1. INTRODUCTION:

Bilastine, 2-[4-(2-(4-(1-(2-ethoxyethyl)-1H-benzimidazol-2-yl) piperidin-1-yl) ethyl) phenyl]-2-methylpropionic acid, (Fig. 1) is a new generation of highly selective antihistamine for the H1 histamine receptor in both in vivo and in vitro studies, and with no clear affinity for other receptors, it has a rapid onset and prolonged period of action¹. It has been shown to be fully sustained and safe in the healthy population at all doses^2-4, which has not shown any calming or cardio-toxic effects in clinical trials and in postmarketing expertise¹.

Fig. 1: Chemical structure of bilastine

Bilastine is an antiallergenic agent, which helps alleviate allergic symptoms such as nasal inflammation and urticarial by combining and preventing H1 receptor activation. Bilastine has decreased the severity of allergic effects due to histamine release from mast cells ⁵. In most European countries, bilastine has recently been awarded branding approval in the form of solid unit-dose preparations (tablets), at a dose of 20mg per unit. Bilastine is a non-compendia drug, and both the European and United States Pharmacopeia still have no compendia methods for testing. However, a few studies have considered the effectiveness, health, and pharmacokinetics of bilastine and its evaluation in biological materials^6-10. A review of the literature showed that only few chromatographic and spectrophotometric techniques have been studied for determining bilastine in dosage forms^1,2,8,11-13. Thus, this study developed a simple, fast, efficient, accurate, reproducible, time-effective, and reliable new validated chromatographic method for analyzing bilastine in Pharmabilast^(R) 20 mg tablet. The procedure mentioned throughout this study was been checked by evaluating these analytical variables: specificity, linearity, precision (repeatability and intermediate precision), accuracy, robustness, and ruggedness as recommended in ICH guidelines^14,15

2. EXPERIMENTAL:

2.1 Reagents and chemicals:

Bilastine (< 99.4% purity) and Pharmabilast tablets were purchased from Pharma Cure for Pharmaceutical Industries. Acetonitrile was HPLC grade from J.T. Baker, methanol (Merck), hydrochloric acid (HCl 38%) (Sigma Aldrich), potassium dihydrogen phosphate (KH₂PO₄), orthophosphoric acid, and all other chemicals were of analytical grade. Bi-distilled water was used as a solvent for all preparations and dilution processes during the study.

2.2 Apparatus:

Agilent HPLC system with an isocratic pump (G1311C), a DAD detector system (G1315D), and a thermostatted column (G1316A) compartment was used for chromatographic analysis. The data were reported using chemstation software version 1.25. A digital pH meter (HANNA) was used for pH measurements, and degradation tests were performed using water bath (JSWB-22T model) in acidic, alkaline, and neutral conditions. Dry air oven (Titanox, Italy) was used to evaluate the impact of dry heat. The chromatographic separation was performed using C18 BDS Hypersil thermo column (250 × 4.6mm i.d), 5 µm particle size. An Agilent Technology 1260 series HPLC system with UV visible detector and an auto sampler was used. During the study, an analytical balance (Shimadzu AUW220 balance, Japan), Vacuum filtration assembly (TID 15, Mumbai, India), and ultrasonic bath were used.

2.3 Chromatographic parameters:

Column: BDS Hypersil thermo, C18, 5µm 4.6 × 250mm or equivalent.

Rate of flow: 1.5ml per min.

Detector: UV at λ 220nm.

Temperature: 40°C.

Injection volume: 20µl.

Mobile Phase: Buffer pH 6 (50%): Methanol (25%): Acetonitrile (25%)

Run Time: 6 Min

2.4 Buffer solution:

Weigh about 6.8gm potassium dihydrogen phosphate reagent, and then add 1.0ml triethylamine and 300ml bi-distilled water to dissolve. Adjust the pH to 6.0±0.05 using phosphoric acid and complete volume to 1000ml by bi-distilled water, then filter using a membrane filter 0.45μ.

