Impurity profiling of multicomponent cough syrup containing Brompheniramine, Dextromethorphan and Phenylephrine by RP-HPLC with PDA detector

Impurity profiling of multicomponent cough syrup containing Brompheniramine, Dextromethorphan and Phenylephrine by RP-HPLC with PDA detector | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impurity profiling of multicomponent cough syrup containing Brompheniramine, Dextromethorphan and Phenylephrine by RP-HPLC with PDA detector Priyanka Raju Pataskar, Gayatri Barabde, Vijay Arjun Bagul, Anand Radheshyam Tiwari This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7427571/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract A key aspect of pharmaceutical analysis that guarantees the efficacy, safety, and quality of medication items is impurity profiling. It involves the Identification and quantification of Impurities that may arise during synthesis. In this research a Novel RP-HPLC method was developed and validated including separation of Impurities and quantification of Brompheniramine Maleate. The separation of all three main components and Impurities was achieved using Gradient mode with the use of UV and PDA detector at 260nm wavelength. In port A of mobile phase 100% Potassium di-hydrogen phosphate buffer pH 3.0 was kept and in port B 100% Acetonitrile was kept. Separation was achieved by using Sunniest C18 column having dimensions 150 x 4.6mm, 3µm. 1.0ml/min flow rate was set with column temperature 30°C. Total run time was 40 min showed separation of all three components i.e. Brompheniramine Maleate (BPM), Dextromethorphan HBr (DMP), Phenylephrine HCl (PPN) and BPM’s impurities. A linear relationship (r = 0.99) revealed for all known analyte with concentration range of LOQ to 150%. Recovery study specifies the accuracy of the method. The repeatability determined that the method is precise enough within acceptance limit. Excellent linearity, accuracy, specificity, precision, robustness, LOD, LOQ and system applicability results are shown by the proposed approach. Moreover, the study on forced degradation showed that the method was stable suggesting. Method development and validation Related substances Brompheniramine Maleate Impurity study HPLC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The concurrent administration of multiple therapeutic agents in fixed-dose combinations (FDCs) is commonly used to treat upper respiratory tract infections and allergic conditions. A prominent combination includes BPM (an H₁-antihistamine), DMP (a non-opioid antitussive), and PPN (a sympathomimetic decongestant), which together provide effective relief from cough, nasal congestion, and allergy symptoms¹⁻³. However, analytical challenges arise due to the need for simultaneous quantification of active pharmaceutical ingredients (APIs) along with process- and degradation-related impurities, particularly Chlorpheniramine related compound B (Impurity A), Chlorpheniramine (Impurity B), and Pheniramine (Impurity C), which are structurally related to Brompheniramine⁴⁻⁶. (Figure I) Brompheniramine, a chiral alkylamine derivative, can easily undergo degradation or epimerization, forming isomeric or process impurities such as Chlorpheniramine, Chlorpheniramine Related Comp B or Pheniramine⁴⁻⁵. These impurities must be quantitatively controlled due to their potential pharmacological activity⁶. Dextromethorphan acts by antagonizing NMDA receptors and agonizing sigma-1 receptors. It is extensively metabolized via CYP2D6, exhibiting interindividual variability in pharmacokinetics⁷⁻⁸. Phenylephrine, a selective α1-adrenergic agonist, has inconsistent bioavailability and is sensitive to matrix interference, necessitating robust detection in multi-analyte systems⁹. Although various methods have been reported for individual or dual drug combinations, literature addressing the simultaneous estimation of all three drugs along with Brompheniramine-related impurities using a single HPLC method remains limited¹⁰⁻¹². This study aims to develop and validate a precise and selective RP-HPLC method capable of quantifying these APIs and impurities in complex FDCs. The method is validated according to ICH Q2(R1) guidelines for parameters including specificity, linearity, precision, accuracy, and robustness¹³. This analytical approach not only improves efficiency in pharmaceutical quality control but also meets regulatory expectations for impurity profiling 16 – 19 and stability testing¹⁴⁻¹⁵. Brompheniramine Maleate Dextromethorphan HBr Phenylephrine HCl Chlorpheniramine related comp B Chlorpheniramine Pheniramine Figure I: Chemical structure of BPM, DMP, PPN & Impurity A, B & C 2. Experimental 2.1 Instrument used Thermo Scientific Ultimate 3000 HPLC were used for the analysis with UV and PDA detectors with Chromeleon 7.2.10 ES, pH meter of Lab India, Analytical balance of Sartorius, Vertex (Cyclo mixer) for mixing of REMI were instruments used for the analysis. 2.2 Chemicals and Reagents Analytical grade reference standards of BPM, DMP, PPN and Brompheniramine-related impurities were obtained from commercial sources. Dimetapp syrup was used for sample which was brought and gifted by my cousin from US. HPLC-grade acetonitrile, potassium dihydrogen phosphate (KH₂PO₄), and orthophosphoric acid were also procured in analytical reagent (AR) grade. Milli-Q water was used for the preparation of all aqueous solutions. 2.3 Preparation of Buffer: Phosphate buffer pH 3.0 was prepared by weighing 6.8g of Potassium dihydrogen orthophosphate in 1000ml of water. Dissolved well with the help of sonicator and then added 5ml of TEA i.e. Triethylamine in it. Further pH 3.0 adjusted with concentrated OPA. 2.4 Preparation of standard: Weighed 20 mg of BPM standard and transfer it in 50 ml volumetric flask. Added adequate amount of methanol. Sonicated to dissolve and then make up to the mark with methanol (BPM stock I 400PPM). Similarly weighed 20 mg of PPN and 20 mg DMP and transferred in separate 50 ml volumetric flask. Stock I of PPN and DMP of 400 PPM prepared. Further diluted 1 ml of BPM stock I, 2.5 ml of PPN stock I and 5 ml of DMP stock I all together in 100 ml volumetric flask and diluted with diluent. (Stock II of all standards) Further dilute 0.5 ml of stock II in 10 ml volumetric flask with diluent. 2.5 Preparation of analytical formulation sample: Weighed and transferred 10 ml sample in 20 ml volumetric flask. Added 15 ml of diluent and stir at vertex for 3–5 minutes to mix and dissolve it properly. Then make up to the mark with diluent. Filter this solution through 0.45µm nylon syringe filter and inject. Note : Placebo Solution also prepared as a sample. 2.6 Preparation of Impurities: Weigh and transfer 5mg of chlorpheniramine maleate impurity in 50ml volumetric flask. Added 30ml of Methanol sonicate to dissolve. Then make up to the mark with Methanol. Similarly, Pheniramine Impurity and Chlor related compound B Impurity prepared of PPM 100. Further 1ml from each stock solutions of impurities were taken and diluted to 50ml with diluent. (2 PPM). From above 2PPM solution 2.5ml of Impurity stock taken and diluted to 10ml with diluent to get 0.5PPM solution. 