Simultaneous Determination of Fexofenadine Hydrochloride and Montelukast Sodium Using New Pencil Graphite Electrode in Their Pure, Synthetic Mixtures, and Combined Dosage Form

Simultaneous Determination of Fexofenadine Hydrochloride and Montelukast Sodium Using New Pencil Graphite Electrode in Their Pure, Synthetic Mixtures, and Combined Dosage Form Dania Nashed (  Dania.nashed92@gmail.com ) University of Aleppo Faculty of Pharmacy https://orcid.org/0000-0003-0029-8153 Imad Noureldin University of Aleppo- faculty of pharmacy Amir Alhaj Sakur University of Aleppo- faculty of pharmacy Research article Keywords: Graphite sensors, potentiometric, fexofenadine hydrochloride, montelukast sodium, molybdate ammonium, cobalt nitrate DOI: https://doi.org/10.21203/rs.3.rs-50184/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract This paper introduces a new electrochemical approach for the concomitant determination of fexofenadine hydrochloride and montelukast sodium by constructing three new graphite electrodes coated with a polymeric membrane. The rst electrode was constructed using ammonium molybdate reagent as an ion pair with fexofenadine cation for the determination of fexofenadine drug, the second electrode was constructed using cobalt nitrate as an ion pair with montelukast anion for the determination of montelukast drug, the third electrode was prepared by incorporating the two previously mention ion pairs in the same graphite sensor, which make this sensor sensitive to each fexofenadine and montelukast drug. The coating material was a polymeric lm comprises of Poly Vinyl Chloride (PVC), Di-butyl phthalate as a plasticizer (DBP), ion pairs of drugs with previously mentioned reagents. The electrodes showed a Nernstian response with a mean calibration graph slopes of [58.97, 28.43, (59.048 , 28,643 )] mv.decade-1 for the three pencil electrodes respectively. The electrodes work effectively over pH range (2-4.5) for fexofenadine hydrochloride and (5-9.5) for montelukast sodium. The in uence of the proposed interfering species was negligible. The effectiveness of the electrodes continued in a period of time (45-69) days. The suggested sensors demonstrated useful analytical features for the determination of both drugs in bulk powder, in laboratory prepared mixtures and their combined dosage form. We have validated the method in accordance with ICH protocol. 1. Introduction Fexofenadine hydrochloride (FEX.HCl) gure (1, a), is a selective antagonist for histamine H1- receptor, it is an effective metabolite of terfenadine. Its chemical name is (RS)2-[4 [1-Hydroxy-4[4-(hydroxy-diphenylmethyl)-piperidyl]butyl]phenyl]-2-methylpropanoic acid(1), fexofenadine described as a second or thirdgeneration antihistamine, on 25 February 2000 FDA approved the utilization of fexofenadine for the handling of periodical allergic rhinitis and chronic urticaria. It restrains the exacerbation of coryza and urticaria and reduces the stringency of the signs associated with those conditions such as sneezing, runny nose, irritating eyes(2). Montelukast Sodium (MON.Na) gure(1,b), is chemically 1-[[[(R)-m-[(E)-2-(7chloro-2-quinolyl) vinyl]--[o-(1-hydroxy-1-methyl ethyl) phenethyl] benzyl]thio]methyl]cyclopropaneacetate(3), (MON.Na) is an antagonist of cysteinyl leukotriene receptor, on 20/2/1998 FDA approved the utilization of MON for chronic handling of asthma, preventing airway edema, smooth muscle contraction and enhanced secretion of thick, viscous mucus(4). Literature showed several analytical methods for the estimation of each drug individually, Fexofenadine HCl was estimated individually by some analytical methods such as HPLC (5–7)– HPTLC(8)- spectrophotometric (9–12) - uorimetry(13) – capillary electrophoresis(14) – potentiometry(15). Similarly, montelukast sodium (MON.Na) was determined using some analytical techniques such as HPLC(16–18), UV