2.5 Preparation of standard solution:

24mg bilastine was precisely weighed and passed into a 100ml volumetric flask. Add 30ml of solvent mixture (50% acetonitrile with 50% bi-distilled water) and sonicated for 5 min and then complete to mark with the same solvent. Transfer 5ml of this solution to 50ml volumetric flask and complete the volume with the same solvent. This concentration (24μg/mL) was considered a 100% concentration. A solution containing five various concentrations of bilastine (60%, 80%, 100%, 120%, and 140% of target concentration) was similarly provided.

2.6 Pharmabilast^(R) sample solution preparation:

Ten tablets of Pharmabilast^(R) 20mg were weighed separately and their average weights were determined. A quantity of the finely ground powder equivalent to one tablet was carefully weighed and transferred into a calibrated 100ml volume flask containing 30ml of the solvent mixture. The flask content was sonicated for approximately 5 min and then the solvent mixture was used to complete the volume. Then transfer 6ml of this solution to 50ml volumetric flask and complete the volume with the same solvent to reach the final working sample concentration of 100% of target concentration. The solution was filtered through a 0.45µm milli-pore filter (Gelman, Germany) to isolate the insoluble excipients, and discard the initial filtrate segment.

2.7 HPLC method progress and validation:

This study was conducted to obtain a novel, safe, cost-effective, and flexible method for evaluating bilastine in tablet dosage form with HPLC. The approach has been accepted according to ICH Harmonized Tripartite Guideline. The method was tested for these variables system suitability, specificity, linearity, precision, accuracy, LOD, LOQ, and robustness^14,16-26.

2.7.1 System Suitability:

Six replicate analyses of the solution containing 100% target concentration of bilastine were performed to test the system suitability. Different chromatographic variables such as retention time, peak area, tailing factor, and column theoretical plates were calculated and the method was validated.

2.7.2 Specificity:

The impact of excipients on the testing result was evaluated. A standard sample of bilastine was first injected to determine the specificity of the method, after which market product, blank and excipients solutions were conducted using the instrument one after another.

2.7.3 Forced degradation studies:

The forced degradation reactions were performed using the drug concentration of 24µg/ml to prove the stability of the analytical method for assay. The sample was subjected to stress at different effects of acid hydrolysis, base hydrolysis, oxidation, heat, and UV photolytic as reported in ICH Q1A (R2) as follows: Drug heat and UV light products were examined in a solid state while solutions were prepared for acid, base hydrolytic, and oxidative degradation. Due to free solubility, all solutions used in forced degradation studies were given by dissolving drug product in a small volume of solvent mixture, and then hydrochloric acid, sodium hydroxide, or hydrogen peroxide was added. All solutions were diluted with a solvent mixture to achieve a final concentration after degradation. Without effect: Assay was prepared as mentioned before in sample preparation. Acid hydrolysis: The sample was stressed by 5ml of 1 M HCl at 80°C for 1 h, then cooled at room temperature, neutralized with 5ml of 1M NaOH, then the volume was completed with a solvent mixture. Photolytic effect (using a mixture of cool white and ultraviolet fluorescent lamps): The sample was exposed under light for 24 h, then the volume was completed with a solvent mixture. Base hydrolysis: The sample was stressed by 5 ml of 1 M NaOH at 80°C for 1 h, cooled at room temperature, neutralized with 5ml of 1.0 M HCl, and then the volume was completed with a solvent mixture. Peroxide degradation: The sample was stressed by 5 ml of 30% H₂O₂ at 35°C for 30 min, then volume was completed with a solvent mixture. Heat degradation: The sample was heated at 80°C for 2 h, and the volume was completed with a solvent mixture. The acceptance criteria were as follows: The existence of satisfying peak purity to prove that the bilastine chromatographic peak was unrelated to more than one component (Bilastine peak is pure) and the percentage of degradation should be (10% – 30%)²⁷. All the prepared diluted solutions were filtered through 0.45µm membrane filter and injected into the HPLC system.

