretical concentration of 50 µg mL-1. The photodegradation degree was evaluated by measuring the percentage of recovered desonide in relation to nonirradiated samples, prepared under the same procedure. The exposure time was chosen in order to simulate the conditions of therapeutic use. 2.4.2. Linearity Five concentrations of desonide reference substance solutions (5, 10, 20, 50 and 100 µg mL-1) were analyzed in three independent assays. The solutions were obtained from the dilution of methanolic standard stock solutions (500 µg mL1 ) with diluent. Linearity was evaluated by linear regression and deviation analyses, which were calculated by the leastsquares method. 2.4.3. Accuracy Accuracy was determined by the recovery of known amounts of desonide reference substance solution added to sample solutions. A sample solution was prepared by accurately weighing an amount equivalent to 2.5 mg of desonide, which was dissolved with diluent in order to obtain a 250 µg mL-1 solution. An accurate amount of 1.0 mL of this solution was transferred to four 10 mL volumetric flasks, named A, R1, R2 and R3. Then, 2.0, 5.0 and 8.0 mL of desonide reference substance solution (50 µg mL-1) were added to R1, R2 and R3, respectively. The diluent was added to achieve concentrations of 35, 50 and 65 µg mL-1, corresponding to 70, 100 and 130 % of the nominal concentration. This procedure was performed in triplicate. 2.4.4. Precision Precision was evaluated by repeatability and intermediate precision and was expressed as relative standard deviation (RSD). To evaluate the repeatability, six samples were prepared as described in section 2.3, in the same conditions (day and analyst). Intermediate precision was determined by the comparison of the results obtained by another analyst on a different day (n=6). 2.4.5. Robustness Robustness was evaluated by deliberately modifying the operating conditions of the method. Variations in the mobile phase, including pH value (± 0.3 pH unit around the fixed value of TEA solution) and composition (content of methanol and TEA solution) were applied. In each tested condition, chromatographic parameters such as retention time, symmetry and theoretical plates were evaluated. Additionally, samples were analyzed to assess any impact on assay results (n=2/condition). 2.5 Photostability Study In order to determine the photostability of desonide hair lotion 0.1% and desonide API, portions were exposed to UVA radiation (352 nm) in the light chamber, as described below. The temperature inside the chamber was daily monitored and kept between 25 and 30 ºC; control samples pro- 2.5.2. Photostability of Desonide API Desonide API was placed in a glass dish, spread across the surface to give a layer thickness not exceeding 3 mm [14], for a period of 72 h. Along this period, samples at 18, 24 and 72 h were removed and diluted to a theoretical concentration of 50 µg mL-1 (n=3/time). The photostability was evaluated by measuring the percentage of recovered desonide in relation to non-irradiated desonide API, prepared under the same procedure. 2.5.3. Kinetic Calculations The degradation rate kinetic of desonide hair lotion 0.1% was determined by plotting concentration versus time, log of concentration versus time and reciprocal of concentration versus time (zero-order, first-order and second-order process, respectively). The best fit to model, indicated by regression coefficient (r), suggested the reaction order. Parameters such as degradation rate constant (k) and t90 (time to decrease the drug content to 90%) were then calculated. The kinetics model can be represented as C= Co – kt and t90% = 0.1 Co/k (zero-order reaction); ln C = ln Co – kt and t90% = 0.106/k (first-order reaction); 1/C = 1/Co+ kt and t90% = 1/9k Co (second-order reaction), where Co is the concentration at time zero, C is the remaining drug concentration at time t and k is the reaction rate constant [9]. 3. RESULTS AND DISCUSSION LC methods remain as the most widely applied methods in pharmaceutical analysis, mainly due to their sensibility, high-resolution capacity and specificity. In this study, a RPLC method was developed to quantify desonide in hair lotion, since there are no pharmacopeial monographs of desonide as bulk or dosage forms. The analytical conditions of the developed method were established based on chromatographic parameters (symmetry, theoretical plates, resolution) and run time. The mobile phase adopted (methanol: acetonitrile: triethylamine solution 0.3%, pH 4.0 (50:10:40), at a flow rate of 1.0 mL minute-1 and isocratic elution, showed to be appropriate to detect desonide in a short time (around 8.5 minutes), with good resolution from peaks of degradation products or excipients (R > 2.0) and acceptable values of theoretical plates (around 6.000) and symmetry (around 1.1). The wavelength selected (244 nm) corresponded to λmax of desonide. The chromatograms of the reference substance and sample solutions at 662 Current Analytical Chemistry, 2013, Vol. 9, No. 4 Dalla Santa et al. Fig. (2). (A) Chromatograms of desonide reference substance 50 µg mL-1. (B) Desonide hair lotion 50 µg mL-1. Peaks: (1) acetone, (2) methylparabene, (3) propylparabene, (4) desonide. nominal concentration of the method (50 µg mL-1) are shown in Fig. (2). The attribution of peaks related to excipients was based on the individual analysis of each one (chromatograms not shown). 