INTRODUCTION:

Bilastine is a piperidine class of antihistamine drug that binds with H₁ receptor and prevents the activation of H₁ receptor by histamine¹. Bilastine (Figure1) is used for the treatment of allergic disorders such as rhinoconjunctivitis and urticaria². It is chemically named as 2-[4-(2-{4-[1-(2-ethoxyethyl)-1H-1, 3-benzimidazol-2-yl]piperidin-1-yl}ethyl)phenyl]-2-methylpropanoic acid. Bilastine is a white crystalline powder with molecular formula C₂₈H₃₇N₃O_3, molecular mass of 463.61g/mole and melting point greater than 195⁰ C³. It is useful for allergic patients receiving treatment for other concomitant diseases such as with renal/ hepatic dysfunction⁴.

Bilastine can be administered orally and is available as a tablet. Peak plasma concentration can be attained within 1.3 hours after administration and have prolonged duration of action⁵. From the review of literature, it was found that very few methods such as LC-MS/MS⁶, HPLC-fluorescence⁷in biological sample, RP-HPLC^8-9, HILIC¹⁰and UV- spectrophotometry^11-12 are available for estimation of Bilastine. The aim and objective of the present work was to develop a simple, fast, accurate, precise, economic and stability indicating method for quantification of Bilastine in both bulk and pharmaceutical formulation, which can be used for routine analysis in QC laboratories. The method was validated as per ICH guidelines^13-14.

Figure 1: Structure of Bilastine

MATERIALS AND METHODS:

The chromatography analysis was performed using Shimadzu Model CBM-20A/20 Alite HPLC system (Shimadzu Co., Kyoto, Japan) equipped with SPD M20A prominence photodiode array detector. The method was developed using C18 column (150 mm × 4.60 mm i.d. 5µm particle size) maintained at room temperature. The mobile phase used was a mixture of formic acid and methanol (50:50).

Chemicals and reagents

Pure analytical sample of Bilastine was used in the present work. Bilastine marketed formulations, Bilasure (20 mg) (Sun Pharmaceutical Industries Pvt.Ltd) and Bilanta (20 mg) (Ajanta Pharma Ltd) were purchased from local pharmacy.

Preparation of Bilastine drug solution

Accurately weighed 25 mg of Bilastine was taken in a 25mL volumetric flask and the volume was made up to the mark with HPLC grade methanol (1000 μg/mL). Further, dilutions were made with mobile phase and the solutions were filtered through 0.45 μm membrane filter prior to injection.

Method optimization

Mobile phase (Formic acid: Methanol)	Flow rate	Reten tion Time (min)	Theo retical Plates	Tailing Factor	Comments
40:60	0.8 mL	1.815	2264	1.495	Rt< 2
45:55	0.8 mL	1.929	2227.963	1.444	Rt < 2
50:50	0.8 mL	2.167	2096.682	1.079	Method optimized

Method validation

Linearity

Linearity was performed by preparing a series of Bilastine solutions (5–100 μg/mL) from the stock solution and diluted with mobile phase. Each concentration was injected in triplicate into the system. The mean peak area of Bilastine was calculated from the obtained chromatograms. The acquired data was checked by plotting a calibration graph taking the concentration of the Bilastine solutions on the x-axis and the corresponding mean peak area values on the y-axis.

Precision

Precision means repeatability of results for a particular method. Each stage of precision was investigated by three replicates of injections with concentrations of 10, 20 and 50 μg/mL. The peak area of these solutions were noted individually thrice within a day and the mean peak area was calculated (Intraday precision). Similarly, the peak areas of these solutions were individually taken thrice in three days and recorded (Interday precision). The precision was expressed by % RSD.

Accuracy

Accuracy of the methods was analyzed by the percentage recovery of the standard drug that was added to the fixed concentration of sample solutions. The study was carried out by adding three different percentage levels of standard drug i.e. 50%, 100%, 150% to the sample concentration. Each solution was prepared in triplicate and the mean peak area was noted to find out the percentage recovery.