2.7 Method development The objective of the work was to develop a new RS method for the identification and measurement of Brompheniramine, Dextromethorphan HBr, Phenylephrine HCl, and Brompheniramine related compounds that have close elution, i.e., Chlorpheniramine, Chlor rel comp B, and Pheniramine. For this reason, several methodologies have been used, considering parameters such as column choice, mobile phase mix, and elution mode; the optimization process is shown in Table I. Table I: Method Development details with different variations in chromatographic conditions Trial No Column Mobile phase composition Elusion mode Result 1 Inertsil ODS 3V Buffer pH 6.0: ACN Isocratic Impurities peak shape distorted 2 Water Symmetry Buffer pH 4.0: Methanol Isocratic Peak shape was not proper 3 Water Symmetry Buffer pH 3.0: Methanol Gradient Impurity A and Brompheniramine merged 4 Inertsil ODS 3V Buffer pH 3.0: ACN Isocratic Peak of PPN was not eluted Potassium hydrogen phosphate buffer having pH 3.0 and Acetonitrile were used for the analysis after trying various buffer compositions. Buffer having different pH also tried as per the pKa value of BPM, DMP and PPN but buffer with pH 3.0 reflect the best peak shape of main peaks and impurities. Different columns such as Inertsil ODS 3V, Waters symmetry C8/C18, phenomenex were used for separation since Sunniest C18 150mm x 4.6, 3µm showed best resolution between main peaks and Impurities also avoid peak drifting problems. At 260nm drug showed maximum absorption. Retention time of BPM, DMP and PPN was 23.7 min, 25.5 min and 3.7 min. 50µL injection volume were chosen with flow rate of 1.0 ml/min. Column temperature was set at 30°C and Auto sampler temperature was set at 25°C. Wavelength was kept 260nm. Gradient program were used for the analysis. Diluent composition was 0.1M OPA: Methanol (90: 10). Refer Table II for mobile phase compositions. Table I: Mobile phase composition Time (min) Flow (ml/min) Mobile Phase %A (Buffer pH 3.0) Mobile Phase %B (ACN) 0.00 1.00 97 3 1.00 1.00 97 3 20.00 1.00 75 25 26.00 1.00 65 35 30.00 1.00 50 50 35.00 1.00 97 3 40.00 1.00 97 3 2.8 Method Validation To confirm that the optimized method performs accurately, consistently, and reliably for its intended application while fulfilling regulatory requirements, it was validated in accordance with ICH Q2(R2) guidelines. 3. Result and Discussion The chromatographic method developed in the current study for the organic impurities of Brompheniramine was validated in line with the ICH guidelines for specificity, linearity, quantification limit, precision, accuracy, solution stability and robustness. The following sections discuss the validation study. 3.1 Specificity & System suitability Specificity is a critical validation parameter that defines the ability of the analytical procedure to quantitate the analyte of interest with precision in the presence of other constituents that could be present in the sample, e.g., impurities, excipients, or degradation products. Standard containing brompheniramine and known impurities was used to determine reference peaks. The test material was the actual sample to determine the API and any impurities present. The placebo containing all excipients except the active ingredient and impurities was used to determine the lack of interfering signals from the formulation matrix. The blank containing the diluent only was used to determine if no unwanted peaks were observed at the retention times of interest. All these tests ensured that the analytical procedure distinctly differentiated brompheniramine and impurities from other ingredients, and thus determined method specificity (Refer figure II, III, IV, V & VI). No interference was observed at the retention time of BPM, DMP, PPM and all 3 impurities of Brompheniramine establishing the developed method with ability to separate the analyte from other components in the formulation and provide accurate quantitation under normal conditions. The peak purity requirements tested for each analyte peak were within acceptable limits. (Table III) Table III: Results of the specificity study Sample No Name of Active Ingredients & Impurity RT (Min) RRT (w.r.t. BPM) Peak Purity 1 BPM 23.643 1.00 1.000 2 DMP 25.223 1.07 0.999 3 PPN 3.667 0.16 0.999 4 Impurity A 22.813 0.96 0.999 5 Impurity B 11.057 0.47 0.999 6 Impurity C 14.957 0.63 0.999 RT, Retention time RRT, Relative Retention time w.r.t., with respect to 3.2 Linearity For organic impurities, we assessed the test method's linearity by preparing a series of linearity solutions containing BPM, DMP, PPN, and BPM’s impurities at five different concentrations. These ranged from the detection limit LOQ to 150% of the target concentration. This included Impurity A, B & C at concentration range from 0.0504–0.7554 µg/ml. We plotted the calibration curve for each impurity, along with BPM, DMP, and PPN, using the peak area against the actual concentration. The regression analysis gave a correlation coefficient greater than 0.999, indicating excellent linearity over the tested range. (Refer table IV & Figure II) We calculated the relative response factor for each impurity by dividing the slope of the impurity by the slope of the main active analyte. This confirms that the method is suitable for accurate quantification across the concentrations usually found in quality control and content uniformity analysis. Table IV: Results of linearity for the determination of BPM, DMP, PPN and BPM’s Impurity Name Concentration Range(µg/ml) Correlation coefficient % Y Intercept Slope of regression line Residual sum of square RRF BPM 0.0200-0.2995 0.9989 0.1934 51.8295 230.07 NA DMP 0.0998–1.4976 0.9990 -0.0506 4.1880 0.14 NA PPN 0.0504–0.7560 0.9991 0.4597 21.4645 0.81 NA Impurity A 0.0504–0.7554 0.9997 0.4214 36.2234 066 0.70 Impurity B 0.0504–0.7554 0.9998 0.7292 99.1438 3.62 1.91 Impurity C 0.0504–0.7554 0.9989 4.1389 41.4007 7.44 0.80 3.3 Accuracy The accuracy of the test method was demonstrated by performing recovery experiments at three different concentration levels, i.e. QL 10% (0.05 µg/ml), 100% (0.5 µg/ml) and 150% (0.75 µg/ml), for each impurity. A standard stock solution of all three impurities was prepared at a concentration of 100 µg/ml for each impurity and three spiked sample solutions were made at each accuracy. The percentage recovery, mean percentage recovery and RSD were calculated from the data obtained from the liquid chromatography (Table IV). The recovery values obtained from the accuracy study were found to be in a range of 90–110% (limit 80.0–120.0%) from all preparations (n = 9) and were considered for the method precision study. Also, the RSD results of the accuracy study confirmed that the developed test method is accurate and precise. Table V: Result of Accuracy study for impurities of BPM & all active API Impurity Mean Recovery Overall mean (n = 9) RSD (%) (n = 9) 0.05 µg/ml 0.5 µg/ml 0.75 µg/ml Impurity A 97.6 101.5 99.8 99.6 3.0 Impurity B 96.8 103.2 98.5 99.5 2.1 Impurity C 98.2 102.4 97.9 99.5 1.9 BPM 102.6 100.2 103.4 102.1 2.3 DMP 97.2 98.6 99.1 98.3 1.8 PPN 99.6 101.3 102.8 101.2 1.5 3.4 Method Precision & Intermediate Precision (MP & IP) Reproducibility of the test procedure established was proven by individual analysis of six spiked sample solutions at target concentration level of 0.2%, w/w from homogeneous samples of the same batch of Dimetapp syrup. The amount of the impurity in each sample was then accurately determined. The recovery values for each impurity (80.0-120%) based on six replicate sample analyses were calculated, and the relative standard deviation (RSD; limit ≤ 10.0%), and these are shown in Table VI. Table VI: Results of MP & IP of known impurities of BPM & all active API Component name MP at limit level IP at limit level % Recovery RSD (%) % Recovery RSD (%) Impurity A 96.5 2.3 97.1 2.1 Impurity B 104.6 1.5 103.8 1.6 Impurity C 103.9 2.6 104.1 2.3 BPM 101.4 1.8 100.9 1.9 DMP 96.6 2.0 97.1 2.0 PPN 98.1 1.3 98.4 1.4 3.5 Range The range of analysis determines the range of upper and lower concentrations of the analyte for which the methodology has been shown to be within acceptable linearity, accuracy, and precision. The analytical procedure showed consistent performance within the validated range, including concentration levels of interest in products and regulatory limits. This ensures the usability of the procedure for stability testing and routine testing of antihistamine drugs. 3.6 Limit of Detection (LOD) The LOD is the minimum concentration of an analyte that can be accurately determined, if not quantitated, by the technique. The LOD was computed from the response standard deviation and the calibration curve slope. The obtained value was low enough to allow trace levels of the antihistamine drugs or impurities to be detected, which is of great relevance in impurity profiling and trace contaminant monitoring of medicines. 