Development and Validation of Dissolution Test Method for Determination of Bilastine from its Formulation using HPLC

Bilastine is a second generation antihistamine medication which is used in the treatment of allergic rhino-conjunctivitis and urticaria (hives). It exerts its effect as a selective histamine H1 receptor antagonist, and has effectiveness similar to cetirizine, fexofenadine. Chemically it is 2-[4-[2-[4-[1-(2-ethoxyethyl)benzimidazol-2-yl]piperidin-1-yl]ethyl]phenyl]-2-methylpropanoic acid. The molecular formula & molecular weight of bilastine is C??H??N?O? and 463.622 g/ mol.

Dissolution testing is a key analytical test used for detecting in an active pharmaceutical ingredient (API) and in formulated product. And High-performance liquid chromatography (HPLC) is an excellent technique for measuring average drug content in tablets and capsules.

Fig. 1. Structure of Bilastine

Literature survey reveals that no instrumental method is reported for estimation of percent release of bilastine from tablet. Hence an attempt has been made to develop an ideal dissolution method for determination of percent release of drug from their dosage form by modern chromatographic method (HPLC). Hence, the aim of existing work to develop a dissolution test method using HPLC method for analysis of bilastine in tablet and to validate dissolution method accordance with ICH guidelines.

2. MATERIAL AND METHOD

Chemicals and reagents

The Bilastine was obtained as a gift sample from Hetero Labs Ltd., Baddi, solan, Himachal Pradesh. Commercial tablets containing Bilastine 20mg was procured from the local chemist shop manufactured by Hetero Labs Ltd. Baddi, Solan, Himachal Pradesh. Acetonitrile and Methanol used was of HPLC grade. Double distilled water was used for preparing dissolution media and HPLC mobile phase. 0.1 N HCl, acetate buffer pH 4.5, Phosphate Buffer pH 6.8 was prepared as per the pharmacopoeia. Other chemicals include ortho phosphoric acid, potassium dihydrogen phosphate, hydrochloric acid and glacial acetic acid of GR grade were used.

Instruments

Uv-Spectrophotometer: Jasco-V-630, HPLC: Shimadzu HPLC 1100 series chromatograph equipped with isocratic pump LC-10ADVP, PDA – SPD M20A detector, Rheodyne injector with 20μL capacity. Dissolution Apparatus: Electrolab Tablet Dissolution tester-TDT-06P, Weighing balance: Shimadzu AUX 220, pH-meter: Digital pH Meter (model 111E), Sonicator: PCI Mumbai 3.5L 100H.

Determination of working wavelength

The standard solutions of Bilastine was prepared in methanol having concentration 10 µg/ml and subjected to U.V. spectrophotometeric study to determine λ max of drug over the wavelength range 200-400 nm. Bilastine shows maximum absorption at 270 and 276 nm, the wavelength selected was 276 nm.

Preparation of standard stock solution

A standard stock solution having concentration 1000 µg/mL of Bilastine was prepared in methanol.

Preparation of working standard solution

A 1.0 mL of the above standard solution was diluted upto 10.0 mL to prepare a solution having concentration 100 µg/mL of Bilastine, from this solution further 2.0 mL was pipetted and diluted upto 10.0 mL to get working standard solution having concentration 20 µg/mL of Bilastine.

Chromatographic condition

Chromatography was achieved on a Waters Symmetry C₈ 5 µm (4.6 × 150 mm) column. The mobile phase was a mixture of Phosphate buffer of pH 3.0 and Acetonitrile (60:40 % (v/v)). All analysis was performed at room temperature at a flow rate of 1 mL/min. The detection was carried out at wavelength 276 nm and the injection volume was 20 µL.

Dissolution Test Conditions

Drug dissolution tests were carried out with USP apparatus II (paddle type) at 50 rpm with dissolution volume of 900mL. Thermostatic bath was used to maintain the temperature of the cell at 37°C±0.5°C. Various dissolution media’s were tried out of which 0.1 N HCL (1.2 pH) was selected. Weighed and dropped 1 tablet in each of the six dissolution vessel containing 0.1 N HCL (pH 1.2) for the drug under analysis. Aliquots of 5.0 mL were withdrawn at 5, 10, 15, 20, 25, 30 and 45 min time interval, used as sample and replaced with an equal volume of the fresh medium to maintain a constant total volume. After the end of each time point, sample aliquots were filtered and chromatographed. The percentage drug release was estimated by validated HPLC method at each time point.