2.7.4 Linearity:

Construction of calibration curves determined the linearity of the system. For this reason, a standard solution of various concentrations of bilastine (60%, 80%, 100%, 120%, and 140%) was used. Measurements were performed triply, and the chromatogram peak areas were plotted against the concentrations to obtain the calibration curves and correlation coefficients.

2.7.5 Accuracy (Recovery Studies):

Accuracy is the percentage of analyte retrieved from a fixed added quantity by test. Retrieval studies were conducted using the standard addition method triply at each grade (80%, 100%, and 120%) to monitor the degree of accuracy of the technique. Fixed concentrations of bilastine standard were added to preanalyzed tests and submitted to the presented HPLC separation method.

2.7.6 Precision:

Precision could be considered as repeatability (intra-day precision) and intermediate precision (inter-day precision). The precision of a set of measurements has been generally described as standard deviation.

2.7.7 Limit of detection (LOD) and limit of quantitation (LOQ):

LOD and LOQ were calculated according to ICH guidelines¹⁴ as follows:

LOD = 3.3 × SD of blank/Slope

LOQ = 10 × SD of blank/Slope

2.7.8 Robustness of Technique:

Limited systematic deviations in parameter of optimized system were performed to estimate the effectiveness of an HPLC separation technique. The impact of flow rate, temperature, mobile phase ratio (organic change), and different column on the retention time and tailing factor was examined. The technique has shown its suitability during normal usage.

3. RESULTS AND DISCUSSION:

The chromatographic conditions for determining bilastine in dosage form were optimized during the developmental phase of the method when it has investigated the influence of different parameters as column type, pH and type of solvent, and composition of the mobile phase. Finally, optimized chromatographic conditions have been reported in chromatographic parameters section.

3.1 System suitability parameters:

Outcomes of system suitability have been reported in Table 1. The standard solution was injected six times and % RSD was established not more than 2.0% for system suitability parameters: retention time (Rt) and peak area of 0.09% and 0.22%, respectively. Moreover, theoretical plates 6777 and tailing factor 1.07 were obtained, indicating a good system for analysis.

Table 1: System suitability data obtained from HPLC chromatograms:

Test No.	Peak area	R.T (Min.)
1	620.0	3.95
2	623.5	3.95
3	622.3	3.95
4	623.4	3.95
5	623.8	3.96
6	623.1	3.95
Average	622.7	3.95
% RSD	0.22%	0.09%

3.2 Specificity:

The specificity test showed that the standard (Fig. 2 A) and sample (Fig. 2 B) are comparable with respect to retention time as in Fig. 2.

3.3 Forced degradation studies:

Pharmabilast^(R) 20mg tablet was stressed under different stress conditions and all the obtained results satisfied the peak purity factor. Results for the assay of bilastine for the above-mentioned drastic conditions are presented in Table 2. Results showed that bilastine is susceptible to the thermal, alkaline, acidic, and H₂O₂states; however, it has been fixed in light stress condition.

Table 2: Bilastine stability in experimental Pharambilast^(R) 20mg tablet preparation after stress testing on different stress conditions

Condition	% Recovery	Peak Purity Factor
Without effect	101.35%	0.999
Photolytic effect	100.26%	0.999
Thermal effect 80°C 2 Hrs	87.63%	0.999
1M HCl 5ml effect + 80°C 1 Hrs	89.46%	0.999
1M NaOH 5ml effect + 80°C 1 Hrs	87.84%	0.999
30% Oxidative effect (5ml)	88.88%	0.999

3.4 Linearity, Range, LOD, and LOQ:

Linearity covered the concentration range of 14.4 µg/ml to 33.6µg/ml at 220nm with a significantly high correlation coefficient value of R²= 0.999 (Fig. 4). As shown in Fig. 4, a linear relationship was obtained between concentrations and the area response of peak (calibration curve) in the limit of 60%–140% of the nominal concentration. The obtained LOD, LOQ, and all statistical data are summarized in Table 3.