3.7 Limit of Quantification (LOQ) LOQ is the minimum quantity of analyte that can be quantitated with acceptable precision and accuracy. LOQ was set based on calibration curve data, and analysis in the LOQ level concentration was repeated to ensure that the method provides reproducible and reliable quantitation. Low LOQ reflects the appropriately precise determination of trace impurities and degradants as well as low levels of active ingredients. 3.8 Robustness Test procedure robustness was shown by changing column temperature, (30°C ± 2°C) and buffer solution pH (pH 3.0 ± 0.2) intentionally so that system suitability does not change on passing the acceptance criteria (Table VII). RT and RRT of all spiked sample impurities and resolution values of spiked samples showed that there is no variation with changing column temperature, and buffer pH on BPM, DMP, PPN and all the four impurities. Impurity estimation deviation was within ± 10%. Change was not noticed by making variations, which proves robustness of method regarding column temperature and buffer pH variation. Table VII: Result of Robustness study of BPM, DMP, PPN & BPM’s Impurity Name & Variable Original Condition Column Temp 28°C Column Temp 32°C Buffer pH 2.8 Buffer pH 3.2 BPM RT 23.75 23.541 23.801 23.432 23.405 RRT 1.00 1.00 1.00 1.00 1.00 % Recovery 102.5 96.5 98.1 103.8 102.3 DMP RT 25.537 25.214 25.705 25.598 25.277 RRT 1.08 1.07 1.08 1.09 1.08 % Recovery 101.2 96.2 98.0 99.4 101.1 PPN RT 3.750 3.496 3.808 3.984 3.782 RRT 0.16 0.15 0.16 0.17 0.16 % Recovery 98.5 98.7 97.6 98.1 99.2 Impurity A RT 22.470 22.014 22.531 22.451 22.204 RRT 0.95 0.94 0.95 0.96 0.95 % Recovery 97.1 101.6 100.7 97.4 97.5 Impurity B RT 14.577 14.124 14.602 14.451 14.288 RRT 0.61 0.60 0.61 0.62 0.61 % Recovery 103.5 100.4 99.1 94.7 96.8 Impurity C RT 10.690 10.315 10.814 10.841 10.416 RRT 0.45 0.44 0.45 0.46 0.45 % Recovery 102.9 99.5 98.7 95.4 95.7 4. Conclusion A simple and accurate chromatography method was developed for the separation, identification, detection and quantitative estimation of Brompheniramine, Dextromethorphan, Phenylephrine and organic impurities of brompheniramine i.e. Chlorpheniramine, Chlorpheniramine related compound B, Pheniramine. The chromatogram run time was chosen as 40 min in which all active pharmaceutical ingredients and impurity peaks were well resolved with a resolution of minimum 1.5 without any change in retention time, drifting or interference. The proposed HPLC method was validated according to the ICH Q2(R2) guidelines. The developed HPLC method was found to be very effective in separating all the impurity peaks and active pharmaceutical ingredient peak from excipients of syrup formulation. The developed method can be used in quality control labs for quantitative estimation of organic impurities of Brompheniramine in routine release testing and stability study of Brompheniramine, Dextromethorphan, Phenylephrine drug substances and drug products. Declarations Declarations Ethical approval and consent to participate Not applicable. All participants provided voluntary, informed consent. Consent for publication: Participants agreed to the publication of anonymized information. Competing interests: The authors declare no competing interests. Clinical Trial Number: Not Applicable Country Affiliation : Affi1 Department of Chemistry, The Institute of Science, HBSU Mumbai-400032, Maharashtra Country Name India Funding: This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution I, Priyanka Raju Pataskar, conceptualised and performed the research work on Brompheniramine RS method development and validation, carried out experimental studies, analysed the data, and prepared the manuscript draft. Dr. Gayatri Barabde, with over 25 years of research experience, supervised the work as PhD guide, provided expert guidance throughout the study, and critically revised the manuscript. Vijay Bagul and Anand Tiwari contributed to experimental support, data interpretation, and provided valuable technical assistance. All authors reviewed and approved the final version of the manuscript. Acknowledgement: The author sincerely acknowledges Dr. Gayatri Barabde for her insightful mentorship and continuous guidance, which played a pivotal role in shaping the research. Deep appreciation is extended to Mr. Vijay Bagul and Mr. Anand Tiwari for their unwavering technical assistance and commitment throughout the course of the study. The author is also grateful to the Institute of Science, Mumbai; and Chemclues laboratory for providing critical infrastructure, instrumentation, and resources that significantly facilitated the successful execution of this work. Data availability: Data is provided within the manuscript or supplementary information files. References Dagariya RK, et al. Stability Indicating Method Development and Validation for Simultaneous Estimation of Dextromethorphan, Phenylephrine, and Chlorpheniramine in Syrup. Trop J Pharm Life Sci. 2021;8(3):1–14. Jain V, et al. Validated RP-HPLC method for simultaneous estimation of bromhexine hydrochloride, chlorpheniramine maleate, and guaifenesin. Saudi Pharm J. 2016;24(3):306–11. Rosireddy V et al. An ecologically sustainable RP-HPLC method for simultaneous estimation of Phenylephrine HCl, Chlorpheniramine Maleate and Dextromethorphan. Int J Green Pharm. 2024. Ali A, et al. 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Quantification Of (4-Bromophenyl) {Pyridine-2-Yl} Acetonitrile Impurity (4-BPPA) by HPLC in Brompheniramine Maleate. 2017. Innovare Acad. Stability Indicating UPLC Method for Simultaneous Estimation of Chlorpheniramine and Dextromethorphan. 2019. Patel H, et al. Review on simultaneous drug estimation. J Chromatogr B. 2022;1198:123193. Singh R, Sharma A. Method validation in pharmaceutical QA/QC. Indian J Pharm Sci. 2016;78(3):379–87. Bagul VA, Ambadekar SR, Tiwari AR. A comprehensive stability indicating method for clonidine hydrochloride impurities profiling and degradation analysis in tablet formulations: green metric study. Discov Chem. 2025;2:160. https://doi.org/10.1007/s44371-025-00238-4 . Development and Validation of New RP-HPLC Analytical Method for Simultaneous Estimation of Impurities Present in Nortriptyline and Pregabalin Combined Pharmaceutical Dosage Form by Vijay Arjun Bagul, Dr. Sushama Raju Ambadekar and Anand Radheshyam Tiwari Published in Urban India journal (ISSN No: 0970–9045), Volume 43 Issue (II). December 2023, Pg 188–197. Tiwari AR, Ambadekar SR, Bagul VA. Sensitive and sustainable stability-indicating RP-HPLC method for simultaneous assay and impurity profiling of amitriptyline HCl: green metric, RGB model and forced degradation study. Discov Chem. 2025;2:157. https://doi.org/10.1007/s44371-025-00218-8 . A Comprehensive Stability-Indicating Analytical Method for Escitalopram Impurity analysis by RP-HPLC; Force Degradation Study by Anand Tiwari, Dr. Sushama Ambedkar and Vijay A. Bagul. Published Int J All Res Educ Sci Methods (IJARESM) ISSN: 2455–6211.12, Issue 2 (February 2024). Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":84960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure I: Chemical structure of BPM, DMP, PPN \u0026amp; Impurity A, B \u0026amp; C\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/40ac49f1323c6de68d3f3a73.png"},{"id":94024999,"identity":"6ab85aaa-7f77-45a5-8f48-114b82a2b0bb","added_by":"auto","created_at":"2025-10-21 13:17:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19166,"visible":true,"origin":"","legend":"\u003cp\u003eFigure II: Chromatogram of Blank\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/fe16e6ba2dc6e09f5caea7a4.png"},{"id":94025001,"identity":"9df3b175-0c98-44c1-9fdf-6deb5a736785","added_by":"auto","created_at":"2025-10-21 13:17:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36164,"visible":true,"origin":"","legend":"\u003cp\u003eFigure III: Chromatogram of Placebo\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/5e78a4bf03f958d24615ae4a.png"},{"id":94025034,"identity":"398704ad-75ab-485b-85a3-8e903f8b2738","added_by":"auto","created_at":"2025-10-21 13:17:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29073,"visible":true,"origin":"","legend":"\u003cp\u003eFigure IV: Chromatogram of Standard solution\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/cf63f41c48a7f822dff50818.png"},{"id":94025877,"identity":"be111d4e-ac51-4663-a7d6-d5a90e4f43b9","added_by":"auto","created_at":"2025-10-21 13:25:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36625,"visible":true,"origin":"","legend":"\u003cp\u003eFigure V: Chromatogram of Sample\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/e508a577e6f38d4df93e5e9e.png"},{"id":94025027,"identity":"247c3dde-6c26-48f7-a98f-7c91288d344d","added_by":"auto","created_at":"2025-10-21 13:17:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39373,"visible":true,"origin":"","legend":"\u003cp\u003eFigure VI: Chromatogram of Sample spiked\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/730d26a0777f7e129456e02f.png"},{"id":94025036,"identity":"7f1d7a01-1832-4e8b-b7d0-6638f4312969","added_by":"auto","created_at":"2025-10-21 13:17:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":60047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure II: Linearity Plot of BPM, DMP, PPN and Brompheniramine’s Impurity\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/93615b1967e113dd2214f6f5.png"},{"id":94027466,"identity":"036bac87-437a-4157-9c8d-52bf80395d83","added_by":"auto","created_at":"2025-10-21 13:49:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1617954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7427571/v1/632d9f60-2ad4-466c-8a12-8a982da296a2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impurity profiling of multicomponent cough syrup containing Brompheniramine, Dextromethorphan and Phenylephrine by RP-HPLC with PDA detector","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe concurrent administration of multiple therapeutic agents in fixed-dose combinations (FDCs) is commonly used to treat upper respiratory tract infections and allergic conditions. A prominent combination includes BPM (an H₁-antihistamine), DMP (a non-opioid antitussive), and PPN (a sympathomimetic decongestant), which together provide effective relief from cough, nasal congestion, and allergy symptoms\u0026sup1;⁻\u0026sup3;. However, analytical challenges arise due to the need for simultaneous quantification of active pharmaceutical ingredients (APIs) along with process- and degradation-related impurities, particularly Chlorpheniramine related compound B (Impurity A), Chlorpheniramine (Impurity B), and Pheniramine (Impurity C), which are structurally related to Brompheniramine⁴⁻⁶. (Figure I)\u003c/p\u003e\u003cp\u003eBrompheniramine, a chiral alkylamine derivative, can easily undergo degradation or epimerization, forming isomeric or process impurities such as Chlorpheniramine, Chlorpheniramine Related Comp B or Pheniramine⁴⁻⁵. These impurities must be quantitatively controlled due to their potential pharmacological activity⁶. Dextromethorphan acts by antagonizing NMDA receptors and agonizing sigma-1 receptors. It is extensively metabolized via CYP2D6, exhibiting interindividual variability in pharmacokinetics⁷⁻⁸. Phenylephrine, a selective α1-adrenergic agonist, has inconsistent bioavailability and is sensitive to matrix interference, necessitating robust detection in multi-analyte systems⁹.\u003c/p\u003e\u003cp\u003eAlthough various methods have been reported for individual or dual drug combinations, literature addressing the simultaneous estimation of all three drugs along with Brompheniramine-related impurities using a single HPLC method remains limited\u0026sup1;⁰⁻\u0026sup1;\u0026sup2;. This study aims to develop and validate a precise and selective RP-HPLC method capable of quantifying these APIs and impurities in complex FDCs. The method is validated according to ICH Q2(R1) guidelines for parameters including specificity, linearity, precision, accuracy, and robustness\u0026sup1;\u0026sup3;. This analytical approach not only improves efficiency in pharmaceutical quality control but also meets regulatory expectations for impurity profiling\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and stability testing\u0026sup1;⁴⁻\u0026sup1;⁵.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBrompheniramine Maleate Dextromethorphan HBr Phenylephrine HCl\u003c/p\u003e\u003cp\u003eChlorpheniramine related comp B Chlorpheniramine Pheniramine\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure I: Chemical structure of BPM, DMP, PPN \u0026amp; Impurity A, B \u0026amp; C\u003c/b\u003e\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1 Instrument used\u003c/h2\u003e\n\u003cp\u003eThermo Scientific Ultimate 3000 HPLC were used for the analysis with UV and PDA detectors with Chromeleon 7.2.10 ES, pH meter of Lab India, Analytical balance of Sartorius, Vertex (Cyclo mixer) for mixing of REMI were instruments used for the analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2 Chemicals and Reagents\u003c/h2\u003e\n\u003cp\u003eAnalytical grade reference standards of BPM, DMP, PPN and Brompheniramine-related impurities were obtained from commercial sources. Dimetapp syrup was used for sample which was brought and gifted by my cousin from US. HPLC-grade acetonitrile, potassium dihydrogen phosphate (KH₂PO₄), and orthophosphoric acid were also procured in analytical reagent (AR) grade. Milli-Q water was used for the preparation of all aqueous solutions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3 Preparation of Buffer:\u003c/h2\u003e\n\u003cp\u003ePhosphate buffer pH 3.0 was prepared by weighing 6.8g of Potassium dihydrogen orthophosphate in 1000ml of water. Dissolved well with the help of sonicator and then added 5ml of TEA i.e. Triethylamine in it. Further pH 3.0 adjusted with concentrated OPA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4 Preparation of standard:\u003c/h2\u003e\n\u003cp\u003eWeighed 20 mg of BPM standard and transfer it in 50 ml volumetric flask. Added adequate amount of methanol. Sonicated to dissolve and then make up to the mark with methanol (BPM stock I 400PPM). Similarly weighed 20 mg of PPN and 20 mg DMP and transferred in separate 50 ml volumetric flask. Stock I of PPN and DMP of 400 PPM prepared.\u003c/p\u003e\n\u003cp\u003eFurther diluted 1 ml of BPM stock I, 2.5 ml of PPN stock I and 5 ml of DMP stock I all together in 100 ml volumetric flask and diluted with diluent. (Stock II of all standards)\u003c/p\u003e\n\u003cp\u003eFurther dilute 0.5 ml of stock II in 10 ml volumetric flask with diluent.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e2.5 Preparation of analytical formulation sample:\u003c/h2\u003e\n\u003cp\u003eWeighed and transferred 10 ml sample in 20 ml volumetric flask. Added 15 ml of diluent and stir at vertex for 3\u0026ndash;5 minutes to mix and dissolve it properly. Then make up to the mark with diluent. Filter this solution through 0.45\u0026micro;m nylon syringe filter and inject. \u003cstrong\u003eNote\u003c/strong\u003e: Placebo Solution also prepared as a sample.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e2.6 Preparation of Impurities:\u003c/h2\u003e\n\u003cp\u003eWeigh and transfer 5mg of chlorpheniramine maleate impurity in 50ml volumetric flask. Added 30ml of Methanol sonicate to dissolve. Then make up to the mark with Methanol. Similarly, Pheniramine Impurity and Chlor related compound B Impurity prepared of PPM 100.\u003c/p\u003e\n\u003cp\u003eFurther 1ml from each stock solutions of impurities were taken and diluted to 50ml with diluent. (2 PPM).