INTRODUCTION:

Bilastine is Histamine and cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators involved in both Seasonal Allergic Rhino Conjunctivitis (SARC) and Asthma.¹ It is 2-[4-[2-[4-[1-(2-ethoxyethyl)benzimidazole-2-yl]piperidin-1-yl]ethyl]phenyl]-2-methylpropanoic acid as shown in Figure 1² By binding and preventing to the activation receptor, the reduce event of allergic indication because of discharge of a tissue cell.³

Figure 1: Structure of Bilastine

Montelukast is a potent, selective and orally active antagonist of the cysteinyl, CysTL1, leukotriene receptor used for the treatment of asthma in children and adults.⁴ It is 2-[1-[[(1R)-1-[3-[(E)-2-(7-chloroquinoline-2-yl) ethenyl] phenyl]-3-[2-(2-hydroxypropanyl) phenyl] propyl]sulfanaylmethyl] cyclopropyl] acetic acid as shown in Figure 2.⁵ Montelukast blocks the action of leukotriene D4 on the cysteinyl leukotriene receptor CysLT1 in the lungs and bronchial tubes by binding to it⁶.

Figure 2: Structure of Montelukast

Bilastine is an antihistamine that is used to relieve the symptoms of allergic rhino conjunctivitis (sneezing, itchy nose, nasal secretion, nasal congestion and red, streaming eyes) and other forms of allergic rhinitis. Montelukast is used for a number of conditions including asthma, exercise induced bronchospasm, allergic rhinitis, and Urticaria.

Bilastine Tablet: 20mg Tablet, Orally disintegrating: 10mg Solution: 2.5mg/ml and Montelukast Granule: 4mg.

Tablet, chewable: Oral: 5.2mg Tablet, Film coated: Oral: 10mg.

The purpose of study I hypothesized that Bilastine with Montelukast is superior to Bilastine monotherapy in reducing SARC symptoms after 4 week and in improving asthma quality of life over a longer time period.

Several analytical methods have been reported in the literature for the analysis of Bilastine and Montelukast individually and combined with other drugs. The analytical methods determination of Bilastine includes UV Spectrometric^7-8, Quality by Design⁹, HPLC^10-13 and Stability Indicating UPLC¹⁴. The analytical methods determination of Montelukast includes Derivative UV Spectrometric^15-16, HPTLC¹⁷, HPLC¹⁸ with single or combination with other drugs. The UV Spectrometric²⁹ and HPLC²⁰had been reported for the Bilastine and Montelukast in combined pharmaceutical dosage form.

However, as per our best knowledge, no stability indicating HPTLC method for Bilastine and Montelukast had been developed. The International Conference on Harmonization (ICH) stability test guideline requires that analytical test procedures used for samples should be stability indicating and validated. The present work describes new method for stability indicating HPTLC method for Bilastine and Montelukast.

MATERIALS AND METHODS:

Instrumentation:

A Camag HPTLC system (Switzerland) comprising of Camag Linomat V semiautomatic sample applicator, Camag TLC Scanner 3, Camag (Muttenz, Switzerland) flat bottom and twin trough developing chamber (10x10 cm), UV cabinet with dual wavelength UV lamp, Camag win CATS software, Hamilton syringe (100μl), analytical balance (wensar), were used in the study.

Materials

Bilastine was obtained from Gift sample procured from Kimia Bioscience (Delhi) and Montelukast was Purchase sample from Balaji Industry (Surat) also Tablet formulation (Bilargic-M)) Procured from local market.

All Chemicals and Reagents used were of Analytical Grade and HPTLC Grade.

Double distilled water was used throughout the experiment which was generated in-house.

Chromatographic Conditions

The plate was dried in air and developed up to 75mm using mixture of Toluene: Methanol (6.5:3.5 v/v) as mobile phase in Camag twin trough chamber previously saturated with mobile phase for 30 minutes. The plate was removed from the chamber, dried in hot air oven standard zones were scanned and quantified at 243nm.

Preparation of Standard Solution

A 1000µg/ml stock solution of Bilastine was prepared by dissolving 100mg in 50ml volumetric flask. A 200 µg/ml of Bilastine solution was prepared by diluting 5ml stock solution up to 25ml with methanol.

A 500µg/ml stock solution of Montelukast was prepared by dissolving 50mg in 100ml volumetric flask. A 100 µg/ml of Montelukast solution was prepared by diluting 5ml stock solution up to 25ml with methanol.