Fig. 4 Plot graph of concentration against the average peak area responses (calibration curve).

Table 3: Bilastine statistical results obtained from the calibration curve.

Parameters	Bilastine
Regression equation	Y = 27.01X + 5.50
Linearity range (µg/ml)	14.4 to 33.6
Slope	27.01
Intercept	5.50
Standard deviation	0.29
Correlation coefficient (R²)	0.999
Limit of detection (LOD) µg/ml	0.04
Limit of quantification (LOQ) µg/ml	0.11

3.5 Accuracy:

The average of retrieval percentage at three grades, 80%, 100%, and 120% after mixing with standard ranged from 98.44% – 101.87%, which is within the accepted limits between 98%–102%²⁸ (Table 4). An excellent approval was obtained between spiked and measured values, confirming the accuracy of the method.

Table 4: Bilastine accuracy (% Recovery) results.

Sample No.	Spiked amount (mg)	Recovered amount (mg)	% Recovery	% Average recovery
1	16	16.28	101.72%	99.99%
2	16	16.30	101.85%
3	16	16.30	101.87%
4	20	19.77	98.87%
5	20	19.69	98.44%
6	20	19.74	98.69%
7	24	23.77	99.05%
8	24	23.85	99.36%
9	24	24.01	100.03%

3.6 Precision:

Outcomes of intraday and interday contrast are tabulated in Table 5. Relative standard deviation (% RSD) values of peak areas were counted for various samples (Table 5). From these results, it can be concluded that this method is extremely exact because percent peak area RSD is not more than 2% for total samples.

Table 5: Bilastine ruggedness and intermediate precision results.

Inter-day
Sample (n)	1^st analyst 1^st day	2^nd analyst	2^nd day
1	101.71%	98.11%	101.11%
2	98.20%	100.30%	99.03%
3	98.85%	101.22%	98.01%
4	98.41%	101.57%	100.43%
5	98.07%	101.03%	98.38%
6	99.38%	99.61%	99.57%
Mean (%)	99.10%	100.31%	99.42%
RSD%	1.37%	1.28%	1.20%
Intraday mean values % (n=18)
Mean (%)	99.61%
RSD (%)	1.32%

3.7 Robustness of the method:

The outcomes of durability of the proposed technique showed a slight alteration in the rate of flow, mobile phase ratio (buffer ratio change) and different columns, and temperature did not produce significant alterations in the analytical outcomes, showing that the technique is durable (Table 6).

Table 6: Results for robustness test of bilastine

Parameters	Changes	RT (min)	USP tailing	USP Plate
Column	1	3.9	1.07	6777
Column	2	3.9	1.22	6566
Flow rate (ml/min)	1.575	3.8	1.14	5628
Flow rate (ml/min)	1.425	4.0	1.12	5884
Temperature ºC	42	3.9	1.15	5837
Temperature ºC	38	3.9	1.12	6310
Buffer ratio change %	55	4.3	1.08	6558
Buffer ratio change %	45	3.6	1.11	6400

4. CONCLUSION:

The HPLC separation technique showed rapid detection of an antiallergenic agent (Bilastine) as a active pharmaceutical ingredient only or mixed with excipients in Pharmabilast® 20mg tablet. The adequate exactness and regularity have been assured in evaluating the minimal concentricity of medication in its strength by HPLC separation technique. The technique has demonstrated several advantages as an easy, specific, low cost, and accurate method. It could be appropriate for routine evaluation at laboratories and convenient for quality management of raw materials, formulations, and dissolution study, and could be used for a bioequivalence study with identical formulation.

5. ACKNOWLEDGMENT:

The authors would like to acknowledge Pharma Cure for pharmaceutical industries for providing equipment and accessories.

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Received on 08.10.2020 Revised on 15.01.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):57-62.

DOI: 10.52711/2231-5675.2021.00011