INTRODUCTION:

High performance liquid chromatography (HPLC), sometimes called high pressure liquid chromatography, is a separation technique based on a solid stationary phase and a liquid mobile phase.¹ Most of the drugs in multi component dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC method eliminates tedious extraction and isolation procedures.²

Bilastine is a newer oral highly selected second generation H1receptor antagonist for the treatment of sympathomimetic of allergic rhino-conjunctivitis and chronic urticaria in adult patients. Rhino-conjunctivitis and urticarial respond to antihistamine treatment and Bilastin is an antihistaminic agents whose action is inhibition of immune system reaction mediated histamine receptor.^3-5

Bilastine, or 2-[4-(2-(4-(1-(2-ethoxyethyl)-1H– Benz imidazole– 2-yl) piperidine-1–yl) ethyl) phenyl]-2-methyl propionic acid, is a new next-generation antihistamine. (Fig. 1) It is a new piperidine molecule and belongs to the same chemical group as many new antihistamines.⁶ Within this group, it is chemically close to the piperidine-benzimidazole subgroup, which also includes molecules such as norastemizole and mizolastine.

Figure 1: Chemical Structure of Bilastine

The drug Bilastine is CDSCO approved for symptomatic treatment of allergic rhino-conjunctivitis. On literature survey, it was found that few U.V. spectroscopy, HPTLC and RP-HPLC methods, Degradation study are available for the determination of Bilastine in tablet dosage form. ^7-15 According to literature survey, the method development approaches specifically focused on pharmaceutical development in a have not been discussed It was planned to develop simple, rapid and sensitive RP-HPLC method for estimation of Bilastine in tablet dosage form. Applying the more robust method which produce consistent, reliable and quality data throughout the life cycle and in turn will leads to less method incidents when used in routine environment.^16-18 Stress study was carried out under the condition of acid hydrolysis, base hydrolysis, oxidation degradation, thermal degradation, and photolytic degradation, as mentioned in ICH Q1 A (R2) guidelines.¹⁹Method was validated as per ICH guideline Q2 (R1).²⁰Forced degradation study is a powerful tool for the development of stability indicating method.²¹ that studies are used to gain a better understanding of active pharmaceutical ingredients and drug product.²² It is to specify the specificity of the stability indication methods and provide insight into degradation pathways and degradation products of the drug substance.²³

MATERIALS AND METHODS:

Chemical and reagents:

Bilastine sample was obtained as a gift from Shiva health care mahesana. HPLC-grade methanol was purchase from Merck Life Science Private Limited, Mumbai, India and HPLC-grade water were purchased from Merck Life Science Private Limited, Mumbai, India. Bilastine tablet (BYLOZA 20 mg) was purchased for the analytical purpose. All the other chemicals and reagents used were of AR grade and purchased from Avantor Performance Material India Limited, Thane, India.

Chromatographic conditions and Equipment:

Analysis was carried out on a Shimadzu LC_2010 CHT HPLC with UV detector. The output signal was monitored and processed using LC solution software. The chromatographic column used was C18 (150mm × 4.6mm, 5µ). Gradient elution process was adopted throughout the analysis. Mobile phase used was 85:15% v/v (ACN: Ammonium acetate (pH 5.0 adjusted with glacial acetic acid).

Instrumental parameters:

The gradient flow of mobile phase was maintained at 1.0 ml/min. The injection volume was 20µL. Eluted sample was monitored at 215nm and run time was 10 min. The retention time of the sample was about 2.519 min.

Preparation of standard and test solutions:

Preparation of stock solution:

Stock solution for method validation were prepared by accurately weighing 100mg of Bilastine and dissolving it in 100ml of methanol by gentle stirring to yield final concentration of 1000µg/ml.

Preparation of working standard solutions:

Working standard solution was prepared by accurately transferring the (0.25, 0.50, 0.75, 1.0, 1.25, 1.5ml) aliquot of the standard stock solution in a series of 10 volumetric flask. The volume was made up to mark mobile phase to obtain concentration of 25-150µg/ml.

Preparation of sample solutions:

Twenty tablets of marketed formulation, BYLOZA (20 mg) were taken and weight of average content was determined. Weight equivalent to 20mg Bilastine was transferred to 100ml volumetric flask and dissolve in methanol. Solution was sonicated and filtered through Whitman filter paper. Average weight of tablet was calculated 113.2mg.

