Simultaneous Estimation of Bilastine and Montelukast in Bulk by Rp-hplc and Assessment of Its Applicability in Marketed Tablet Dosage Form

Aim: This study proposes to develop and validate the RP-HPLC method for Bilastine and Montelukast and to substantiate the RP-HPLC analysis bestowing to ICH validation guideline Q2R1. Place and Duration of Study: Y. B. Chavan College of Pharmacy, Aurangabad, MS, India, between January 2020 and October 2021. Methodology: The mixture of drugs was subjected to optimization by trial runs with different chromatographic parameters, viz. flow rate, λ in nm, etc. The system suitability was performed by repeated injections of Bilastine (200μg/mL) and Montelukast (200μg/mL) to confirm the optimization. Furthermore, the demonstrated method was validated as per ICH Q2R1 recommendations for parameters like accuracy, precision, robustness, the limit of detection and quantitation, etc. Results: The outcomes of the method in terms of percent relative standard deviation (%RSD) of retention time (RT) and mean peak area were seen as 0.09, 0.35, and 0.35, 0.56 for Bilastine and Montelukast, respectively. The method was successful in achieving the qualifying criteria entrusted to ICH guidelines. The correlation coefficient, slope, and y-intercept were illustrated to be 0.9971, Original Research Article Andhale and Nikalj; JPRI, 34(3B): 8-25, 2022; Article no.JPRI.79943 9 17595, 217883, 0.998, 35458, and 17147, correspondingly for Bilastine and Montelukast, respectively. The range was seen in the order of 160-260μg/mL and 80-130μg/mL for Bilastine and Montelukast. The precision of the method was established with %RSD of repeatability and intermediate precision was < 2 at three standard levels across the range. The %accuracy of the method was observed in the range of 96.95-101.41 %w/w and 97.37-101.89 %w/w in the order for Bilastine and Montelukast. The robustness of the method displayed the results within the prescribed boundaries. The recovered amount of Bilastine and Montelukast by spike method was observed to be 96.37-98.88 %w/w and 96.11-100.06%w/w. Conclusion: The author has accomplished the predefined goals by successful development and validation of the RP-HPLC method for the quantification of Bilastine and Montelukast as per ICH Q2R1 guidelines.


INTRODUCTION
The efficiency and speed of High-Performance Liquid Chromatography (HPLC) have proved the method's development requirements in the past 30 years [1,2]. Recently, the HPLC has proved to be the most valuable technique for a custom analysis of peptides [3,4]. Hence, HPLC is the first choice of analytical scientists for qualitative and quantitative analysis of drug substances and drug products. Further, HPLC is competent enough to separate the most complex mixtures [5,6].

Fig. 2. Structure of Montelukast
Literature research found the chromatographic, and spectroscopic analysis of Bilastine, Montelukast either alone or in combination with other drug substances. In recent reports, Umesh Chandra et al have developed the RP-HPLC method for in vitro dissolution testing of Bilastine and Montelukast using Hypersil BDS C18 column (100 x 4.6, 3 µm). The elution was achieved employing a blend consisting of triethylamine 0.1% v/v and Acetonitrile [13]. In another report, Saloni Kothari et al have estimated Bilastine and montelukast sodium in combined tablet dosage form. Also, the authors reported the Qabsorbance method in their aforesaid report [14]. Because HPLC is the most widely used method for analyzing drug substances and drug products, there is a further need to explore the more promising methods for quantification of Bilastine and Montelukast in combined dosage forms. Hence this research work is an endeavor to develop and validate the RP-HPLC method for quantifying Bilastine and Montelukast in a mixture using RP-HPLC. Hence, this research work was undertaken.

Materials
Potassium dihydrogen phosphate, tri-ethylamine, and orthophosphoric acid were purchased from a local chemical distributor by Thermo Fisher Scientific India. The Bilastine and Montelukast were purchased from a local vendor in Aurangabad, MS, India. The Bilasure M tablet (Hetero Labs Ltd.) with the strength of 20mg Bilastine and 10mg Montelukast was procured from a local medical store in the aforesaid city. This formulation was used to recover the amount of Bilastine and Montelukast from the pharmaceutical tablet dosage form to ensure the applicability of the method for a custom analysis of the fixed-dose combination.