Limit of Detection (LOD), Limit of Quantification (LOQ)

LOD and LOQ are two different parameters useful for determination of lowest concentration of drug that can be detected and quantified suitably. LOD and LOQ determinations were done using standard deviation of the response (σ) by slope of the calibration curve (S) mathematical values. The σ/S values were multiplied with 3.3 for LOD and 10 for LOQ determinations.

Forced degradation studies

Forced degradation studies were performed as per ICH guidelines to determine the ability of the drug to withstand its properties in the applied stress conditions. Bilastine was exposed to different stress conditions such as acidic hydrolysis, basic hydrolysis, oxidation and thermal treatment.

Acid degradation was performed by treating the drug solution with 0.5 mL of 0.1 N HCl and then the sample was heated at 60ºC for about 30 minutes on a water bath. The stressed sample was then cooled and neutralized with 0.5 mL of 0.1N sodium hydroxide solution. The solution was made up to final volume with the mobile phase. 20 μl of the solution was injected in to the system.

Alkaline degradation was performed by treating the drug solution with 0.5 mL of 0.1 N NaOH and then the sample was heated at 60ºC for about 30 minutes on a water bath. The stressed sample was then cooled, neutralized with 0.5mL of 0.1N hydrochloric acid and diluted with mobile phase. 20 µl of the solution was injected in to the system.

Thermal degradation was performed by heating the drug solution at 60ºC for 30 minutes on a water bath. The solution was then cooled and diluted with mobile phase. 20 μl of the solution was injected in to the system.

Oxidative degradation was performed by adding 0.5 mL of hydrogen peroxide to the drug solution and was heated at 60ºC for 30 minutes on a water bath. The solution was then cooled and diluted with mobile phase. 20 μl of the solution was injected in to the system.

Assay of Bilastine tablets

Two different brands of Bilastine tablets with 20 mg (API) lable claim were used for the assay method. 20 Bilastine tablets were weighed and triturated into powder. Tablet powder weight equivalent to 25 mg was taken into a 25 mL volumetric flask, dissolved in methanol, sonicated for 30 minutes and filtered using 0.45 mm membrane filter. The filtered solution was made upto the volume with methanol. Aliquots of sample solutions were taken and diluted with mobile phase. 20 µL of this solution was injected in to the system. From the resultant chromatogram peak area and retention time were noted.

RESULTS AND DISCUSSION:

A new stability indicating RP-HPLC method was developed for the quantification of Bilastine in tablets. The chromatographic separation was achieved on a C18 column (150 mm × 4.60 mm i.d. 5µm particle size) using formic acid: methanol (50:50 v/v) as mobile phase and a flow rate of 0.8 mL/min (UV detection at 282nm) for the determination of Bilastine. A sharp peak was observed at 2.167 min (Run time 10 min). The optimized chromatographic conditions were shown in Table 1.

Table 1: Optimized chromatographic conditions.

Parameter	Optimized chromatographic conditions
Mobile Phase	Formic acid : methanol (50:50)
Stationary Phase	C18 Phenomenex column (150 mm × 4.60 mm i.d.; 5µm particle size)
Flow Rate	0.8 mL/min
Detection wavelength	282nm
Column temp.	(25°±2°C)
Injection Volume	20 µL
Detector	SPD M20A prominence photodiode array detector
Elution	Isocratic mode
Total Run Time	10 min
Retention time	2.167

Linearity

Bilastine has shown linearity over the concentration range 5–100 μg/mL (Table 2) with linear regression equation y = 15283x + 1079.7 (R² = 0.9999) (Figure 2). The tailing factor for Bilastine was less than 2 and the theoretical plates were more than 2000. The lowest amount of drug that can be detected (LOD) and quantified (LOQ) were found to be 0.08931μg/mL and 0.27063 μg/mL, respectively.

