INTRODUCTION:
The present work was to develop and validate as per ICH guidelines for accuracy, precision, repeatability, reproducibility, robustness and ruggedness.8
Bilastine
MATERIALS AND METHODS:
Materials and reagents:
A gift sample of Bilastine from Sun Pharmaceutical Ind. Ltd, Mumbai used as a standard drug. Bilastine 20mg tablet bought from the local market, other chemicals like Methanol were bought from SD fine chemicals, Mumbai,India. All the reagents used in this method were of analytical grade.
Apparatus And Equipments Required:
UV-Vis double beam spectrophotometer (Model; Shimadzu: 1700S, Japan), Electric Sonicator, Volumetric flasks (10ml,50ml,100ml), Calibrated analytical pipettes, Electronic digital balance (Techno, Mumbai)
Determination of Absorption Maxima:
Stock solution (100µg/ml) of Bilastine was prepared in Methanol. This solution was appropriately diluted with further Methanol to obtain a concentration of 10µg/ml. The solution was kept in cuvette. The spectrum was recorded in the range of 200-400nm on Shimadzu-1800 double beam UV-Visible Spectrophotometer using Methanol as blank solution.
Determination of concentration range (Beer’s limit):
Determination of concentration range which obeys the Lambert and Beer’s law is necessary for accuracy and reproducibility in spectrophotometric analysis for quantitative determination of any drug. For this; Bilastine stock solution (100µg/ml) was prepared using pure drug in methanol. Further dilutions were made using 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml, 1.8ml of above solution was transferred to a series of 10ml volumetric flasks this gave a series of concentrations ranging from 2 to 18µg/ml of bilastine. The final volume was made up to 10ml mark using methanol, sonicated for 5 minutes. The resultant solutions were measured using a double beam uv-vis-spectrophotometer at wave length of 226.2nm against a reagent blank. A calibration curve was plotted with concentration against absorbance. From the graph it was clear that Beer’s law was obeyed in concentration range of 2-12µg/ml and deviation was observed above these concentrations.
Preparation of standard calibration curve:
100mg of pure Bilastine drug was dissolved in little quantity of methanol in a 100ml volumetric flask, the volume was made up to the mark using methanol. The solution was sonicated for 10 minutes. This gave a bilastine solution with concentration of 1mg/ml (1000µg/ml). 10ml of this solution was further diluted 100ml in volumetric flask using methanol to obtain a concentration of 100µg/ml. Further dilutions were made using 0.2, 0.4, 0.6, 0.8, 1.0, 1.2ml solution was transferred to a series of 10ml volumetric flasks (to Obtain a series of concentrations ranging from 2-12 µg/ml of Bilastine). The final volume was made up to 10ml mark using Methanol, sonicated for 5 minutes, the absorbance were measured at 226.2nm against a reagent blank. A calibration curve was plotted with concentration against absorbance.
Estimation of Bilastine in tablet dosage forms:
Twenty tablets were weighed accurately and powdered. The Tablet powder equivalent to 100mg of Bilastine was transferred into a 100ml volumetric flask and dissolved in little quantity of methanol. Then the solution was sonicated for 30 minutes and filtered using whatman filter paper No#41. The filtrate so obtained was diluted with methanol to produce 100ml. Further dilutions were made with methanol to get required concentrations within Beer’s - Lambert limits. The resultant solutions were measured at wave length of 226.2nm against a reagent blank. The concentration of drug was calculated with the help of standard calibration curve.
Analytical Method Validation:
Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for its intended use. All analytical methods that are intended to be used for analyzing any clinical samples will need to be validated. Validation of analytical methods is an essential but time-consuming activity for most analytical development laboratories. It is therefore important to understand the requirements of method validation in more detail and the options that are available to allow for optimal utilization of analytical resources in a development laboratory.
RESULTS AND DISCUSSIONS:
Figure 1 Showing absorption maxima of Bilastine at 226.2nm:
Figure: 2 Showing Beer’s limit for Bilastine pure drug:
Table:1 Showing Beer’s range for Bilastine:
|
Sl.No. |
Concentration in µg/ml |
**Absorbance at λmax 226.2nm |
|
1 |
0 |
0 |
|
2 |
2 |
0.03 |
|
3 |
4 |
0.061 |
|
4 |
6 |
0.088 |
|
5 |
8 |
0.117 |
|
6 |
10 |
0.146 |
|
7 |
12 |
0.172 |
|
8 |
14 |
0.188 |
|
9 |
16 |
0.21 |
|
10 |
18 |
0.232 |
Figure 3 Showing standard calibration curve
for Bilastine
Table 2 Showing absorbance of Bilastine at various concentrations:
|
Sl.No. |
Concentration in µg/ml |
**Absorbance at λmax 226.2nm |
|
1 |
0 |
0 |
|
2 |
2 |
0.030 |
|
3 |
4 |
0.061 |
|
4 |
6 |
0.088 |
|
5 |
8 |
0.117 |
|
6 |
10 |
0.146 |
|
7 |
12 |
0.172 |
Table 3 Showing absorption of drug from tablet dosage form:
|
Volume of stocks solution used |
Amount of drug (label claim) (µg/ml) |
Absorbance at 226.2nm |
Amount of drug found (µg/ml) |
Percentage purity found ±S.D** (%w/w) |
|
0.2ml |
2 |
0.030 |
2.011 |
100. 55±0.14 |
|
0.6ml |
6 |
0.088 |
5.635 |
93.91±1.44 |
|
1.0ml |
10 |
0.146 |
9.977 |
99.77±0.64 |
|
1.4ml |
14 |
0.250 |
13.176 |
94.11±0.49 |
|
1.8ml |
18 |
0.290 |
17.144 |
95.22±1.89 |
(**average of three determinations)
Table 4 Accuracy results for Bilastine:
|
Brands |
Initial amount (µg/ml) |
Amount of pure drug added (µg/ml) |
Amount recovered (µg/ml) |
% Recovery ±S.D** |
|
Bilaxten |
10 |
8(80%) |
8.022 |
100.27±0.140 |
|
10 |
10(100%) |
9.892 |
98.92±0.222 |
|
|
10 |
12(120%) |
12.021 |
100.17±0.412 |
(**Average of six determinations,n=6)
Table 5 Precision results for Bilastine:
|
Sl. |
Conc. in(µg/ml) |
Inter-day absorbance Mean±S.D** |
% C.V |
Intra-day absorbance Mean±S.D** |
% C.V |
|
1 |
8 |
0.408±0.033 |
0.33 |
0.407±0.012 |
0.60 |
|
2 |
10 |
0.507±0.032 |
0.32 |
0.507±0.016 |
0.21 |
|
3 |
12 |
0.604±0.58 |
0.58 |
0.603±0.022 |
0.22 |
**Average of three determinations,n=3