\u003c/p\u003e\n\u003cp\u003eFrom above 2PPM solution 2.5ml of Impurity stock taken and diluted to 10ml with diluent to get 0.5PPM solution.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e2.7 Method development\u003c/h2\u003e\n\u003cp\u003eThe objective of the work was to develop a new RS method for the identification and measurement of Brompheniramine, Dextromethorphan HBr, Phenylephrine HCl, and Brompheniramine related compounds that have close elution, i.e., Chlorpheniramine, Chlor rel comp B, and Pheniramine. For this reason, several methodologies have been used, considering parameters such as column choice, mobile phase mix, and elution mode; the optimization process is shown in Table I.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable I: Method Development details with different variations in chromatographic conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTrial No\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eColumn\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMobile phase composition\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eElusion mode\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eResult\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInertsil ODS 3V\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBuffer pH 6.0: ACN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIsocratic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurities peak shape distorted\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWater Symmetry\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBuffer pH 4.0: Methanol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIsocratic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePeak shape was not proper\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWater Symmetry\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBuffer pH 3.0: Methanol\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGradient\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity A and Brompheniramine merged\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInertsil ODS 3V\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBuffer pH 3.0: ACN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIsocratic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePeak of PPN was not eluted\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePotassium hydrogen phosphate buffer having pH 3.0 and Acetonitrile were used for the analysis after trying various buffer compositions. Buffer having different pH also tried as per the pKa value of BPM, DMP and PPN but buffer with pH 3.0 reflect the best peak shape of main peaks and impurities. Different columns such as Inertsil ODS 3V, Waters symmetry C8/C18, phenomenex were used for separation since Sunniest C18 150mm x 4.6, 3\u0026micro;m showed best resolution between main peaks and Impurities also avoid peak drifting problems. At 260nm drug showed maximum absorption. Retention time of BPM, DMP and PPN was 23.7 min, 25.5 min and 3.7 min. 50\u0026micro;L injection volume were chosen with flow rate of 1.0 ml/min. Column temperature was set at 30\u0026deg;C and Auto sampler temperature was set at 25\u0026deg;C. Wavelength was kept 260nm. Gradient program were used for the analysis. Diluent composition was 0.1M OPA: Methanol (90: 10). Refer Table II for mobile phase compositions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable I: Mobile phase composition\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabb\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTime (min)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFlow (ml/min)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMobile Phase %A (Buffer pH 3.0)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMobile Phase %B (ACN)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e35\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e2.8 Method Validation\u003c/h2\u003e\n\u003cp\u003eTo confirm that the optimized method performs accurately, consistently, and reliably for its intended application while fulfilling regulatory requirements, it was validated in accordance with ICH Q2(R2) guidelines.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Result and Discussion","content":"\u003cp\u003eThe chromatographic method developed in the current study for the organic impurities of Brompheniramine was validated in line with the ICH guidelines for specificity, linearity, quantification limit, precision, accuracy, solution stability and robustness. The following sections discuss the validation study.\u003c/p\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Specificity \u0026amp; System suitability\u003c/h2\u003e\n\u003cp\u003eSpecificity is a critical validation parameter that defines the ability of the analytical procedure to quantitate the analyte of interest with precision in the presence of other constituents that could be present in the sample, e.g., impurities, excipients, or degradation products. Standard containing brompheniramine and known impurities was used to determine reference peaks. The test material was the actual sample to determine the API and any impurities present. The placebo containing all excipients except the active ingredient and impurities was used to determine the lack of interfering signals from the formulation matrix. The blank containing the diluent only was used to determine if no unwanted peaks were observed at the retention times of interest. All these tests ensured that the analytical procedure distinctly differentiated brompheniramine and impurities from other ingredients, and thus determined method specificity (Refer figure II, III, IV, V \u0026amp; VI). No interference was observed at the retention time of BPM, DMP, PPM and all 3 impurities of Brompheniramine establishing the developed method with ability to separate the analyte from other components in the formulation and provide accurate quantitation under normal conditions. The peak purity requirements tested for each analyte peak were within acceptable limits. (Table III)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable III: Results of the specificity study\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabc\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSample No\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eName of Active Ingredients \u0026amp; Impurity\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRT (Min)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRRT (w.r.t. BPM)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePeak Purity\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBPM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.643\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.000\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDMP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25.223\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.999\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.667\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.999\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.813\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.999\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11.057\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.999\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.957\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.999\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eRT, Retention time\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eRRT, Relative Retention time\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003ew.r.t., with respect to\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Linearity\u003c/h2\u003e\n\u003cp\u003eFor organic impurities, we assessed the test method's linearity by preparing a series of linearity solutions containing BPM, DMP, PPN, and BPM\u0026rsquo;s impurities at five different concentrations. These ranged from the detection limit LOQ to 150% of the target concentration. This included Impurity A, B \u0026amp; C at concentration range from 0.0504\u0026ndash;0.7554 \u0026micro;g/ml. We plotted the calibration curve for each impurity, along with BPM, DMP, and PPN, using the peak area against the actual concentration. The regression analysis gave a correlation coefficient greater than 0.999, indicating excellent linearity over the tested range. (Refer table IV \u0026amp; Figure II) We calculated the relative response factor for each impurity by dividing the slope of the impurity by the slope of the main active analyte. This confirms that the method is suitable for accurate quantification across the concentrations usually found in quality control and content uniformity analysis.