Determination of wavelength for measurement

The UV spectra of Bilastine (10-40µg/ml) and Montelukast (5-20µg/ml) solutions were measured in the 200-400nm range using Methanol as a blank. From Overlain spectra it was found that at 243nm both the drug has considerable absorbance. Thus 243nm was selected as wavelength for estimation of both the drug.

Stress Degradation studies:

Acidic Degradation:

A 100mg of Bilastine and 50mg Montelukast was accurately weighed and transferred to a 100ml volumetric flask add 10ml Methanol. To the above prepared solution add 25ml of 1M HCl. The solution was reflux for 2hrs at 60⁰C in water bath. After that cooled and neutralized by 1M NaOH and diluted up to mark with Methanol.

Alkaline Degradation:

A 100mg of Bilastine and 50mg Montelukast was transferred to a 100ml volumetric flask add 10ml methanol. To the above solution 25ml of 0.5M NaOH was added. The solution was reflux for 2hours at 60⁰C in water bath. After that cooled and neutralized by 25ml of 0.5N HCl and diluted up to mark with methanol.

Oxidative Degradation:

A 100mg of Bilastine and 50mg Montelukast was accurately weighed and transferred to a 100ml volumetric flask add 20ml methanol. To the above solution add 10ml of 3% H₂O₂. The solution was reflux for 1hour at 70⁰C in water bath. After that cooled and diluted up to mark with methanol.

Photolytic degradation:

A 100mg of Bilastine and 50mg Montelukast was accurately weighed and transferred to Petry plate with close lead. Plate was exposed to direct sunlight for 24 hrs. Add 20ml methanol in Petry plate and transfer it to 100ml volumetric flask. Wash Petry plate with small aliquots of methanol. Transfer in volumetric flask and dilute up to mark volume with methanol.

Thermal degradation:

A 100mg of Bilastine and 50mg Montelukast was accurately weighed and transferred to Petry plate with close lead. Plate was heated in previously heated hot air oven at 80⁰C for 6 hrs. Remove Petry plate and cooled it. Add 20ml methanol Petry plate and transfer it to 100 ml volumetric flask. Wash Petry plate with small aliquots of methanol. Transfer in volumetric flask and dilute up to mark volume with methanol.

Pipette out 5ml of each degraded solution up to 25ml with methanol to give a solution having concentration 200µg/ml of Bilastine and 100µg/ml of Montelukast. A volume of 1μl of each degraded solution was applied to TLC plate. The plate was dried in air and developed up to 80mm using mixture of Toluene: Methanol (6.5:3.5 v/v) as mobile phase in a Camag twin through chamber previously saturated with mobile phase for 30minutes. The plate was removed from the chamber, dried in hot air oven and standard zones were scanned and quantified at 243nm.

Method validation:

Linearity:

The linearity of response in the concentration range of 200-1200ng/spot of BIL and 100-600ng/spot of MON were determined as plot of spot area vs. concentration.

Precision:

Variations of results within the same day (Intra-day), variation of the results between days (Inter-day) were analyzed and its %RSD for each observation was calculated. For Intra-day and Inter-day precision, drugs containing Bilastine 200-1200ng/spot and Montelukast 100-600ng/spot were determined 3 times a day interval of 1hour and different day, and %RSD was calculated.

Accuracy:

To check accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre analyzed sample solution at three different levels 80, 100 and 120%. Mean percentage recovery was determined.

LOQ and LOD:

The limit of detection (LOD) is smallest concentration of the analyte that gives the measurable response. The LOQ is the smallest concentration of the analyte, which gives a response that can be accurately quantified. LOD and LOQ were calculated using the following formula: LOD = 3.3 σ/S and LOQ = 10σ/S. Where σ is Standard deviation of the response and S is slope of the calibration curve.

Robustness:

The %R.S.D. of the peak areas were calculated for change in mobile phase composition, mobile phase volume and chamber saturation time at a concentration level of 200ng/spot Bilastine and 100ng/spot of Montelukast in triplicate. The low values of % R.S.D. (<2) obtained after introducing small deliberate changes in the developed HPTLC method indicated the robustness of the method. The low value of %RSD indicated that the method is robust in the mentioned conditions.

Analysis of Bilastine and Montelukast in Marketed formulation:

20 Tablets of formulation (Bilargic-M) containing 20 mg of Bilastine and 10mg of Montelukast Sodium were weighed accurately. The average weigh of tablets powdered were found. The tablet powder equivalents to 20mg of Bilastine and 10mg Montelukast was weighed and transferred into 50ml volumetric flask and add a minimum quantity of solvent to dissolve the substance by using Ultra sonicator for 15 minutes and made up to the volume with 200µg/ml of Bilastine and 100µg/ml of Montelukast. The content was filtered through the whatman filter paper No.41. 0.5µL of 200µg/ml of Bilastine and 100µg/ml of Montelukast was applied on TLC plate and analyzed as per the developed method.