Dissolution method parameter optimization for Bilastine

Various dissolutions were performed to optimize the parameters like dissolution apparatus dissolution media, dissolution media volume and rpm, using the optimized chromatographic conditions and the solubility data of the drugs and select a set of parameter that will give maximum % release of the drug.

Study of change in USP apparatus type

To study the effect of change in USP apparatus, 0.1 N HCL (pH 1.2) was selected as dissolution media. A media volume of 900 mL and thermostatic bath temperature was kept constant and the dissolution was performed on two different USP apparatus. The results were evaluated for cyclobenzaprine. The % drug release for two apparatus types were calculated.

Change in the volume of dissolution media

The dissolution media used in the above study was kept constant, 0.1 N HCL (pH 1.2) whereas dissolution media volume was varied from 500 mL, 900 mL and 1000 mL, performed using USP apparatus type II. The % drug release was calculated.

Change in dissolution media (Buffer)

Phosphate buffer (pH 6.8), acetate buffer (pH 4.5) and 0.1 N HCL (pH 1.2) were used as dissolution media, with a media volume of 900 mL was selected. The % drug release was calculated.

Change in speed of rotation (rpm)

The 0.1N HCL was selected as optimized dissolution media with media volume of 900 mL. The speed of rotation (rpm) is varied from 50, 75 and 100 rpm. The % release was calculated.

Method validation

The dissolution test method was validated through the determination of linearity, precision, accuracy, solution stability. Prior to injecting sample solutions, the column was equilibrated for at least 30 min with the mobile phase flowing through the system.

Linearity

The linearity for Bilastine with respect to concentration was demonstrated by considering the label claim of the Bilastine as 100% target concentration [20µg/ml of Bilastine] and preparing the solutions in the mobile phase with concentration ranging from about 5 to 30 % of target concentration.

Accuracy

The accuracy of the proposed method was evaluated by spiking method i.e. adding known amount of Bilastine standard drug (50%-150%) to that of target dissolution concentration as per the label claim of 20 mg Bilastine formulation respectively. To determine the % recovery of the drug, pure drug was added at the selected levels along with 20 mg tablet. Dissolution of the drug was performed using optimized dissolution parameters. Aliquots of 10 mL were withdrawn and analyzed by chromatographic method at spiked concentration levels of 50%, 100 and 150%, respectively. Each concentration was analyzed in triplicate.

Precision

The precision of the method was evaluated by measuring the precision expressed as % RSD. Tablet samples were subjected to dissolution test condition 900 mL of dissolution medium (0.1 N HCL buffer) pre-heated at 37°C±0.5°C, paddle with stirring rate of 50 rpm). The test sample were obtained by performing the dissolution of the drug under analysis using optimized dissolution parameters and were chromatographed by using finalised chromatographic parameters. The study was performed with 3 consecutive studies i.e. Repeatability precision study, Intraday Precision study and Inter-day precision study.

Ruggedness

The ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the samples under a variety of conditions. The method was studied for ruggedness by analysing sample and standard preparation by two different analyst.

Robustness

The robustness of an analytical procedure is a measure of its ability to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The robustness of test method was carried out for following parameters:

a) Change in flow rate, b) Change in detection wavelength, c) Change in pH of mobile phase

3. RESULT AND DISCUSSION

HPLC Method development and dissolution method validation

The optimized chromatographic condition mentioned below was kept constant throughout the experimentation and mobile phase was allowed to equilibrate with stationary phase which was indicated by a steady line.

System: Shimadzu

Stationary Phase: Waters Symmetry C₈ 5 µm (4.6 × 150 mm)

Mobile phase: Phosphate buffer (pH 3.0): ACN (60:40 % v/v)

Detection wavelength: 276nm

Flow rate: 1.0 mL/min

Injection volume: 20 µL

Run time: 10 min

Fig. 2. Chromatogram of standard Bilastine

A 20 μL solution of above mix standard was injected through manual injector and chromatogram was recorded. A standard  chromatogram for Bilastine is shown in Fig. 2

System suitability parameters

The system suitability study was carried out using six replicates injections of standard solution containing 20µg/mL of Bilastine was prepared in mobile phase. A 20µL of solution were injected through manual injector and analyzing the chromatograms for peak area, theoretical plates, % RSD and tailing factor. Results are shown in Table. 1.