spectrophotometric (19)(16), capillary electrophoresis(20), Potentiometric (21, 22), and voltammetric (23). The combination remedy of fexofenadine with montelukast sodium supply enhancing effect thereby reducing the symptoms e caciously(24), the determination of these drugs as combined dosage forms was limited by a few methods like HPLC (25–27), HPTLC (28) and derivative spectrophotometric Page 2/20 methods(29)(30).There was no previous electrochemical method for the determination of fexofenadine HCl combined with montelukast Na. The novelty in this presented work that we have created a new, accurate, sensitive, time and cost-saving potentiometric method for determination of fexofenadine HCl and montelukast sodium simultaneously using pencil graphite electrodes depending on the difference in the active pH range for each sensor. Pencil graphite electrodes consider a developed form of ion-selective electrodes. The advantages of these electrodes are the small size where we can use them in biological systems, their rst response time, long lifetime compared to those traditional ion-selective electrodes(31), in addition to the advantages have known for the ion-selective electrodes such as being simple, accurate, economic, and saving time where there is no need for previous procedures to the sample(32–36). We have successfully applied this method for the determination of the combined dosage form without previous separation and that was our scienti c challenge. (a) (b) Figure 1: Chemical structure of (a) fexofenadine hydrochloride (b) Montelukast sodium fexofenadine act as a cation in that it makes up an ion pair with Molybdate anion, but montelukast act as anion and makes up ion pair with the cationic reagent cobalt nitrate, therefore we can determine each drug separately without interference of the other drug potential. The determination of fexofenadine hydrochloride and montelukast sodium in this presented work relies upon the construction of a pencil graphite electrode coated with a polymer lm, which consists of polymer, plasticizer and ion pair of previous mention drugs and reagents. The ion pairs consider the active part in the electrode, the role of polymer is to provide a mechanical support to other components of membrane lm, which covered the graphite rod, and the plasticizer gives an appropriate pliancy of the coating lm. Among various types of ion-selective electrodes, pencil graphite electrode shows good adsorption, conductivity, high sensitivity, small background current, and simple preparation(37). These electrodes carry on as interface. Thus, the membrane potential in the cell sees as the electric potential difference between the two interfaces in accordance with Nernstian equation E = E0 + 2.303 = RT/ZF log [FEX] 2.1 Apparatus Potentiometric measurements carrying out using Radiometer analytical – ion check 10 pH/mv meter (CEDEX- France), all pH measurements were carried out utilizing Crison pH meter model Glp21/EU (Spain), ultrasonic bath model Power Sonic 405(Korea). All weights were taken by Sartorius balance model 2474 (Germany) its accuracy ± 0.1 mg. 2.2 materials and chemicals Page 3/20 High pure fexofenadine hydrochloride and Montelukast sodium was obtained by Sigma Aldrich, analytical grade ammonium molybdate, cobalt nitrate (BDH chemicals, England), high molecular weight PVC (SABC. KSA), tetrahydrofuran solvent (MERCK 99.5%), di- butyl phthalate (MERCK 99%). 2.3 Standard drug solutions 2.3.1 FEX stock standard solution (10− 2 mol L− 1) The FEX stock solution was prepared by dissolving accurate weight in bi-distilled water, and then the volume was made up to the mark into a 50-mL volumetric ask. 2.3.2 MON stock solution (10− 2 mol L− 1) The MON stock solution was prepared by dissolving accurate weight in bi-distilled water, and then the volume was made up to the mark into a 50-mL volumetric ask. 2.3.3 working solutions A series of working solutions their concentrations varying (1 × 10− 7 − 1 × 10− 3 mol L− 1) were prepared by serial dilutions from the stock solutions using bi-distilled water. 