Stability of an Aspirin in the Aspirin+Curcumin Admixture at Different Storage Conditions

Aims: The pure stability of aspirin in the aspirin (100 μg/mL) only and aspirin (100 μg/mL) +curcumin (600 μg/mL) admixture without any ingredient under two solvents (distilled water, DW and normal saline, NS), three storage temperatures (25oC, 4oC and -20oC) and periods (1, 3 and 7 days) was evaluated. Study Design: The injectable DWand NS-aspirin containing solutions in the laboratory cap polyethylene bottle were stored and evaluated at controlled temperature (25oC, 4oC and -20oC) during 7 days. Methodology: Effects of admixture compounds, periods of storage and temperature of storage on the concentrations of active compound (aspirin) were analyzed. The concentration of aspirin in each solution was determined by stability-indicating highperformance liquid chromatography (HPLC)-ultraviolet (UV) detection. A 1.0 mL volume of each sample was withdrawn and reconstituted with 3.0 mL of ethanol and directly injected into HPLC system immediately after filtration at 1, 3 and 7 days for analysis. The stability of the solutions was determined by calculating the percentage of the initial aspirin Others Article British Journal of Pharmaceutical Research, 3(4): 830-838, 2013 831 concentrations remaining at each test condition and periods. Stability was defined as the retention of at least 90% of the initial aspirin concentration. Results: The concentration of aspirin of the aspirin only and aspirin+curcumin admixture solutions remained at least 90% of original without any color change or precipitation in the DW and NS solution at 4oC and -20oC throughout 7 day period and showed instability that decreased gradually below 90% of original concentrations after 1 day at 25oC in the two solutions. Conclusion: Two kinds of solutions of only aspirin and aspirin+curcumin admixture, in DW and NS, showed different stability dependence on temperature of storage that means maintained stability at 4oC and -20oC and did not show effect of admixture of curcumin on aspirin stability during 7 days except 25oC.


INTRODUCTION
The chemical stability of a drug is of great importance since it becomes less effective as it undergoes degradation. Also, drug decomposition may yield toxic by-products that are harmful to the patient. Acetylsalicylic acid (ASA) decomposes rapidly in solutions of ammonium acetate or of the acetates, carbonates, citrates or hydroxides of the alkali metals. ASA is stable in dry air, but gradually hydrolyses in contact with moisture to acetic and salicylic acids. In solution with alkalis, the hydrolysis proceeds rapidly and the clear solutions formed may consist entirely of acetate and salicylate [1]. Daily Aspirin's effect on cancer has been widely studied, particularly its effect on colorectal cancer. Multiple meta-analyses and reviews have concluded that regular use of aspirin reduces the long-term risk of CRC incidence and mortality [2][3][4][5]. In addition, experiments were carried out to measure the synergistic effects of a combination of aspirin with other chemopreventive agents for the anticancer benefit [6][7][8][9][10]. Therefore, it is necessary to evaluate the physical and chemical stabilities of aspirin in order to determine the formulation of optimal dosage form that balances chemopreventive efficacy with safety of aspirin in combination with other agents. The purpose of this study is to provide information about the physical and chemical stability of aspirin in aspirin and curcumin of admixture under two solvents, various storage temperatures and periods. Thus, the study evaluated the stability of aspirin in the aspirin (100 μg/mL) only and aspirin (100 μg/mL) + curcumin (600 μg /mL) admixture without any ingredient under two solvents (distilled water, DW and normal saline, NS), three storage temperatures (25ºC, 4ºC and -20ºC) and periods (1 st , 3 rd and 7 th days)

Sample Preparation
Solutions of the designated samples (aspirin 2.5 mg, aspirin 2.5 mg and curcumin 15 mg) and diluents (to final volume of 25 mL with normal saline and distilled water) were prepared ( Table 1). At the time of preparation of each test solutions, 1 mL of samples were collected at specified time intervals; 0, 1 st , 3 rd and 7 th day, and added in 15 mL falcon tubes and stored in RT(25 ± 2ºC), CT(4 ± 2ºC) and FT (-20 ± 2ºC) until analyzed. A 1.0 mL volume of each sample was withdrawn and reconstituted with 3.0 mL of ethanol and directly injected into HPLC system immediately after filtration at 1 st , 3 rd and 7 th days for analysis.