Preparation of standard stock and working solution
20mg of Bilastine and 10 mg of Montelukast and transferred to the same 10mL volumetric flask containing a mixture of Acetonitrile: Phosphate buffer (pH 6.8) (60:40). The volume was made up to the mark with the help of the mobile phase. The consequential stock solutions of Bilastine (2000μg/mL) and Montelukast (1000μg/mL) were filtered through a 0.45µ membrane filter and ultrasonicated for three cycles each of 10 min. 1.0 mL of stock solution was diluted to 10ml to obtain the working solution of 200 μg/mL and 100 μg/mL for Bilastine and Montelukast respectively. The resulting working solution was injected into a given set of chromatographic conditions to observe the response. Initially, the chromatographic conditions were varied as per the response observed after each injection. The details of the protocol that followed were as illustrated in Table 1.

System suitability testing
The identical standard solution of Bilastine 200μg/mL and Montelukast 100μg/mL in the blend was injected in the RP-HPLC column with succeeding (Table 2) optimized chromatographic parameters and the chromatogram was recorded. The chromatogram was analyzed to estimate retention time, peak area, number of theoretical plates, tailing factor, etc. The obtained results were compared with limits given in ICH guidelines Q2R1. The correspondent procedure was adopted an added five times and outcomes were noted in each case of the chromatogram seen. The mean retention time and mean area were calculated accordingly.

Precision
Three standard solutions of the mixture of Bilastine and Montelukast were used across the given range (160 to 260μg/mL of Bilastine and 80-130 μg/mL of Montelukast to establish the precision of the method. The repeatability was recognized by repeated measurements of standard solutions on the same day. A three hours interval of the same day was the interchangeable condition to study repeatability. However, intermediate precision (system precision) was established on different days in the series. The three standards, viz., 170, 210, and 250μg/mL for Bilastine and 85, 105, and 125μg/mL for Montelukast was injected, and the mean peak area was integrated from the chromatograms. The %RSD was calculated in each case and compared with the prescribed standards for its compliance.

%Accuracy
The accuracy of the method was established by using three standard solutions of Bilastine and Montelukast in the mixture (API mixture) as cited in the precision study. The solutions were injected in triplicate and the corresponding concentration was estimated by extrapolation on the calibration curve. The %accuracy was then estimated using the following formula : % Accuracy = x 100

Robustness
The robustness of the method was studied by deliberate variations in the three method parameters, viz., organic concentration of the mobile phase, mobile phase flow rate in 'mL/min', and detector wavelength in 'nm'. The study design was as prescribed in Table 3.

Concentrations of Bilastine (200μg/ml) and
Montelukast (100μg/ml) were injected as a mixture of the solution to previously optimize chromatographic situations in triplicate at each level of change, and chromatograms observed were noted. From the chromatograms that resulted, the mean peak area was calculated. The %RSD was then calculated and assessed for its compliance as per ICH guidelines.

%recovery studies and assessment of the applicability of the method for a custom analysis of Bilastine and Montelukast as Fixed-Dose Combination (FDC) in marketed tablet dosage form
Preparation of Stock from API: Accurately weighed 20mg of Bilastine and 10mg of Montelukast (API) and were transferred to an identical 10 ml volumetric flask having an indistinct quantity of mobile phase. The volume of the latter was made up to the mark with the mobile phase to attain a concentration of 2000μg/mL for Bilastine and 1000 μg/mL for Montelukast. 1.0 mL of this standard stock solution was further diluted to 10 mL to get the working concentration of 200 μg/mL and 100 μg/mL for Bilastine and Montelukast respectively. The identical procedure was repeated another two times to get three working standard concentration solutions as above. The resulting solution was filtered through a 0.45μ membrane filter and ultra-sonicated for three cycles each of 10 min. These (three) solutions were injected into a fitting chromatographic system in triplicate, and the mean peak area in each case was estimated. The peak area was noted and kept aside.

System suitability testing
The study was performed with six repeated measurements of BIL and MTL in the mixture at 100% concentration, viz., 200μg/mL & 100μg/mL for BIL and MTL, respectively. The chromatograms observed were integrated to determine peak area, standard deviation (SD), and %RSD. The results observed are tabulated in Table 4 for BIL and Table 5 for MTL. The representative chromatogram of the study is shown in Fig. 3.
The other parameters (as listed below) were compared for their compliance as per ICH guideline Q2R1.
 Several theoretical plates or Efficiency (N).  Capacity factor (K).  Separation or Relative retention (α).  Resolution (Rs).  Tailing factor (T).  Relative Standard Deviation (RSD).   The %RSD observed for BIL for RT and mean peak area were 0.09 and 0.53 respectively. Whereas, the %RSD of MTL for RT and mean peak area were 0.35 and 0.56 respectively. The NOP of BIL and MTL were 3118 and 6567 respectively. The asymmetry factor was observed to be 1.69 and 1.38 in that order.