Forced degradation study:

Forced degradation studies of the drug were carried out under conditions of acid hydrolysis, alkali, neutral, oxidative, thermal, photolytic degradation.

Acid Hydrolysis:

Forced degradation in acidic media was performed by taking 2ml stock solution of Bilastine to 10ml volumetric flask. Add 2ml of 2 N HCL in volumetric flask and kept 80º C for 30 min. Then neutralized it with 2 N NaOH and diluted up to the mark with mobile phase. Solution strength of 100µg/ml.

Alkali Hydrolysis:

Forced degradation in basic media was performed by taking 2ml stock solution of Bilastine to 10ml volumetric flask. Add 2ml of 2 N NaOH in volumetric flask and kept at room temp. 80ºC for 2 hrs. Then neutralized it with 2 N HCL and diluted up to the mark with mobile phase. Solution strength of 100µg/ml.

Neutral Hydrolysis:

Forced degradation in neutral degradation, was performed by taking 2ml stock solution of Bilastine to 10ml volumetric flask. Add 2ml of HPLC grade water in volumetric flask and kept at 80ºC for 2 hrs. Diluted up to the mark with mobile phase. Solution strength of 100 µg/ml.

Oxidative Degradation:

Forced degradation in oxidative condition was performed by taking 2ml stock solution of Bilastine to 10ml volumetric flask. Add 2ml of 10% H₂O₂in volumetric flask and kept at 80ºC for 30 min. Diluted up to the mark with mobile phase. Solution strength of 100µg/ml.

Thermal Degradation:

Forced degradation in thermal degradation, 10mg accurately weighed amount of Bilastine was exposed to 70ºC for 8 hrs. After this exposure, the drug powder was mixed and transferred in to 10ml volumetric flask, dissolved in methanol and diluted up to mark with diluent. Final dilution was done with sample diluent to make final concentration of 100µg/ml.

Photolytic Degradation:

Forced degradation in photolytic degradation, 10mg accurately weighed amount of Bilastine was exposed to 254nm for 24 hrs. After this exposure, the drug powder was mixed and transferred in to 10ml volumetric flask, dissolved in methanol and diluted up to mark with diluent. Final dilution was done with sample diluent to make final concentration of 100µg/ml.

Then 20μl solution of above solutions were injected into HPLC system and analyzed under the chromatographic condition described earlier

Method validation:

Linearity and Range:

Aliquots of working standard solution (0.25, 0.50, 0.75, 1.0, 1.25, and 1.5ml) were transferred into series of 10 ml volumetric flask and diluted up to mark with methanol to obtain final concentration of 25-150µg/ml and mixed properly. 20µl aliquots of each solution were chromatographed three times and analysis was performed by optimized method. The regression equation was derived from the plot of average area of Bilastine peak against the concentration of Bilastine.

Precision:

Intra-day Precision:

Intraday precision was determined by analyzing of Bilastine standard solutions in the range 50, 75 and 100 µg/ml of it triplicate in a day. Percentage RSD for Bilastine was calculated.

Inter-day Precision:

Inter-day precision was determined by analyzing of SAR standard solutions in the range 50, 75 and 100µg/ml of it in three different days. Percentage RSD for Bilastine was calculated.

Accuracy

Accuracy was determined by calculating recovery of Bilastine by the standard addition method. The known amounts (4.0, 5.0 and 6.0ml) of working standard solutions of Bilastine (100µg/ml) were added to 2ml sample solution of Bilastine (100µg/ml) in 10ml of volumetric flask and diluted up to mark with methanol. Each solution was injected triplicate and recovery was calculated from regression equation of calibration curve by measuring peak areas.

Limit of Detection and Limit of Quantification:

LOD and LOQ of the drug were calculated using following equations according to ICH guidelines.

LOD = 3.3 σ/s and LOQ = 10 σ/s were found.

Where, σ is the SD of the response

S is the slope of the calibration curve.

Robustness:

The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by changing four small changes like Flow rate (1.0 ± 0.2 ml/min), Organic phase (70± 5ml), Injection Volume (20 ±5µL), pH (5.0±0.5). After each sample solution was injected and area, HETP, tailing factor and retention time were checked.

An aliquot of 20µL from sample solution was injected under a chromatographic condition and peak area was measured and % assay was calculated from regression equation. Response was an average of six determinations.

RESULTS AND DISCUSSION:

Selection of wavelength for measurement:

To determine Wavelength for measurement, standard spectra of Bilastine was scanned between 200-400nm. It is evident that Bilastine show an absorbance at 215nm respectively. The chematogram for standard and test bisltine were shown in figure 2 and 3.