3.1 Validation of the Analytical Method 3.1.1 Specificity To be specific, a method should demonstrate that it can quantify the analite from a drug mixture, degradation products and excipients, without interferences. The specificity of the proposed method was assessed using sample solutions submitted to forced degradation and running a placebo solution. The chromatograms obtained in specificity tests are shown in Fig. (3A-D). The analite peak showed good resolution from adjacent peaks (R>2.0) in all the stress conditions, and the analysis conducted by the PDA detector indicated suitable peak purity index (> 0.9999), confirming that no other substances coeluted with desonide. Additionally, excipients showed no interference in the desonide detection and quantitation. Among the stress conditions performed, basic medium proved to be the most important for desonide instability. The drug content decreased to 16% after 30 minutes of contact with sodium hydroxide 1M, with heating, and at least three degradation products were produced (Fig. 3-A). On the other hand, in acidic medium, the results indicated higher drug stability, since under conditions similar to that of basic medium (time, reagent power and heating), the desonide content remained around 99%. In this condition, one degradation product was observed (Fig. 3-B). The exposure of desonide to oxidant medium induced no degradation (Fig. 3-C). Under exposure to UVA radiation, the desonide content decreased to 93%, highlighting to be an interesting result, considering the low energetic power of this radiation. Despite the Assay and Photostability Study of Desonide Hair Lotion Current Analytical Chemistry, 2013, Vol. 9, No. 4 663 Fig. (3). Chromatograms of desonide hair lotion after (A) basic stress, NaOH 0,1M/2h/ 50 ºC; (B) acid stress, HCl 0,1M/2h/ 50 ºC; (C) oxidative treatment - H2O2 30% /2 h/ 50ºC; (D)UVA radiation, 2h. Peaks: (1) Acetone, (2) Methylparabene, (3) Propylparabene, (4) Desonide, (5) Hydrogen peroxide. 664 Current Analytical Chemistry, 2013, Vol. 9, No. 4 decrease being observed in the desonide content, no extra peaks were observed under UVA light (Fig. 3-D). This indicated that desonide is converted into degradation products, which do not present chromophore group or that it decomposes to low molecular weight fractions. Placebo runs confirmed no interference from excipients in the desonide detection and quantification at the wavelength chosen to the analysis (chromatograms not shown). 3.1.2. Linearity The analytical curves constructed for desonide demonstrated good linearity in the range of 5 to 100 µg mL-1, which corresponded to 10% to 200% of nominal sample concentration of 50 µg mL-1 (usual concentration of the method). The correlation coefficient value (r = 0.9999) showed good relationship between concentrations and analytical response and the statistical evaluation (ANOVA) demonstrated significant linear regression (P > 0.05) and non-significant linearity deviation (P < 0.05), with a linear regression equation y=37774x + 10295, where x is the concentration and y is the absolute peak area (mAU). 3.1.3. Accuracy and Precision Accuracy was assessed from the technique of standard additions, in order to determine recovery of spiked analyte. Three replicate determinations of solutions containing 35, 50 and 65 µg mL-1 of desonide, corresponding to 70, 100 and 130% of the method nominal concentration were analyzed. Accuracy criteria for an assay method are that the mean recovery will be 100±2% of the target concentration, at each level over the range evaluated [15]. Recovery values ranged from 98.33 to 101.34% and the mean recovery was 100.26% (RSD 1.24%), indicating the accuracy of the method. The precision was determined by repeatability (intraday assay) and intermediate precision (different day and analyst) and was expressed as relative standard deviation (RSD) of a set of results. The intraday RSD values were 0.45% (n=6, analyst 1, day 1) and 1.76% (n=6, analyst 2, day 2), respectively. The intermediate precision RSD value was 1.23% (n=12). These results confirm the absence of random errors and the precision of the analytical procedure. High values of desonide were recovered from the hair lotion (120.56 ± 1.48, mean assay (%) ± SD). We believed that this can be assigned to the concentration of the pharmaceutical form during batch production, justified by high amounts of acetone, which is the main excipient. 3.1.4. Robustness In order to establish the robustness of the developed method, deliberate modifications in pH and proportion of components from mobile phase were performed, the results of which are shown in Table 1. In each condition, effects on chromatographic parameters were monitored. All of them remained in accordance with recommended values [16]. None of the factors under study affected the chromatographic parameters significantly, and all of them were in accordance with recommended values [16]. As expected, the retention time increased to approximately 14 minutes Dalla Santa et al. when the methanol content reduced to 45%, turning the run too long. Despite the changes observed in retention time due to mobile phase composition, no significant changes in the desonide assay were observed (P-value = 0.4007). 