Linearity and range
The linearity of the method was assessed by injecting a series of standard solutions of BIL (160-260μg/mL) and MTL (80-130μg/mL) into the mixture. The mean peak area was integrated from the chromatogram observed. The peak area corresponding to each standard solution of BIL and MTL was as illustrated in Table 6.
The calibration curve was plotted from the mean peak area and standard concentrations of BIL and MTL. The calibration curve obtained was as shown in Figs. 4 and 5 for BIL and MTL respectively. From the calibration curve, the equation of the line, slope, and y-intercept were calculated. The further regression coefficient was estimated in each case and found to be 0.9971 and 0.998 for BIL and MTL, respectively.
The equation of line was observed to be y = 17595x + 217883 and y = 35458x -17147 for BIL and MTL respectively.

Precision
The precision experiment was performed in two ways.
 Repeatability: precision under interchangeable working conditions, a similar analyst over a quick period.  Intermediate precision: the system is assessed on an array of days.
The ICH guidelines advocate that repeatability should be documented appropriately using a minimum of nine determinations through the standard range for the method (e.g., three concentrations / three replicates each) or a minimum of six repeated measurements at 100% of the assessment concentration. In this case, it was performed by the prior method.
The three standard solutions at three levels across the range of the method of the BIL and MTL in the mixture were injected and chromatograms were recorded. The peak area was integrated and subjected to statistical analysis to determine the mean peak area, SD, and %RSD. The results of the %RSD observed in the repeatability study of BIL were in the range of 0.44-1.97. The %RSD for intermediate precision was in the range of 1.05 to 1.68 ( Table 7). The outcomes are seen within the boundaries prescribed.  From the outcomes of this study, it was concluded that the presented method successfully passed the precision experiment as per ICH guidelines.

%accuracy
As per ICH guideline Q2R1, the accuracy of the method should be established at three levels with three repeated measurements at each level across the range of the method. The accuracy was estimated by using three standard concentrations of BIL and MTL at three levels across the range. The results observed for accuracy were as seen in Tables 9  and 10.

Robustness
The robustness of the present method was studied by small but purposeful variations in the method parameters, viz., detector wavelength in 'nm', the flow rate of the mobile phase in ''mL/min'', and organic concentration of the mobile phase in '%v/v'. The experimental setup of the robustness experiment was tabulated in the experimental section cited above.
The outcomes of the robustness experiment with deliberate variation in the detector wavelength were as tabulated in Further, the method was subjected to another purposeful variation in the method parameter, i.e., a change in the concentration of the organic phase of the mobile phase. The variation was set at ±5%. The results acquired were as shown in Table 12. From the results acquired in this study, the % assay of BIL was in the range of 98.05-101.59 %w/w. Also, the %assay for MTL was observed in the range of 98.38-99.63 %w/w. The results were seen to conform with the standards.  In addition, the method was also assessed for the effect of small but deliberate changes in the flow rate (±0.15mL/min) of the mobile phase. The measurements were carried out in triplicate and the chromatogram observed was integrated to determine the peak area and mean peak area. The percent assay was calculated in each case of the change for BIL and MTL as illustrated in Table 13. The percent assay for BIL and MTL was found to be in the 95.33-103.77%w/w and 98.38-103.57%w/w ranges, respectively. The method was observed to be robust even at deliberate variations in the flow rate of the mobile phase.