RESULTS AND DISCUSSION:

Optimization of HPTLC method²¹

TLC procedure was optimized with a view to develop a stability-indicating assay method. The drug reference standards were spotted on the TLC plates and developed in different solvent systems. Different mobile phases were tried to resolve Bilastine and Montelukast. The best suited mobile phase was found to be Toluene: Methanol in the ratio of 6.5:3.5v/v. The optimized mobile phase were selected was Toluene: Methanol, (6.5:3.5v/v). Developed mobile phase resulted in resolution for two drugs with Rf 0.31±0.01 and 0.61± 0.01 for Bilastine and Montelukast respectively. Well defined spots were obtained when the chamber was saturated with the mobile phase at room temperature.

Validation of the method^22-24:

Linearity:

Linearity responses for Bilastine and Montelukast were assessed in the concentration range 200-1200ng/spot and 100-600ng/spot, respectively. The linear equations for the calibration plots were for Bilastine was y = 8.4512x +129.3 and for Montelukast was y = 5.6208x+2179.9, with correlation coefficient (r²) being 0.998 and 0.993 respectively. Range was established with five replicate readings of each concentration. The 3D Overlain chromatogram was shown in Figure 3.

Figure 3: Overlain 3D chromatogram of linearity of Bilastine (200-1200 ng/spot) and Montelukast (100-600 ng/spot)

Precision:

Precision of the method was determined in the terms of intraday and interday variations (%RSD). Intraday precision (%RSD) was assessed by analysing standard drug solutions within the calibration range, three times on the same day, %RSD was found to be 0.78 – 1.21% for Bilastine and 0.24 – 0.72% for Montelukast.

Interday precision (%RSD) was assessed by analysing standard drug solutions within the calibration range on three different days over a period of week. %RSD was found to be 0.79 – 0.95% for Bilastine and 0.26 – 0.91% for Montelukast. This indicates that adequate preciseness of the method.

LOQ and LOD:

The LOQ and LOD were determined based on signal-to-noise ratios, using an analytical response of 10 and three times of the background noise, respectively. The LOD and LOQ were found to be 26.26ng/spot for Bilastine and 33.34ng/spot for Montelukast and 79.60ng/spot for Bilastine and 99.64ng/spot for Montelukast respectively.

Accuracy:

The proposed method when used for extraction and subsequent estimation of Bilastine and Montelukast from pharmaceutical dosage forms after spiking with 80,100 and 120% of additional drug afforded recovery of 100.14±0.90 - 99.49±0.72 for BIL and 100.96±2.25 - 99.27±2.86 for MON respectively as shown in Table 1.

Table 1: %Recovery data for Bilastine and Montelukast

Sr. No.	Amount from Sample		Total Amount of Spiked		Total Amount (ng/spot)		Recovered amount (ng/spot) ± S.D (n=3)		% Recover of Spike amount ± S.D (n=3)
	BIL	MON	BIL	MON	BIL	MON	BIL	MON	BIL	MON
BLANK	20	10	̵	̵	100	50	99.71	49.30	̵	̵
	20	10	̵	̵	100	50	100.78	49.83	̵	̵
	20	10	̵	̵	100	50	100.66	50.90	̵	̵
Mean							100.38 ± 0.58	50.01 ± 0.82	̵	̵
80%	20	10	16	8	180	90	180.18	89.16	100.58	101.41
	20	10	16	8	180	90	179.23	89.33	98.06	98.74
	20	10	16	8	180	90	180.53	92.00	99.84	102.74
Mean							179.98 ± 1.29	90.40 ± 2.04	99.49 ± 0.72	100.96 ± 2.25
100%	20	10	20	10	200	100	200.05	98.94	100.34	99.27
	20	10	20	10	200	100	201.71	100.89	100.93	102.12
	20	10	20	10	200	100	199.82	99.11	99.16	96.43
Mean							200.53 ± 0.90	99.65 ± 2.85	100.14 ± 0.90	99.27 ± 2.86
120%	20	10	24	12	220	110	219.81	190.25	100.08	99.93
	20	10	24	12	220	110	220.41	110.50	99.69	101.11
	20	10	24	12	220	110	219.81	111.75	99.30	101.41
Mean							220.01 ± 0.93	110.50 ± 0.78	99.69 ± 0.18	100.82 ± 0.71

Robustness:

No significant effect was observed on system suitability parameters such as capacity factor, resolution and theoretical plates of respective components, when small but deliberate changes were made to chromatographic conditions.