3.2. Photostability Study Chemical and physical degradation of pharmaceutical forms and drugs may change their pharmacological effects, resulting in altered therapeutic effects as well as toxicological consequences. Quality should be maintained under the various conditions faced by pharmaceuticals, such as those observed during production, transportation, storage and during treatment. Therefore, understanding the factors that change the stability of pharmaceuticals and identifying ways to guarantee their stability are important [9]. It is known that glucocorticosteroids have to be protected from light, and their photoreactivity has been explored both in solution and in the solid state [9]. Regarding desonide stability, its photostability in organic solutions was evaluated and it was found that desonide converts into three degradation products [10]. However, there are no data about the desonide stability in aqueous medium or in pharmaceutical products. To study the photostability behavior of desonide, hair lotion 0.1% and desonide API were submitted to UVAradiation, in a light chamber. The desonide hair lotion 0.1% evaluated showed to be very unstable under exposure to UVA-radiation, since after 15 h, the remaining desonide content was around 39%. It must be emphasized that only after two hours of irradiation, the residual content was less than 90%, which is considered, for most of pharmaceutical products, the lowest drug content permitted during expiration date. The results about the desonide hair lotion 0.1% stability are shown in Table 2, and the chromatograms at time zero and 15 hours are shown in Fig. (4). Temperature did not influence the degradation behavior, since temperature of 30 ºC was maintained inside the chamber, and the desonide content in samples protected from light remained around 99.9%. Desonide API showed lower degradation rate, and the content after 72 hours of exposure was around 96.5% (Table 2). Despite the small decrease, the value observed appeared lower than the compendia limits assumed for most active pharmaceutical ingredient. So, it can be suggested that desonide API must be protected from light during storage. The degradation kinetics was determined for hair lotion, through the drug concentration decrease over time. Evaluation of the correlation coefficients obtained by plotting the drug concentration versus time, log of concentration versus time and reciprocal of concentration versus time, indicated that the degradation process of desonide in hair lotion can be described by first-order kinetics, under the experimental conditions under study (y = -0.0616x + 4.5778, r = 0.9935). From the slope of the straight line, it was possible to determine the first-order rate constant (k) and then, the t90 (time for 10% decomposition). The value of t90 obtained (1.7 hours) was in accordance with the experimental data, and indicated that in a very short period of time, the deson- Assay and Photostability Study of Desonide Hair Lotion Current Analytical Chemistry, 2013, Vol. 9, No. 4 ide concentration got lower than that considered as shelflife for most pharmaceutical products. As a consequence high levels of degradation products are present in contact with the skin. It is important to emphasize that the manufacturer establishes the application of the product during the day, and there is no special recommendation or advice about its photostability in the label. Table 1. 665 Data obtained from photostability study of pharmaceutical products, performed during the formulation development, provide a basis to establish storage conditions and specific recommendations, such as special care during sun exposure, in order to minimize adverse effects [17]. Thus, our findings suggest that the concomitant use of sunscreen could be a way to avoid the degradation of the desonide hair lotion 0.1%. Evaluation of Robustness of Analytical Method. Chromatographic Parameters Parameters Changed RTc Ad Ne Assay (%) 8.60 1.07 6970 119.14 8.72 1.07 6906 119.28 50:10:40 8.56 1.07 6921 120.06 55:10:35 5.98 1.09 6258 119.72 45:10:45 14.08 1.05 7660 118.72 pH of mobile phase a pH 3.7 pH 4.3 Composition of mobile phase b a methanol: acetonitrile: triethylamine solution 0.3%, pH in the cited range; b methanol: acetonitrile: triethylamine solution 0.3%, pH 4.0, being 50:10:40 the original condition; retention time; d asymmetry; e theoretical plates. Table 2. Results of Desonide Assay in Hair Lotion and Desonide API after photodegradation (UVA, 352 nm), Using LC Method. Sample Time (Hours) Theoretical Concentration (µg mL-1) Measured Concentrationa (µg mL-1) / RSD Measured Concentration (%) Hair lotion 0 50.0 50.05 / 0.71 100.1 1 47.42 / 1.27 94.8 2 41.57 / 1.58 83.1 4 36.30 / 1.19 72.6 6 34.69 / 1.26 69.4 8 28.71 / 1.52 57.4 15 19.71 / 2.34 39.4 49.95 / 0.52 99.9 18 49.53 / 2.54 99.1 24 49.81 / 2.14 99.6 72 48.23 / 0.73 96.5 Desonide API a 0 Each value is the mean of three independent samples. 50.0 c 666 Current Analytical Chemistry, 2013, Vol. 9, No. 4 Dalla Santa et al. Fig. (4). Chromatograms of desonide hair lotion at zero time (A) and after 15 hours of exposure to UVA radiation (B). Peaks: (1) Acetone, (2) Methylparabene, (3) Propylparabene, (4) Desonide. CONCLUSION REFERENCES The developed LC-method showed to be a stabilityindicating method, able to be used in routine quality control and stability studies. 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