%Recovery / Estimation of the applicability of the method for a custom analysis of BIL and MTL as FDC in marketed pharmaceutical dosage form (tablets)
The drug content in the drug product can be studied by the percent recovery method. This also ascertains the accuracy of the method. Moreover, if the study is carried out using a marketed dosage form, it also illustrates the applicability of the method for a custom analysis of that marketed formulation(s) used for the study. In this method, the percent recovery was studied by using the marketed combined tablet dosage form of BIL and MTL.
The percent recovery experiment to determine the drug content of BIL and MTL and to ascertain the accuracy of the method was performed at three levels, viz., 80, 100, and 120% of the 100% test concentration. The percent recovery was performed by the spike method. The known amount of standard solution of a mixture of BIL and MTL was spiked into each sample solution (prepared from the tablet combined dosage form). The final test solution was injected into a given set of chromatographic conditions in triplicate and the chromatograms were recorded. The sample peak area was calculated in each case by deducting the peak area corresponding to the standard concentration spiked. The percent accuracy was then calculated by using the formula as given in the experimental section (2.2.3.5). The results observed for percent recovery of the BIL and MTL were as depicted in Tables 14 and 15. The representative chromatogram is shown in Fig. 6.    Further, the retention time for Bilastine and Montelukast was found to be 2.950min.and 7.040min. respectively. The above positions of retention time for BIL and MTL in tablet dosage form were observed in conformity with that of the API mixture. As a result, the method was found to be sensitive for detecting BIL and MTL as FDC in marketed tablet dosage forms in the presence of permissible tablet excipients. Also, no supplementary peaks were seen in the chromatogram, which further confirmed the sensitivity of the method.

LOD and LOQ
In this method, the LOD and LOQ were calculated by the standard deviation of the responses obtained for all standard concentrations of Bilastine and Montelukast in the linearity investigation.
Also, the following formulae were used to calculate the LOD and LOQ of Bilastine and Montelukast. The results obtained were as tabulated in Table  16. As shown in Table 16, the LOD and LOQ for BIL were 7.43 and 22.53μg/ml respectively. LOD and LOQ for MTL were noted as 3.06 and 9.28μg/ml respectively.

Discussion
Extensive literature was explored before designing the present research work, which entitles simultaneous estimation of Bilastine and Montelukast as a mixture of API and assessment of its applicability. Peethal Pratyusha et al reported a UV spectroscopic method for the determination of BIL in 2020 and claimed that Beer's law was obeyed between 10-140µg/mL of BIL. The author also studied zero-order and firstorder kinetics [15]. Another UV method with an experimental design for robustness in 0.1 mol/liter HCl as a solvent was reported. The author claimed the precise, linear, specific, and exact [16]. Peethal Pratyusha et al. also reported the RP-HPLC method for the determination of BIL. The separation was achieved using formic acid and methanol in 50:50%v/v. The RT of BIL was noted to be 2.167min [17]. Pardeshi P P et al also reported the RP-HPLC method for analysis of BIL. Methanol and orthophosphoric acid buffer (70:30%v/v) were used as a mobile phase [18]. Firdous et al developed the UPLC method for estimation of BIL. The separation was achieved using buffer: methanol: acetonitrile as a mobile phase. The method has good precision and accuracy [19].

