DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING ASSAY FOR AZILSARTAN KAMEDOXOMIL IN SOLID DOSAGE FORMS

Mohamed A. Kassem 1 , Magdy I. Mohamed 2 and Asmaa A. Mohamed 3* . Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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Base degradation:--40 mg of azilsartan kamedoxomil powder was accurately weighed in a 100 ml volumetric flask containing about 50ml acetonitrile: water 50:50, 10 ml 0.01 N NaOH was added heated on water bath at temperature of 70 °C for 5 min then transfer and was neutralized by acid and completed to volume with the mobile phase.10ml was diluted to 100 ml with mobile phase.
-40 mg of azilsartan kamedoxomil powder was accurately weighed in a 100 ml volumetric flask containing about 50ml acetonitrile: water 50:50, 10 ml 0.1 N NaOH was added heated on water bath at temperature of 70 °C for 5 min then transfer and was neutralized by acid and completed to volume with the mobile phase. 10ml was diluted to 100 ml with mobile phase.
Peroxide degradation:--40 mg of azilsartan kamedoxomil powder was accurately weighed in a 100 ml volumetric flask containing about 50ml acetonitrile: water 50:50, 5ml of 30% (w/v) H2O2 was added heated on water bath at temperature of 70 °C for 2 min then transfer and completed to volume with the mobile phase.10ml was diluted to 100 ml with mobile phase.
Photolytic degradation:-Azilsartan kamedoxmil solution of 100 μg/ml to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain 40μg/ml and 50 μl were injected into the system and the chromatograms were recorded to assess the stability of sample (4,5) Method validation:-The developed method was validated to establish the specificity, precision, linearity, accuracy and robustness according to ICH guidelines, USP (6)(7) .

Linearity:-
The peak area chromatogram of azilsartan kamedoxomil in the range of (10-70 µg.ml -1 ), The calibration curve at 254 nm to the drug concentrations was constructed the regression equation was calculated for different concentrations of azilsartan kamedoxomil standard solution using acetonitrile: water 50:50 and diluted obtain final dilution corresponding to the following concentrations: 10, 20, 30, 40, 50, 60 and 70 µg.ml -1 using the mobile phase, the regression equation, the correlation coefficient, slope of the regression line, and residual standard deviation (SD) were calculated Specificity:-The terms specificity and selectivity are often used interchangeably as both the USPand the ICH (6) currently use the term specificity, it will also be used here to avoid any confusion. The USP (7) defines specificity as the ability to measure accurately and specifically the analyte of interest in the presence of other components in the sample matrix. These components may include other active ingredients, excipients, impurities, and degradation products. According to the ICH, (6) the validation procedure should be able to demonstrate the ability of the method to assess unequivocally the analyte in the presence of impurities, matrix components, and degradation products. It was done by preparing placebo {all contents of tablets without adding active ingredient} was injected, and peak area should not appear at the retention time specified for azilsartan kamedoxomil.

Accuracy
Accuracy is the measure of how close the experimental value is to the true value. It should be established across the specified range (that is, line of working range) of the analytical procedure.
Assay percentage to the labeled claim: The analytical method procedure was performed by preparing triplicates of 3 different test solution concentrations 80% , 100% , 120% as FDA (8) recommended (in this assay were 32, 40, 48 µg.ml -1 ), and % Azilsartan kamedoxomil was calculated .The data should be calculated as percent of label claim, and the mean of the replicates along with % Relative Standard Deviation (RSD) for each level reported to demonstrate accuracy and sample analysis precision.
Percentage recovery: Determination of the percentage recovery of added known amount of pure azilsartan kamedoxomil reference standard to the test solution sample to prove the accuracy of the analytical method. After the addition of (10, 15, 20, 25, 30 µg.ml -1 ), the final tests concentrations were respectively (50, 55, 60, 65, 70 µg.ml -1 ),

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Triplicates of these test concentrations were prepared and assayed and % recovered were computed. The data should be calculated as percent of label claim, and the mean of the replicates along with % RSD for each level reported to demonstrate accuracy and sample analysis precision.

Precision:-
Precision is the measure of how close the data values are to each other for a number of measurements under the same analytical conditions or "the degree of agreement among individual tests results obtained by repeatedly applying the analytical method to multiple samplings of homogenous sample" Repeatability:-6 test solutions having all 100% of test or target concentrations were prepared following the test preparation procedure in the analytical method and having all the same concentrations of (40 µg.ml -1 ). Results obtained stored and computed. RSD should be not more than ±1 as FDA recommended.

Ruggedness (Intermediate precision):-
Intermediate precision expresses within-laboratory variations. This was previously evaluated as part of ruggedness. This attribute evaluates the reliability of the method. The data obtained from the interday and intraday confirm the ruggedness of the used analytical method.

Interday:-
The previous procedure was repeated three times on three different days for the analysis of the concentrations ,The concentrations were calculated from the corresponding regression equation.

Intraday:-
The previous procedure was repeated three times on different time intervals on the same day for the analysis of the concentrations, the concentrations were calculated from the corresponding regression equation.

Robustness:-
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in some parameters and provide an assurance of its reliability during normal usage. The robustness of the method is investigated by varying some or all conditions, e.g., organic composition of the mobile phase, pH, ionic strength, column temperature, age of column, column type. ICH (6) guidelines recommend that robustness studies be performed during the method development stage. Robustness can also be partly assured by good system suitability specification. Therefore, it is important to set tight but realistic system suitability specifications. the method was assured that it is robust by making slight change Robustness of the method was investigated by varying the instrumental conditions such as flow rate (±10%), column oven temperature (±5%), wave length of detection (±5nm), organic content in mobile phase (±2%) and pH of buffer in mobile phase (±0.2 units).