2.4 procedure 2.4.1 preparation of FEX.Mol ion pair The ion pair of fexofenadine cation with molybdate anion was prepared by mixing 1 mmol of fexofenadine hydrochloride with 1 mmol of molybdate ammonium, an off-white precipitate was formed, then the precipitate was ltered and washed several times by bi-distilled water. The conductivity of the ltrate was checked to be ≤ 2 µs/cm which con rmed the disposal of all obstructive ions. 2.4.2 preparation of MON.Co ion pair The ion pair of Montelukast anion with cobalt cation was prepared by mixing of 1 mmol of Montelukast sodium with 2 mmol of cobalt nitrate, a pink precipitate was formed, then the precipitate was ltered and washed several times by bi-distilled water. The conductivity of the ltrate checked to be ≤ 2 µs/cm which con rmed the disposal of all obstructive ions. 2.4.3 Fabrication of FEX pencil graphite coated electrode The coating solution was prepared by mixing 0.45 g PVC with 0.9 g DBP, then 0.15 g of ion Pair (FEX.Mol) was added, all the components were dissolved in a small volume of THF. In this previous solution, a graphite rod was immersed several times to get a homogeneous layer of the coating material on the graphite rod. The coated graphite electrode was activated before beginning to measure the potential, by dipping it in 10− 3 mol/l FEX solution for 24 hrs. 2.4.4 Fabrication of MON pencil graphite coated electrode Page 4/20 The coated solution was prepared by mixing 0.6 g PVC with 1.2 g DBP, then 0.2 g of ion Pair (MON.Co) was added, all the components were dissolved in a small volume of THF. In this previous solution, a graphite rod was immersed several times to get a homogeneous layer of the coating material o the graphite rod. The coated graphite electrode was activated before beginning to measure the potential, by dipping it in 10− 3 mol/l MON solution for 24 hrs. 2.4.5 fabrication of FEX&MON pencil graphite electrode (the combined electrode) The preparation of this electrode was done by mixing 0.2 g of IP1 + 0.2 g of IP2 with 0.7 g PVC and 0.9 g DBP, all the components were dissolved in a small volume of THF. In this previous solution, a graphite rod was immersed several times to get a homogeneous layer of the coating material o the graphite rod. The coated graphite electrode was activated before beginning to measure the potential, by dipping it in (10− 3 mol.L− 1 ) FEX and MON solutions separately for 24 hrs. in each solution. 2.4.6 Direct potentiometric determination of fexofenadine hydrochloride A standard series of fexofenadine hydrochloride (10− 7-10− 2) mol.l− 1 was prepared accurately and all the potentiometric measurements carried out using (1and 3) graphite coated electrodes in junction with Ag/AgCl reference electrode. The potential produced by the proposed electrodes was recorded for each concentration to get the regression equations, which used to determine this drug. 2.4.7 Direct potentiometric determination of Montelukast sodium A standard series of Montelukast sodium (10− 7-10− 2) mol.l− 1 was prepared accurately and all the potentiometric measurements carried out using the (2 and 3) graphite coated electrodes in junction with Ag/AgCl reference electrode. The potential produced by the proposed electrodes was recorded for each concentration to get the regression equations, which used to determine this drug. 2.4.8 Effect of pH The effect of pH on the potential response of the two sensors was studied over the pH ranges of (2–6) for fexofenadine and (3–11) for montelukast. This was obtained by adding diluted aliquots of (0.1 mol L− 1 ) hydrochloric acid or sodium hydroxide solutions to the (1.00 × 10− 3 and 1.00 × 10− 4) mol L− 1 drug solutions. The potential obtained at each pH value was recorded. 