Rana et al. explored the RP-HPLC method for simultaneous estimation of Montelukast and
Ebastine. Methanol, acetonitrile, ammonium acetate in the ratio of 80:10:10 %v/v/v was used in a mobile phase to attain the separation of the components. Also, the method was successfully employed for Montelukast and Ebastine in commercially available marketed tablet dosage forms [20]. Sharma H K et al. determined the impurities of MTL sodium using RP-HPLC. The degradation was observed in acid and oxidative environments, whereas, it was found to be stable in other stress conditions. The separation was achieved using gradient elution [21]. Singh et al. estimated MTL by RP-HPLC. Acetonitrile: 1mM sodium acetate at pH 6.3 was used in the ratio of 90:10 %v/v on the C 18 stationary phase and the detection was achieved at 285nm [22]. Gholve et al. further explored the quantification of the MTL using a mobile phase consisting of methanol:acetonitrile: water in a ratio of 60:30:10 %v/v/v. The eluent was monitored at 344nm with RT 3.582 [23]. Murlidharan et al. furthered continued the work and developed HPLC and UV spectroscopic methods for estimation of MTL. One-way ANOVA was employed to analyze the results statistically. The method was successfully applied to the dosage form [24]. Barnabas et al developed a novel stability-indicating method for the determination of related substances of MTL in a pharmaceutical dosage form using RP-HPLC. The separation was achieved in a gradient mode with triethylamine and acetonitrile in various combinations at pH 6.6. The method showed an excellent regression coefficient of 0.999 [25].
Two reports recently published showed the simultaneous estimation of Bilastine and Montelukast in combined dosage forms. The RP-HPLC method reported by Umesh Chandra et al [13] was particularly aimed to estimate the percentage release of both drugs in dissolution medium. The elution was carried in gradient mode. Further, the RP-HPLC method reported by Saloni Kothari et al [14] was aimed to estimate the Bilastiene and Montelukast in combined tablet dosage forms. Hence, this research work was planned to provide a competitive method for a custom analysis of BIL and MTL for pharmaceutical tablet dosage forms commercially available in the market.
The separation of BIL and MTL was achieved on C18 (250x4.6mm), 5µm id stationary phase by using acetonitrile: disodium hydrogen phosphate buffer in a proportion of 60:40 %v/v. The elution was monitored at 254nm with a flow rate of 0.6mL/min. The analysis was carried out at 25 ºC with a run time of 15min. The optimization of the method was done by using various combinations of method parameters as shown in Table 1. The final selection was as depicted above. In the above blend of mobile phases, the RT noted for BIL and MTL were 2.95 and 7.13, respectively.
The system suitability test was carried out to ensure the appropriate working of the system and it was observed that the results in terms of %RSD of mean peak area and mean RT. The %RSD was observed at <0.5 for RT and <2 for mean peak area in the case of both, i.e., BIL and MTL. Also, other parameters were observed to comply with the standards prescribed in ICH guidelines Q2R1. The linearity of the method was observed by injecting a series of standard concentrations of BIL MTL into the mixture. Linear regression was observed for BIL and MIL with regression coefficients of 0.9971 and 0.998 respectively. The linearity of the method was observed in the range of 160-260μg/mL and 80-130μg/mL with the equation of lines Y = 17595x + 217883 & Y = 35458x -17147 for BIL and MTL correspondingly. The method was observed to be linear in the given concentration range depicted above.
The precision of the method was established using two methods, viz., repeatability and intermediate precision. The study was carried out using three standard solutions at three levels across the range. The values %RSD observed in repeatability were in the range of 0.44-1.97 and 0.44-1.95 for BIL and MTL in that order. However, the %RSD observed for intermediate precision was found in the range of 1.05-1.68 and 0.90-197. All outcomes of the precision experiment showed the %RSD values within the prescribed limits (<2). Hence, the method was proved to be precise for the quantification of BIL and MTL.
The robustness of the method was carried out by purposeful variations in the method parameters, viz., detector wavelength, the organic concentration of the mobile phase, and flow rate. The results were reported in terms of percent assay of BIL and MTL and were found to conform with the standards prescribed in the compendia. Further, it was demonstrated that the small and deliberate alterations in the method parameters could not affect the method performance for the quantification of drug substances as well as drug products. Hence, the method was proved robust.
The method was observed to be accurate for the estimation of BIL and MTL in the mixture at three concentration levels across the range. The percent assay results were seen in agreement for both BIL and MTL.
The %recovery of the method was established at three recovery levels of 100% test concentrations of BIL and MTL in the mixture. The % recoveries of BIL and MTL at 80, 100 and 120% levels were noted to be 98.88, 97.25, 96.37 5w/w and 97.70, 96.11, 100.06 %w/w correspondingly. All results noted were in agreement with the limits prescribed. Further, the respective peaks of the BIL and MTL were observed at the same position as those seen in the API mixture. This suggests that the method remains unaffected by commonly used excipients (sample matrix) in the formulation of the tablet dosage form. Also, no supplementary peaks were observed in the chromatogram. This indicated method was specific to the selected drug combination of BIL and MTL. In addition, this study confirmed the effective applicability of the presented method for a custom analysis of BIL and MTL in pharmaceutical tablet dosage forms commercially available on the market.
The developed method was seen as superior in terms of resolution and cost. As the flow rate used in the presented method is less as compared to both reported methods by Umesh Chandra et al and Saloni Kothari et al. Further, the method of the previous author was particularly aimed to determine the percent drug release whereas in this method the complete validation protocol for a custom analysis of BIL and MTL is explored. Minimum injection volume with the maximum resolution is the specialty of this method. Further, successful recovery of the BIL and MTL from the marketed tablet dosage form entrusted the applicability of the method for routine analysis of marketed pharmaceutical products of fixed-dose combinations. Also, the method proved to be economic because of the minimum consumption of the mobile phase (at a low flow rate of 0.6 mL/min) and the minimum loading of the sample on the column.
The low LOD and LOQ values (7.43 & 22.53 µg/mL for BIL and 3.06 & 9.28 µg/mL for MTL, respectively) further proved the method's sensitivity for detection and quantification of Bilastine and Montelukast.