Table. 1. Results of system suitability parameters

Optimization of dissolution method parameters

Various dissolutions were performed to optimize the parameters like dissolution media, dissolution media volume, apparatus and rpm, using the optimized chromatographic condition and the solubility data the drug to select a set of parameter that will give maximum % release of drug.

Change in USP apparatus

The % release of drug is shown in Table. 2. From the table it was observed that the release of drug in USP I was slo as compared to USP II. Therefore, USP II was selected as one of the optimized dissolution parameter and was further used in the experimentation.

Table. 2. Results showing effect of change in USP apparatus

Change in volume of dissolution media

The result of % release of drug is shown in Table. 3. From the table it as observed that the percent drug release in a media volume using 500 mL was found to be less as compared to a media volume of 900 mL and faster in 1000 mL as compared to 900 mL. Hence media volume of 900 mL was selected as one of the optimized dissolution parameter and was further used in the experimentation.

Table. 3. Results showing effect of change in volume of dissolution media

Change in dissolution media (Buffer)

The result of % release of drug is shown in Table. 4. From the Table, it was observed that the release rate of the drug obtained was less in Phosphate Buffer (pH 6.8) as well as in Acetate Buffer (pH 4.5) as compared to 0.1 N HCl. Therefore, 0.1N HCl was selected as optimized dissolution media and further used in the experimentation.

Table. 4. Results showing effect of change in dissolution media

Change in speed of rotation (rpm)

The result of % release of drug is shown in Table. 5. From the Table, it was observed that the release of drug at 50 rpm was found to be optimum as compared to other rpm condition. Hence, 50 rpm mL was selected as one of the optimized dissolution parameter and was further used in the experimentation.

The finalized dissolution parameter selected for the dissolution test method of BILASTINE are shown in Table. 6 and percent release of drug under final chromatographic and final dissolution parameters are shown in Table. 7 for BILASTINE.

Table. 5. Results showing effect of change in speed of rotation (rpm)

Table. 6. Final dissolution method parameters

Table. 7. Percent release of drug under final chromatographic and final dissolution parameters

Table. 8. Observations of linearity responses of bilastine

The peak area of linearity solutions noted was plotted against the corresponding concentrations to obtain the calibration graph. The correlation coefficient were found to be 0.9993. Fig. 3. The calibration data is presented in Table. 8.

Fig. 3. Graph showing linearity of bilastine at different concentrations

The results of accuracy data at different spike level are shown in Table. 9. From the accuracy studies the mean % recovery of drug at each accuracy level was found to be 99.39, which is found to be in the acceptance range of 95% - 105%. It was proved that the method is accurate.

The precision of the dissolution study carried out as repeatability, intra-day and inter-day variations. The % release as found to be in acceptance level and % RSD of drug from repeatability study was found to be 0.59, the % RSD of intra-day study was found to be 0.91 and the % RSD for inter-day study was found to be 0.90 respectively ascertaining the precision of method. The observation and the results of precision study for drug is summarized in Table. 10, 11 and 12.

The observation and results of ruggedness study for drug is summarized in the Table 13. Ruggedness of the method is evaluated by analysis of aliquots from homogenous slot by to different analyst. The RSD between two different analysts should not be more than 2.0 %. The proposed method was found to be rugged.

The robustness of the method as evaluated by injection of the sample at deliberately varying the chromatographic conditions i.e. change in flow rate by 0.2mL/min, change in wavelength and pH by ±5. The method was found to be robust. The results obtained by robustness study are given in Table 14.

Table. 9. Observations of Recovery Study

Table. 10. Results of Repeatability Study

Table. 11. Results of Intra-day Precision Study

Table. 12. Results of Inter-day Precision Study

Table. 13. Observations and Result of Ruggedness study

Table. 14. Observations and Result of Robustness Study