Table 2: Linearity of Bilastine.

Conc. (µg/mL)	*Mean peak area
5	75903
10	151807
20	303615
25	388313
50	765434
75	1159341
100	1519867

Figure 2: Calibration curve of Bilastine

Precision

Intra day and inter day precisions were found to be 0.034-0.136 and 0.045-0.112 which was within the limit (NMT 2.0%). The results of precision were shown in Table 3a and Table 3b.

Table 3a: Intraday precision study of Bilastine.

Conc. (µg/mL)	*Mean peak area	Statistical Analysis
Conc. (µg/mL)	*Mean peak area	Mean ± SD (% RSD)*
10	151807	151615.7±206.3621 (0.1361)
10	151643
10	151397
20	303615	303282±316.9527 (0.1045)
20	303247
20	302984
50	765434	765224.3±263.8983 (0.03448)
50	765311
50	764928

*Mean of three replicates

Table 3b: Inter day precision study of Bilastine.

Conc. (µg/ mL)	*Mean peak area			*Mean ± SD (% RSD)
Conc. (µg/ mL)	Day 1	Day 2	Day 3	*Mean ± SD (% RSD)
10	151615.7	151923	151894	151810.7± 170.0716 (0.1120)
20	303282	303561	302989	303277.3± 286.0286 (0.0943)
50	765224.3	765816	765199	765413.1± 349.1509 (0.0456)

*Mean of three replicates

Accuracy

Three concentrations of 50%, 100%, 150% were injected in a triplicate manner and the mean% recovery was found to be 99.8% which was in the acceptance limit of 98.0 to 102.0%. The accuracy data was represented in Table 4.

Assay of Bilastine tablets

Two different brands of Bilastine tablets with 20 mg (API) lable claim were used for the assay method. The amount of Bilastine was found to be 99.87-99% (Table 6) and there was no interference of excipients.

Table 6: Assay of Bilastine tablets

S.No.	Brand name	Label claim (mg)	Observed amount (mg)	% Recovery*
1	Brand I	20	19.97	99.87
2	Brand II	20	19.8	99

*Mean of three replicates

CONCLUSION:

The RP-HPLC technique was validated as per ICH guidelines and was found to be simple, precise, accurate and useful for quantification of Bilastine tablets.

ACKNOWLEDGEMENTS:

The authors are thankful for the management of GITAM (Deemed to be University), Visakhapatnam, Andhara Pradesh, India, for providing necessary facilities to carry out the research work.

REFERENCES:

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Received on 08.03.2019 Modified on 28.05.2019

Research J. Pharm. and Tech 2020; 13(6): 2849-2853.

DOI: 10.5958/0974-360X.2020.00507.7

Spiked Conc. (µg/mL)	Formulation (µg/mL)	Total Conc. (µg/mL)	*Conc. Obtained (µg/mL) ±SD (%RSD)	% Recovery
10 (50%)	20 20 20	30 30 30	29.92±0.0916 (0.306)	99.7
20 (100%)	20 20 20	40 40 40	39.90±0.2645 (0.662)	99.75
30 (150%)	20 20 20	50 50 50	49.91±0.1 (0.2004)	99.8

Stress condition	Rt (min)	Peak area	% Recovery*	% Drug degradation	Theoretical Plates (>2000)	Tailing factor (<1.5)
Standard drug	2.167	303615	100	----	2469.475	1.167
Acidic degradation 0.1N HCl/ 60⁰ C/ 30 min	2.294	298103	98.18	1.81	4454.689	0.913
Alkaline degradation 0.1N NaOH/ 60⁰C/ 30 min	2.299	294352	96.94	3.06	13441.569	0.830
Oxidative degradation H₂O₂/ 60⁰C/ C 30 min	2.157 2.289	296654	97.70	2.3	2489.016	---
Thermal degradation /60⁰C /30 min	2.141	293916	96.80	3.2	2373.726	1.194