2.4.9 selectivity of the electrodes The sensitivity of the constructed sensors was studied in the presence of some obstructive ions and excipients, which may exist with the drug material. The selectivity was studied using the matched potential method. In this method, the selectivity coe cient is characterized as the activity ratio of the essential and the interfering ion that exhibits the equal potential change(38). Page 5/20 K= ( α'A- αA) / αB Where; K is the selectivity coe cient, α'A is the activity of the primary ion, αA is the xed activity of the primary ion, αB is the activity of interfering ion. 2.4.10 determination of FEX and MON in laboratory prepared mixtures Different ratio mixtures of FEX and MON solutions were prepared, for that, different volumes of the stocks solutions for both drugs were mixed to get a speci c concentration of each drug which must be within the linearity range. Each drug was determined using its proposed sensor in the presence of the other drug, depends on the effective pH range for each electrode. 1. 2.4.11 Preparation of test solutions 1. a. the determination of FEX.HCl in its pharmaceutical dosage form For the determination of FEX.HCl in its pharmaceutical dosage form as a single drug. 20 tablets of Fexofenadine drug were nely powdered; exact weight proportionate to one tablet was taken, dissolved with bi-distilled water, and sonicate the solution in the ultrasonic bath for 5 minutes. Then the solution was ltered, an appropriate volume was taken from the ltrate and diluted with bi-distilled water in a 25 ml volumetric ask to get 10− 4 mol.l− 1 of drug solution. b. the determination of MON.Na in its pharmaceutical dosage form For the determination of MON.Na in its pharmaceutical dosage form as a single drug, 20 tablets of Azmalir drug were nely powdered; exact weight proportionate to one tablet was taken, dissolved with bidistilled water, and sonicate the solution in the ultrasonic bath for 15 minutes. Then the solution was ltered, an appropriate volume was taken from the ltrate and diluted with bi-distilled water in a 25 ml volumetric ask to get 10− 4 mol.l− 1 of drug solution. c. The determination of FEX& MON as a combination form According to the common combination ratio of FEX&MON formulation, the binary mixture was prepared in ratio 12:1. precisely weighed (120 mg) FEX and (10 mg) MON then, common excipients that are used in the tablet formulation were added, the mixture was transferred to a 50 ml volumetric ask and diluted to the mark by bi-distilled water. For 20 minutes the solution was sonicated and ltered. From the ltrate, 10 ml was taken and diluted to 25 ml in volumetric ask by bi-distilled water to get the sample solution. 3. Results And Discussion 3.1. Calibration of the electrodes Page 6/20 The constructed electrodes were dunked into a standard series solution of each drug; their concentration ranging (10− 7 – 10− 1) mol.l− 1, the potential of each solution was recorded, then a calibration graphs were plotted between the potential and the minus logarithm of drug concentration as shown in gure (2) and (3). The validations rules were applied according to ICH recommendations and the results are shown in table (1). The sensors showed to be active for 69 days for FEX.Mol, and 45 days for MON.Co sensor, during these days the slope of the regression equation was measured and found to be almost stable, but after this duration the slope was decreased obviously. Table 1 Response characteristics and the validation data of the constructed sensors Figure 2: Potentiometric pro le of FEX.Mol sensor parameter Figure 3: Potentiometric pro le of MON.Co sensor FEX.Mol MON.Co The Combined sensor FEX MON Slope ± SD (mV.decade− 1) -59.227 ± 0.05 28.43 ± 0.09 -59.048 ± 0.7 28.643 ± 0.22 Intercept (mV) 435.11 -45.628 439.19 -44.342 Correlation coe cient 0.9991 0.9998 0.9999 0.9998 Response time (seconds) 20 27 29 32 pH range (2-4.5) (5-9.5) (2-4.5) (5-9.5) Linearity range (mol.l− 1) (10− 2-10− 5) (10− 2-10− 5) (10− 2-10− 5) (10− 2-10− 5) Life time (days) 69 45 45 Recovery a % 99.84 ± 0.51 100.92 ± 0.21 99.76 ± 0.50 100.55 ± 0.71 Repeatability b 1.59 1.18 1.70 1.63 Reproducibility c 1.73 0.29 1.91 1.99 Lodd (µM) 0.014 0.021 0.025 0.019 Loq (µM) 0.043 0.063 0.076 0.059 1. a. Average of three determinations. 