Lower Limit of Detection (LLOD):-
Lower limit of detection (LLOD) is the lowest concentration of the analyte that can be detected, but not necessarily quantitated, under the stated experimental conditions. It is a parameter of limit test and specifies whether or not an analyte is above or below a certain value. Determination of limit of detection is described for instrumental and noninstrumental methods. For instrumental methods, one determines the signal-to-noise ratio by comparing test results from samples with known concentration of analyte with those of blank samples and establishes the lowest concentration at which analyte can be reliably detected. A signal-to-noise ratio of 2:1 or 3:1 is required. Another approach is to calculate the standard deviation for analysis of a number of blank samples. The standard deviation multiplied by a factor, usually 2 or 3, gives an estimate of limit of detection.
The detection limit may be calculated based on the standard deviation of the residuals (SD) and slope (S) of the calibration curve (a specific curve should be generated by using samples containing analyte in the range of detection limit), according to the formula: LLOD = 3.3X SD/ S Where SD is standard deviation of residuals, S is slope of line of calibration curve Lower Limit of Quantitation (LLOQ):-Lower limit of quantitation (LLOQ) is the lowest concentration of the analyte that can be determined with Acceptable precision and accuracy under the stated experimental conditions of method, this I a parameter of the quantitative assay for low concentrations of compounds in sample matrices such s degradation product in the finished product. USP LLOQ is similar to LLOD, is expressed as conc. of the analyte in the sample, and precision and accuracy are also reported. LLOQ is dependent on the type of procedure, i.e., instrumental or noninstrumental. For instrumental, sometimes signal to noise ratio of 10:1 is used to determine LLOQ. ICH lists the same two options that can be us to determine LLOQ. The evaluation for instrumental or noninstrumental; the latter method based on standard deviation of the response and the slope. LLOQ = 10X SD / S Where SD is standard deviation of residuals, S is slope of line of calibration curve System Suitability:-The system suitability specifications are parameters that provide assistance in achieving this purpose. According to the ICH (6) and the USP (7) . System suitability tests are performed prior to analysis of actual samples. These parameters are studied by analysis of a system suitability sample that is a mixture of main active drug and expected by-product or a known impurity. Parameters required such as tailing factor (T) should be for azilsartan kamedoxomil not be more than 2, Resolution (R S ) should be more than 2 if there is more than one peak ], capacity factor (k') should be more than 2 and plate count should be not less than 2000 (8)(9)(10) Stability of solutions:-Many solutes readily decompose prior to chromatographic investigations, for example, during the preparation of the sample solutions, extraction, cleanup, phase transfer or storage of prepared vials (in refrigerators or in an automatic sampler). Under these circumstances, method development should investigate the stability of the analytes and standards.
The term system stability has been defined as the stability of the samples being analyzed in a sample solution. It is a measure of the bias in assay results generated during a preselected time interval, for example, every hour up to 46 hours, using a single solution. System stability should be determined by replicate analysis of the sample solution. System stability is considered appropriate when the RSD, calculated on the assay results obtained at different time intervals, does not exceed more than 20 percent of the corresponding value of the system precision. If, on plotting the assay results as a function of time, the value is higher, the maximum duration of the usability of the sample solution can be calculated.
Stability of standard solution checked by analyzing solutions prepared according to the standard preparation described in test method, each containing (the concentration of standard) at different time interval for 48 hr at room temperature and comparing to freshly prepared standard solution. Stability of test solution checked by analyzing solutions prepared according to the test preparation described in test method, each containing (the concentration of test) at different time interval for 48 hr at room temperature and comparing to freshly prepared standard solution

Results and Discussion:-
When Azilsartan medoxomil were subjected to chromatographic analysis in mobile phases of different strengths and compositions, it was found that mobile phase consisting of 0.025 M ammonium acetate (pH: 5.0): acetonitrile (30:70 v/v) gave adequate retention at a flow rate of 2 ml/min. The wavelength at which detection was carried out was 254 nm. The retention time for Azilsartan medoxomil was 5.011 min. A linear calibration curve was obtained in the concentration range (10-70µg.ml -1 ) representing the relationship between concentration and the response (peak area) as shown in Fig. (2) and table (1). Y = 41489X + 717.17, r 2 = 0.9999 Where Y is the peak area at 254 nm, X is the concentration in µg.ml -1 and r 2 is correlation coefficient.
The validity of the developed method was further assessed by applying the standard addition technique for the analysis of Edarbi 40 mg tablets. The method found to be linear over range 10-70 µg.ml -1 , the correlation coefficient (r 2 ), standard deviation (SD) and slope were found to be 0.9999, 717.17 and 41489, respectively (table 1and Fig.10). Mean accuracy was 99.594%±0.78; with mean percent recovery was found to be 100.18% ± 1.084 as shown in tables (2.a, 2.b). The method was shown to be selective and specific. The method was shown to be precise since repeatability mean percent resulted was 99.981% ±0.41, RSD=0.41%, table (3). The method also was found to be rugged and RSD did not exceed 0.5% as shown in tables (4.a, 4.b).
The method was found robust as slight changes(±0.2) in flow from 2 ml/min to 2.2 ml/min, 1.8 ml/min and the pH of buffer of mobile phase changed (±0.2) from 5.5 to 5.3 and 5.7 did not affect the results significantly as RSD was less than 1%. Results are shown in table (5). Lower limit of detection (LLOD) was found to be 0.832 μg/ml and Lower limit of quantitation (LLOQ) was found to be 2.521 μg/ml. System was suitable as tailing was 0.89 [Tailing should be not more than 2], plate count was found to be more than 5967 and capacity factor was found to be 5.940 [limit not less than 2] The solutions of both standard and test were found to be stable as the RSD of stability were found to be 0.033% and 0.025%, respectively where the values were found to be less than 20% percent of RSD precision of the method.