HPLC Method
The concentration of aspirin in each sample solution was determined using a stabilityindicating HPLC assay method based on several references [11][12][13][14][15][16]. The flow rate of mobile phase was 0.8 mL/min. The method was validated for linearity, precision (inter-day and intra-day), accuracy and selectivity [17]. The experiment was repeated five times on the same day and five consecutive days to determine inter-and intraday precisions [18]. Linearity, accuracy and precision were evaluated by determining five concentrations of aspirin in range of 6.25~100 µg/mL. Linear regression analysis of peak area and concentration yielded a good correlation coefficient ≥0.999. Interand intra-day precision were expressed as the percent relative standard deviation (% RSD). The accuracy was expressed as the percent ratio between the experimental and nominal concentrations for each sample.

Standard Solutions
A 1 mg/mL of stock solution of aspirin was prepared by dissolving in 75% (V/V) ethanol/DW. Standard samples of aspirin were prepared by diluting the stock solution with 75% (V/V) ethanol/DW to concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL. The injection volume was 5 µL. The standard samples were assayed to repeat five times on the same day as an external standard method.

Sample Analysis
Each aspirin sample solution was prepared as described. A 5 µL of sample was injected into the HPLC system, and each sample was assayed in three times a day. Effects of admixture compounds, periods of storage and temperature of storage on the concentrations of active compound (aspirin) were analyzed. The samples were visually inspected for color change and precipitate formation was evaluated on each day of analysis.

Data Analysis
The stability of aspirin in the aspirin only and aspirin+curcumin admixture solutions was determined by calculating the percentage of the initial aspirin concentrations remaining at each test condition and hours. Stability was defined as the retention of at least 90% of the initial aspirin concentration.

Statistical Analysis
Experimental data were expressed as the mean ± standard deviation (S.D.). Differences in the concentrations of aspirin for 7 days investigated at the three different temperatures were performed by one-way ANOVA with scheffe test as a post hoc test using SPSS version 18.0. Statistical significance was set at p<0.05.

RESULTS AND DISCUSSION
The linearity could be established for aspirin in the concentration range of 6.25~100 µg/mL (r 2 = 0.9998, Fig.1A). Table 2 lists the percent relative standard deviation (% RSD) data obtained on analysis of the samples (n=5) on the same day and on consecutive days (n=5). The results were within limits (%RSD <10) shown in Table 2. As evident, the %RSD values were <9.84% and <2.10% for inter-day and intra-day results, respectively. Accuracies were 96.6~102.2% and 98.6~101.0%for inter-day and intra-day, respectively, meaning that the method was sufficiently precise and accurate. The retention time of the intact aspirin and the degradation product were about 4.6 and 5.3 minutes (Fig.1B, Fig. 1C).The aspirin undergoes hydrolysis in solutions at room temperature with the resultant degradation products being salicylic acid and acetic acid [13]. ASA=aspirin, DR= degradation product and CUR= curcumin.
The concentrations of aspirin which were observed at analytic time of each day during 7 days for each of the designated samples (aspirin only and aspirin+curcumin admixture), solution (DW and NS) and storage conditions are listed in Table 3.
In the aspirin in DW solution, the mean values of concentration were statistically (p < 0.05) different from 1 st to 7 th day among temperatures. The difference mean values of 7 th day were higher than the other day. These were values 19.14 ± 0.41 (in 25ºC), 3.38 ± 2.54 (in 4ºC), and 0.55 ± 0.51 (in -20ºC). In the aspirin in NS solution, the mean values were statistically (p < 0. 05 The differences mean values showed that the concentration of aspirin decreased. Therefore, the concentration of aspirin reduced according to change of temperature and time, regardless of solution type and the admixture of curcumin. The concentration of Aspirin remained by a minimum of 22% of the 0 day concentration under RT (25 ± 2ºC), 90% of the 0 day concentration under CT(4ºC ± 2ºC) and 94% of the 0 day concentration under FT (-20ºC ± 2ºC). The results of this study indicate the chemical stability of aspirin solution. Therefore, the concentration of aspirin of the aspirin only and aspirin+curcumin admixture solutions remained at least 90% of original without any color change or precipitation in the DW and NS solution at 4ºC and -20ºC throughout 7 th day period and showed instability that decreased gradually below 90% of original concentrations after 1 st day at 25ºC in the two solutions.

CONCLUSION
The objective of this study was to provide information about the physical and chemical stability of aspirin only and aspirin+curcumin admixture under two solvents, three storage temperatures and periods by HPLC assay method.
This study showed different stability of aspirin that depended on temperature of storage and period. On the hand admixture of curcumin on aspirin stability did not show any effect during 7 days except for 25ºC. However, this study was carried out for only one week. Therefore, further study is needed for the evaluation of chemical stability during a longer period of time.