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable IV: Results of linearity for the determination of BPM, DMP, PPN and BPM\u0026rsquo;s Impurity\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabe\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eName\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eConcentration Range(\u0026micro;g/ml)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCorrelation coefficient\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e% Y Intercept\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSlope of regression line\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eResidual sum of square\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRRF\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBPM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0200-0.2995\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.9989\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.1934\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51.8295\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e230.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDMP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0998\u0026ndash;1.4976\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.9990\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e-0.0506\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.1880\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0504\u0026ndash;0.7560\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.9991\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.4597\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e21.4645\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0504\u0026ndash;0.7554\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.9997\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.4214\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36.2234\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e066\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.70\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0504\u0026ndash;0.7554\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.9998\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.7292\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.1438\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.91\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0504\u0026ndash;0.7554\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.9989\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.1389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e41.4007\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.80\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Accuracy\u003c/h2\u003e\n\u003cp\u003eThe accuracy of the test method was demonstrated by performing recovery experiments at three different concentration levels, i.e. QL 10% (0.05 \u0026micro;g/ml), 100% (0.5 \u0026micro;g/ml) and 150% (0.75 \u0026micro;g/ml), for each impurity. A standard stock solution of all three impurities was prepared at a concentration of 100 \u0026micro;g/ml for each impurity and three spiked sample solutions were made at each accuracy. The percentage recovery, mean percentage recovery and RSD were calculated from the data obtained from the liquid chromatography (Table IV). The recovery values obtained from the accuracy study were found to be in a range of 90\u0026ndash;110% (limit 80.0\u0026ndash;120.0%) from all preparations (n\u0026thinsp;=\u0026thinsp;9) and were considered for the method precision study. Also, the RSD results of the accuracy study confirmed that the developed test method is accurate and precise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable V: Result of Accuracy study for impurities of BPM \u0026amp; all active API\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabf\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eImpurity\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMean Recovery\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eOverall mean (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eRSD (%) (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.05 \u0026micro;g/ml\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.5 \u0026micro;g/ml\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0.75 \u0026micro;g/ml\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e103.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBPM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e100.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e103.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDMP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Method Precision \u0026amp; Intermediate Precision (MP \u0026amp; IP)\u003c/h2\u003e\n\u003cp\u003eReproducibility of the test procedure established was proven by individual analysis of six spiked sample solutions at target concentration level of 0.2%, w/w from homogeneous samples of the same batch of Dimetapp syrup. The amount of the impurity in each sample was then accurately determined. The recovery values for each impurity (80.0-120%) based on six replicate sample analyses were calculated, and the relative standard deviation (RSD; limit\u0026thinsp;\u0026le;\u0026thinsp;10.0%), and these are shown in Table VI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable VI: Results of MP \u0026amp; IP of known impurities of BPM \u0026amp; all active API\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabg\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eComponent name\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eMP at limit level\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eIP at limit level\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRSD (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRSD (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e104.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e103.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImpurity C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e103.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e104.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBPM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e100.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDMP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Range\u003c/h2\u003e\n\u003cp\u003eThe range of analysis determines the range of upper and lower concentrations of the analyte for which the methodology has been shown to be within acceptable linearity, accuracy, and precision. The analytical procedure showed consistent performance within the validated range, including concentration levels of interest in products and regulatory limits. This ensures the usability of the procedure for stability testing and routine testing of antihistamine drugs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 Limit of Detection (LOD)\u003c/h2\u003e\n\u003cp\u003eThe LOD is the minimum concentration of an analyte that can be accurately determined, if not quantitated, by the technique. The LOD was computed from the response standard deviation and the calibration curve slope. The obtained value was low enough to allow trace levels of the antihistamine drugs or impurities to be detected, which is of great relevance in impurity profiling and trace contaminant monitoring of medicines.