2. b. Repeatability: the intraday precision (n = 3 × 3), average of three concentrations (5*10− 5, 5*10− 4 and 5*10− 3 mol.l− 1) were repeated three times within the day. 3. c. Intermediate precision: the interday precision (n = 3 × 3), average of three concentrations (5*10− 5, 5*10− 4 and 5*10− 3 mol.l− 1) were repeated three times on two consecutive days. Page 7/20 d. Lod 3.3 SD of intercept/ slope, LOQ = 10*SD/ slope 3.2 Effect of pH The effect of pH on the measured potential was studied. For that, different fexofenadine solutions their pH values range (2–6) were prepared, the potential was measured for each solution using FEX.Mol graphite sensor, we found that the potential stay stable between pH range (2.5–4.5), at pH value more than 4.5 a noticed decrease in potential was found. For MON.Co sensor, different Montelukast solutions their pH values range (3–11) were prepared, and the potential was measured for each solution using MON.Co sensor, the effective pH range was found to be (5-9.5), at pH values less than 5, Montelukast drug participated, and more than 9.5 there was a decrease in the measured potential. It was found that there is no requirement for using any buffer, as buffers may involve some obtrusive substances, and because of the wide range of pH for both sensors (I and II). The obtained results are shown in gures (4) and (5). Figure 4: Effect of pH on potentiometric response for FEX.Mol sensor Figure 5: Effect of pH on potentiometric response for MON.co sensor 3.3 selectivity of the constructed electrodes The potential response of the proposed sensors in the presence of several related substances was studied, and the potentiometric selectivity coe cients were calculated to estimate the selectivity of the electrodes towards the primary drug ion (FEX) in case of sensor I and (MON) in case of sensor II, in the presence of the other drug ion and some obstructive ions which may exist in the drug solution. As shown in table (2), the constructed electrodes exhibit a good selectivity in the presence of the other drug which con rms the ability of determination of each drug in the combination dosage forms. Page 8/20 Table 2 Selectivity coe cients of the coated graphite constructed sensors Interfering B Sensor 1 (FEX.mol) Sensor 2 (MON. co) The combined sensor FEX MON K Fex,B K Mon,B K Fex,B K Mon,B CaCl2 4.88*10 − 3 3.2*10 − 2 4.93*10 − 3 3.4*10 − 2 KCl 1.32*10 − 3 4.6*10 − 3 1.67*10 − 3 4.6*10 − 3 NH4Cl 6.07*10 − 3 2.1*10 − 2 6.78*10 − 3 2.3*10 − 2 NaCl 1.34*10 − 3 3*10 − 3 1.53*10 − 3 3.5*10 − 3 dextrose 7.4*10 − 3 6.1*10 − 3 7.66*10 − 3 6.4*10 − 3 Mg stearate 2.4*10 − 3 8.7*10 − 3 2.67*10 − 3 8.9*10 − 3 Avicel 6.5*10 − 3 5.5*10 − 3 6.72*10 − 3 5.6*10 − 3 FEX …… 3.8*10 − 3 …… 4.2*10 − 3 MON 5.5*10 − 2 …… 5.61*10 − 2 …… 3.4. Potentiometric determination of laboratory prepared mixtures containing different ratios of FEX and MON The potential of the laboratory prepared mixtures containing different ratios of FEX and MON was measured, and the results showed that the proposed sensors FEX.Mol and the combined sensor can be effectively used for selective determination of FEX in the presence of MON, and the proposed sensor MON.Co and the combined sensor can be successfully used for selective determination of MON in the existence of FEX without a need for any previous separation, just we need to adjust the pH of each solution within the effective pH range for each electrode. The results are summarized in Table (3). Page 