Optimized Chromatographic Condition:

HPLC System: LC 2010 CHT (Shimadzu)

Column: C18 Waters (150mm x 4.5mm, 5µm)

Mobile Phase: ACN: Ammonium acetate (pH 5.0 adjusted with glacial acetic acid) (85:15 %v/v)

Flow rate: 1.0ml/min

Detection Wavelength: 215nm

Injection volume: 20µL

Column oven temperature: Room temperature

Run time: 10 min

Diluents: Methanol

Results of forced degradation study:

From degradation study it was found that Bilastine was marginally degraded in acid and oxidative conditions and stable in alkali, neutral, thermal, and photolytic conditions. Results of forced degradation study was shown in table 1.

Table: 1 Result of Forced Degradation Study of Bilastine

Sr. No.	Stress type	Condition	No. of peaks	% Degradation
1	Acid Hydrolysis	2 N HCl at 80ºC for 30 min.	1	7.25
2	Alkali Hydrolysis	2 N NaOH at 80ºC for 2 hr.	-	-
3	Neutral Hydrolysis	H₂O at 80ºCfor 2 hr.	-	-
4	Oxidative Degradation	10% H₂O₂at 80ºC for 30 min.	1	5.24
5	Thermal Degradation	At 70ºC for 8 hr.	-	-
6	Photolytic Degradation	UV 254 nm for 24 hr.	-	-

Linearity and Range:

The linearity range for Bilastine was found to be in the range of 25-150µg/ml. Linearity shown in the “Fig. 4.” And linearity data are shown in table 3.

Figure: 4 Clibration curve of Bilastine

Precision:

The %RSD of Intra-day and Inter-day for bilastine was found to be 0.64 to 0.81 and 0.14 to 0.32 respectively.

Accuracy:

Accuracy of the method was confirmed by recovery study from marketed formulation of bilastine at three level (80% - 120%) of standard addition. Percentage recovery for Bilastine was 99.56 - 101.06%, shown in table 2.

Table: 2 Result of Accuracy study for Bilastine

Level	Test conc. (µg/ml)	Std. conc. (µg/ml)	Amt. of found ± S.D.	% Recovery
80%	50	40	89.69±0.87	99.66
100%	50	50	101.06±0.76	101.06
120%	50	60	109.52±1.10	99.56

* Average of three determinations

Limit of Detection and Limit of Quantitation:

LOD and LOQ was found to be 0.45µg/ml and 1.20 µg/ml respectively.

Robustness:

The typical variations studied under these parameters are flow rate, mobile phase composition, column temperature, change in pH. According to data comparison develop method was robust.

Analysis of marketed formulation:

Applicability of the proposed method was tested by analysising the available tablet formulation BYLOZA.

All method validtion parametre of bilastine results were shown in table 3.

Table 3: Validation Parameter of Bilastine

Sr. No.	Parameter	Bilastine
1.	Linearity Range	25-150 µg/ml
2.	Regression Line equation	Y=27691x+ 914034
3.	Correlation co-efficient	0.996
4.	Precision (%RSD)
	Intra-day Precision	0.637-0.807
	Inter-day Precision	0.140-0.325
5.	Accuracy (%Recovery)	99.78-100.53
6.	Limit of Detection(µg/ml)	0.41
7.	Limit of Quantification(µg/ml)	1.25
8.	% Assay	101.1
9.	Robustness (% RSD of Assay)	0.85

CONCLUSION:

The developed method was found to be simple, sensitive and selective for analysis of Bilastine. Bilastine was marginally degraded in acidic and oxidative conditions and was found to be stable in all other conditions. Percent degradation was calculated by comparing the areas of the degraded peaks in each degradation condition with the corresponding areas of the peaks of the drugs under non degradation condition. In the proposed study, stability-indicating RP-HPLC method was developed and validated as per ICH guidelines for the estimation of Bilastine.

ACKNOWLEDGEMENTS:

The authors are thankful to the Shri Sarvajanik Pharmacy College, Mehsana, Gujarat, India, for providing necessary infrastructure facilities. The authors are also highly grateful to the Dr. C. N. Patel sir for the generous gift sample of pure Bilastine.

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Received on 06.01.2021 Modified on 30.04.2021

Asian J. Pharm. Ana. 2021; 11(3):183-187.

DOI: 10.52711/2231-5675.2021.00031