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7 Limit of Quantification (LOQ)\u003c/h2\u003e\n\u003cp\u003eLOQ is the minimum quantity of analyte that can be quantitated with acceptable precision and accuracy. LOQ was set based on calibration curve data, and analysis in the LOQ level concentration was repeated to ensure that the method provides reproducible and reliable quantitation. Low LOQ reflects the appropriately precise determination of trace impurities and degradants as well as low levels of active ingredients.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e3.8 Robustness\u003c/h2\u003e\n\u003cp\u003eTest procedure robustness was shown by changing column temperature, (30\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and buffer solution pH (pH 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) intentionally so that system suitability does not change on passing the acceptance criteria (Table VII). RT and RRT of all spiked sample impurities and resolution values of spiked samples showed that there is no variation with changing column temperature, and buffer pH on BPM, DMP, PPN and all the four impurities. Impurity estimation deviation was within \u0026plusmn;\u0026thinsp;10%. Change was not noticed by making variations, which proves robustness of method regarding column temperature and buffer pH variation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable VII: Result of Robustness study of BPM, DMP, PPN \u0026amp; BPM\u0026rsquo;s Impurity\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tabh\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eName \u0026amp; Variable\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eOriginal Condition\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eColumn Temp 28\u0026deg;C\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eColumn Temp 32\u0026deg;C\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBuffer pH 2.8\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBuffer pH 3.2\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eBPM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.541\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.801\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.432\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23.405\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e103.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eDMP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25.537\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25.214\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25.705\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25.598\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e25.277\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.08\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ePPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.496\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.808\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.984\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.782\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eImpurity A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.470\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.531\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.451\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22.204\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.95\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e101.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e100.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eImpurity B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.577\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.124\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.602\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.451\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e14.288\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.61\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e103.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e100.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e94.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eImpurity C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.690\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.315\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.814\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.841\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.416\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRRT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.45\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e% Recovery\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e99.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e98.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e95.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e95.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eA simple and accurate chromatography method was developed for the separation, identification, detection and quantitative estimation of Brompheniramine, Dextromethorphan, Phenylephrine and organic impurities of brompheniramine i.e. Chlorpheniramine, Chlorpheniramine related compound B, Pheniramine. The chromatogram run time was chosen as 40 min in which all active pharmaceutical ingredients and impurity peaks were well resolved with a resolution of minimum 1.5 without any change in retention time, drifting or interference. The proposed HPLC method was validated according to the ICH Q2(R2) guidelines. The developed HPLC method was found to be very effective in separating all the impurity peaks and active pharmaceutical ingredient peak from excipients of syrup formulation. The developed method can be used in quality control labs for quantitative estimation of organic impurities of Brompheniramine in routine release testing and stability study of Brompheniramine, Dextromethorphan, Phenylephrine drug substances and drug products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable. All participants provided voluntary, informed consent.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003cp\u003eParticipants agreed to the publication of anonymized information.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eClinical Trial Number:\u003c/h2\u003e\u003cp\u003eNot Applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003e\u003cb\u003eCountry Affiliation\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eAffi1\u003c/strong\u003e\u003cp\u003eDepartment of Chemistry, The Institute of Science, HBSU Mumbai-400032, Maharashtra\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCountry Name\u003c/strong\u003e\u003cp\u003eIndia\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eI, Priyanka Raju Pataskar, conceptualised and performed the research work on Brompheniramine RS method development and validation, carried out experimental studies, analysed the data, and prepared the manuscript draft. Dr. Gayatri Barabde, with over 25 years of research experience, supervised the work as PhD guide, provided expert guidance throughout the study, and critically revised the manuscript. Vijay Bagul and Anand Tiwari contributed to experimental support, data interpretation, and provided valuable technical assistance. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement:\u003c/h2\u003e\u003cp\u003eThe author sincerely acknowledges Dr. Gayatri Barabde for her insightful mentorship and continuous guidance, which played a pivotal role in shaping the research. Deep appreciation is extended to Mr. Vijay Bagul and Mr. Anand Tiwari for their unwavering technical assistance and commitment throughout the course of the study. The author is also grateful to the Institute of Science, Mumbai; and Chemclues laboratory for providing critical infrastructure, instrumentation, and resources that significantly facilitated the successful execution of this work.