9/20 Table 3 Potentiometric determination of laboratory prepared mixtures containing various ratios of FEX and MON Ratio FEX Recovery % MON FEX MON Sensor 1 Sensor 3 Sensor 2 Sensor 3 1 1 98.40 98.22 99.31 98.89 5 1 97.27 97.13 99.97 99.20 10 1 100.92 100.52 101.62 101.12 12 1 101.16 100.99 98.40 98.14 1 12 97.72 97.56 97.58 97.33 99.09 ± 1.82 98.88 ± 1.76 99 ± 1.84 98.94 ± 1.42 Mean ± SD 3.5. Potentiometric determination of the sample solution The prepared sensors in conjunction with the double junction Ag/AgCl reference electrode were soaked separately in the sample solution after the adjusting of pH value of the sample solution within the effective pH range of each electrode. The resulting potential was recorded, the corresponding concentration was calculated from the regression equations for each sensor. We have successfully determined each of fexofenadine and Montelukast drugs in their combination form without any need for any previous separation. The excipients which were added, found to be don’t in uence on the potential response, that approves the ability of the developed method for the determination of fexofenadine and montelukast in their binary dosage form. The results realized were compared with the results obtained by reference UV spectroscopic methods(9)(39), the statistical tests show that there is no signi cant difference in the results was found by applying the two methods as shown in the table (4) Page 10/20 Table 4 Determination of FEX and MON in pharmaceutical preparations using the proposed method and reference methods. Commercial Name Composition Amount found, mga R%±SD tvalueb Fvaluec Fexofenadine Fexofenadine 120 mg 119.37 99.47 ± 1.16 1.06 3.53 Azmalir Montelukast 10 mg ---- ---- ---- ---- Combination form Fexofenadine 120 mg 119.27 99.39 ± 0.87 1.96 3.39 Montelukast 10 mg ---- ---- ---- ---- Fexofenadine Fexofenadine 120 mg ---- ---- ---- ---- Azmalir Montelukast 10 mg 10.05 100.5 ± 1.74 4.07 1.66 Combination form Fexofenadine 120 mg ---- ---- ---- ---- Montelukast 10 mg 9.97 99.71 ± 1.61 2.82 3.20 Fexofenadine Fexofenadine 120 mg 119.53 100.39 ± 0.78 2.26 3.46 Azmalir Montelukast 10 mg 10.12 98.80 ± 1.20 2.77 1.77 Combination form Fexofenadine 120 mg 120.83 100.69 ± 0.69 2.13 2.95 Montelukast 10 mg 9.89 98.88 ± 1.34 2.22 3.30 Sensor 1 FEX.Mol Sensor 2 MON.Co Sensor 3 FEX.MOl + MON.Co a: average of 3 replicates b: t critical 4.302 (0.05) c: f critical 19 (0.05), n = 3 4. Conclusion Page 11/20 This research was the rst electrochemical method for the simultaneous determination of fexofenadine hydrochloride and montelukast sodium. The results showed that the constructed method was accurate, precise and sensitive for the determination of each drug as pure form, laboratory prepared mixtures, and pharmaceutical formulation without any separation steps. Based on the results, it can be concluded that the coated graphite electrodes offered a simple, rapid, eco-friendly, high sensitivity and selectivity alternative method for the simultaneous determination of drugs, so we suggest using this type of electrode in drug analysis. Abbreviations FEX fexofenadine hydrochloride MON montelukast sodium Mol ammonium molybdate Co cobalt nitrate ICH The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use PVC poly venyl chloride DBP di-butyl phthalate THF tetrahydrofuran Declarations Ethic approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request Page 12/20 Competing interests The authors declare that they have no competing interests Funding This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors. Authors’ contributions A.A. Sakur: conceived and designed the experiments. 1. Nashed: performed the experiments and wrote the paper. 2. Noureldin: analyzed and interpreted the data. 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Page 19/20 Figure 5 Effect of pH on potentiometric response for MON.co sensor. Page 20/20