Assay analysis by HPTLC Method:

A single spot at Rf = 0.41 and Rf = 0.71 was observed in the chromatogram of the Bilastine and Montelukast samples extracted from tablets was shown in Figure 4. There was no interference from the excipients present in the tablets respectively. The drug content was found to be 98.82%±0.001 and 99.25%±0.001 for Bilastine and Montelukast was shown in Table 2.

Figure 4: Chromatogram of assay for Bilastine and Montelukast

Table 2: Marketed formulation analysis for Bilastine and Montelukast

Formulation	Labelled added (mg/tab)		Amount found (mg/tab)		Assay (% estimated) (n±3)
Bilargic-M	BIL	MON	BIL	MON	BIL	MON
Bilargic-M	20	10	19.5	9.1	98.82% ± 0.001	99.25% ± 0.001

Stability studies:

All stressed samples in both solid and solution state remained colorless. HPLC studies on Bilastine and Montelukast under different stress conditions using Toluene: methanol (6.5: 3.5) as the solvent system suggested the following degradation behavior in different stress condition.

Acidic condition:

The drug gradually decreased with time on refluxing with 0.1M hydrochloric acid for 2 hr, and 5.64% degradation was observed for Bilastine and 16.72% degradation was observed for Montelukast respectively as shown in Figure 5.

Figure 5: Chromatogram of Bilastine 200 ng/spot and Montelukast 100 ng/spot in acidic condition

Degradation in alkali:

After Refluxing drug solution with 0.5 M NaOH at 60°C for 2 hr, the percentage degradation of Bilastine and Montelukast in alkaline condition was found to be 26.95% and 17.61% respectively as shown in Figure 6.

Figure 6: Chromatogram of Bilastine 200ng/spot and Montelukast 100 ng/spot in Alkaline condition

Oxidative degradation:

After Refluxing drug solution with 5ml of 3 % hydrogen peroxide at 70°C for 1hr, the percentage degradation of Bilastine and Montelukast in oxidative condition was found to be 32.22% and 9.25% respectively as shown in Figure 7.

Figure 7: Chromatogram of Bilastine 200 ng/spot and Montelukast 100 ng/spot in Oxidative condition

Photolytic degradation:

Drug sample was exposed to direct sunlight for 24hr, the percentage degradation of Bilastine and Montelukast in photolytic condition was found to be 14.94 and 15.65% respectively as shown in Figure 8.

Figure 8: Chromatogram of Bilastine 200 ng/spot and Montelukast 100 ng/spot in Photolytic condition

Figure 9: Chromatogram of Bilastine 200 ng/spot and Montelukast 100 ng/spot in Thermal condition

Thermal degradation:

Drug sample was exposed to dry heat 80˚C for 6 hr in hot air oven, the percentage degradation of Bilastine and Montelukast in thermal degradation was found to be 17.54% and 14.65% respectively as shown in Figure 9.

The summary of forced degradation study was shown in Table 3.

CONCLUSION:

A validated stability-indicating HPLC analytical method has been developed for the determination of Bilastine and Montelukast in API. The results of stress testing undertaken according to the International Conference on Harmonization (ICH) guidelines reveal that the method is selective and stability-indicating. The proposed method is simple, accurate, precise, specific, and has the ability to separate the drug from degradation products and excipients found in the tablet dosage forms.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

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Received on 04.07.2022 Modified on 12.11.2022

Research J. Pharm. and Tech 2023; 16(10):4498-4504.

DOI: 10.52711/0974-360X.2023.00733

Force degradation Condition	Stress Condition	Remaining unchanged (%)		Rf value of Degradants		% Degradation
		BIL	MON	BIL	MON	BIL	MON
Acidic Degradation	1N HCl/ 60⁰C/ 2hrs	94.16	83.28	0.52	0.52	5.84	16.72
Alkaline Degradation	0.5 M NaOH/60˚C /2hr	73.05	82.39	0.90	0.33, 0.68, 0.90	26.95	17.61
Oxidative Degradation	3% v/v H₂O₂/70˚C/1hr	67.78	90.75	0.52	0.52, 0.68, 0.91	32.22	9.25
Photolytic Degradation	24 hours in sunlight	82.06	84.35	0.50	0.50, 0.76	14.94	15.65
Thermal Degradation	80˚C/6 hr	85.46	85.41	0.51	0.51, 0.69	17.54	14.65