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDagariya RK, et al. Stability Indicating Method Development and Validation for Simultaneous Estimation of Dextromethorphan, Phenylephrine, and Chlorpheniramine in Syrup. Trop J Pharm Life Sci. 2021;8(3):1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJain V, et al. Validated RP-HPLC method for simultaneous estimation of bromhexine hydrochloride, chlorpheniramine maleate, and guaifenesin. Saudi Pharm J. 2016;24(3):306\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRosireddy V et al. An ecologically sustainable RP-HPLC method for simultaneous estimation of Phenylephrine HCl, Chlorpheniramine Maleate and Dextromethorphan. Int J Green Pharm. 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAli A, et al. Stability-indicating high-performance liquid chromatographic method for simultaneous determination of aminophylline and chlorpheniramine maleate. N Am J Med Sci. 2015;7(9):438\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChao MK. Ion-pair Reversed-phase HPLC Separation and Assay of Cough-Cold Combination Drugs. J Pharm Sci. 1979;68(10):1269\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNezhadali A, et al. Method development for simultaneous determination of three active ingredients. Heliyon. 2019;5(11):e02790.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQ2(R1). Guideline: Validation of Analytical Procedures. International Council for Harmonisation; 2005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHavrylenko O et al. Optimization of HPLC methods for the simultaneous determination of paracetamol, phenylephrine, dextromethorphan, and chlorpheniramine. Pharmacia. 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSahu R, Patnaik A. Analytical determination of drug impurities. Int J Pharm Sci Rev Res. 2015;30(1):10\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRahman N, Ahmad Y. Quantitative analysis of multi-drug cold preparations. Anal Methods. 2014;6:2999\u0026ndash;3005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEMA. ICH Q2(R2) Guideline: Validation of Analytical Procedures. 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIOSR JAC. Quantification Of (4-Bromophenyl) {Pyridine-2-Yl} Acetonitrile Impurity (4-BPPA) by HPLC in Brompheniramine Maleate. 2017.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eInnovare Acad. Stability Indicating UPLC Method for Simultaneous Estimation of Chlorpheniramine and Dextromethorphan. 2019.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatel H, et al. Review on simultaneous drug estimation. J Chromatogr B. 2022;1198:123193.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh R, Sharma A. Method validation in pharmaceutical QA/QC. Indian J Pharm Sci. 2016;78(3):379\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBagul VA, Ambadekar SR, Tiwari AR. A comprehensive stability indicating method for clonidine hydrochloride impurities profiling and degradation analysis in tablet formulations: green metric study. Discov Chem. 2025;2:160. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s44371-025-00238-4\u003c/span\u003e\u003cspan address=\"10.1007/s44371-025-00238-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDevelopment and Validation of New RP-HPLC Analytical Method for Simultaneous Estimation of Impurities Present in Nortriptyline and Pregabalin Combined Pharmaceutical Dosage Form by Vijay Arjun Bagul, Dr. Sushama Raju Ambadekar and Anand Radheshyam Tiwari Published in Urban India journal (ISSN No: 0970\u0026ndash;9045), Volume 43 Issue (II). December 2023, Pg 188\u0026ndash;197.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTiwari AR, Ambadekar SR, Bagul VA. Sensitive and sustainable stability-indicating RP-HPLC method for simultaneous assay and impurity profiling of amitriptyline HCl: green metric, RGB model and forced degradation study. Discov Chem. 2025;2:157. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s44371-025-00218-8\u003c/span\u003e\u003cspan address=\"10.1007/s44371-025-00218-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eA Comprehensive Stability-Indicating Analytical Method for Escitalopram Impurity analysis by RP-HPLC; Force Degradation Study by Anand Tiwari, Dr. Sushama Ambedkar and Vijay A. Bagul. Published Int J All Res Educ Sci Methods (IJARESM) ISSN: 2455\u0026ndash;6211.12, Issue 2 (February 2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Method development and validation, Related substances, Brompheniramine Maleate, Impurity study, HPLC","lastPublishedDoi":"10.21203/rs.3.rs-7427571/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7427571/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA key aspect of pharmaceutical analysis that guarantees the efficacy, safety, and quality of medication items is impurity profiling. It involves the Identification and quantification of Impurities that may arise during synthesis. In this research a Novel RP-HPLC method was developed and validated including separation of Impurities and quantification of Brompheniramine Maleate. The separation of all three main components and Impurities was achieved using Gradient mode with the use of UV and PDA detector at 260nm wavelength. In port A of mobile phase 100% Potassium di-hydrogen phosphate buffer pH 3.0 was kept and in port B 100% Acetonitrile was kept. Separation was achieved by using Sunniest C18 column having dimensions 150 x 4.6mm, 3\u0026micro;m. 1.0ml/min flow rate was set with column temperature 30\u0026deg;C. Total run time was 40 min showed separation of all three components i.e. Brompheniramine Maleate (BPM), Dextromethorphan HBr (DMP), Phenylephrine HCl (PPN) and BPM\u0026rsquo;s impurities. A linear relationship (r\u0026thinsp;=\u0026thinsp;0.99) revealed for all known analyte with concentration range of LOQ to 150%. Recovery study specifies the accuracy of the method. The repeatability determined that the method is precise enough within acceptance limit. Excellent linearity, accuracy, specificity, precision, robustness, LOD, LOQ and system applicability results are shown by the proposed approach. Moreover, the study on forced degradation showed that the method was stable suggesting.\u003c/p\u003e","manuscriptTitle":"Impurity profiling of multicomponent cough syrup containing Brompheniramine, Dextromethorphan and Phenylephrine by RP-HPLC with PDA detector","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 13:17:00","doi":"10.21203/rs.3.rs-7427571/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-13T11:39:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-25T13:07:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T02:26:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-18T12:01:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215530012564552920581707373565345032288","date":"2025-10-13T17:31:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308650069120312780604459314374750043143","date":"2025-10-10T20:00:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139407929997689917702451351515873083898","date":"2025-10-10T03:23:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258136779669985508703009915142584386456","date":"2025-10-08T22:54:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-08T16:31:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-08T12:07:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-23T04:29:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-23T04:29:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2025-08-21T15:08:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a88b40b1-7692-4358-bf2e-ae0ab6955cca","owner":[],"postedDate":"October 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-24T10:23:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-21 13:17:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7427571","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7427571","identity":"rs-7427571","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}