QBD base stability indicating method development and validation of rocecadotril and its formulation

QBD base stability indicating method development and validation of rocecadotril and its formulation | 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 QBD base stability indicating method development and validation of rocecadotril and its formulation Priya Yannawar, Akshata Patil, Dr. Bhushan Baviskar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8172934/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present study aims to develop and validate a QbD-based stability-indicating RP-HPLC method for the estimation of Racecadotril and its pharmaceutical formulation. Using a systematic QbD approach, critical method parameters such as mobile phase composition and flow rate were optimized through Design of Experiments. Chromatographic separation was achieved on a Waters Reliant C18 column (4.6 × 250 mm, 5 µm) using a mobile phase of phosphate buffer and acetonitrile (45:55 v/v), with a flow rate of 1.5 mL/min and detection at 210 nm. The method demonstrated a retention time of approximately 8.5 minutes for Racecadotril. Stress degradation studies under acidic, basic, oxidative, and thermal conditions confirmed the stability-indicating nature of the method. Validation performed in accordance with ICH guidelines established the method’s specificity, accuracy, precision, sensitivity, and robustness. The developed method is suitable for routine quality control analysis of Racecadotril in both bulk drug and finished dosage forms. Reverse- Phase High Performance Liquid Chromatography Design of Experiments (DoE) Forced degradation study Racecadotril Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 INTRODUCTION Racecadotril, commonly referred to as acetorphan, is an antidiarrheal agent that exerts its effect by peripherally inhibiting the enzyme enkephalinase. As a prodrug, it is rapidly converted into its active metabolite, thiorphan, which specifically binds to the active site of the enzyme. Unlike conventional opioid antidiarrheal drugs that primarily slow down intestinal motility, racecadotril reduces intestinal secretion of water and electrolytes, thereby exhibiting an antisecretory mechanism. 1 – 3 Analytical method development involves selecting a suitable procedure to accurately assess the composition of a pharmaceutical formulation. Among various analytical techniques, HPLC stands out as a robust and reliable method for the qualitative and quantitative evaluation of compounds, particularly when the analyte is soluble in a liquid medium. RP-HPLC utilizes a non-polar stationary phase and a polar or moderately polar mobile phase. 4–5 Forced degradation studies are an integral part of stability testing, involving the deliberate exposure of drug substances and formulations to stress conditions. 6 These tests help identify potential degradation products, determine the molecule’s stability profile, and confirm that the method is stability-indicating.. 7 Analytical QbD applies QbD concepts to analytical method development. A central concept in AQbD is the “Design Space,” which refers to the multidimensional range of input variables that have been demonstrated to produce a product of consistent quality. The design space is established through risk assessment and links CQAs with their associated CPPs, ultimately ensuring method reliability and robustness. MATERIALS AND METHODS Instruments and chemicals: Apparatus and Equipment’s – HPLC System specification: Table 1: Instrument and chemical Materials Racecadotril (API) was obtained from Athena Drug Delivery Solutions Pvt. Ltd., Mumbai. The marketed formulation used was Torsec 30 mg. All chemicals and reagents, including acetonitrile and water, methanol, potassium dihydrogen phosphate, and phosphoric acid, were of AR or HPLC grade and used as received. 8 Method Development: 9-13 Selection of solvent: depend upon solubility of the drug solvent methanol use for preparation of solutions. Selection of chromatographic mode: RP-HPLC method use for the separation of the drug as it gives more resolve peak than other liquid chromatography at specific solvent and pH. RP-HPLC method development and optimization: Standard solution of racecadotril was used for HPLC method development trails and then optimized method used to determine racecadotril. Column selection: A Water reliant C18, 250 mm x 4.6mm, 5 μm. Column is versatile column and gives good separation used for the method development with UV detector. Selection of mobile phase: 14-15 There are various factors to be related with the selection of MP such as best separation, peak index, peak summary, theoretical plates etc. MP gives symmetrical and well separate peak in less time. After trying various MP, Buffer: ACN (45:55) was selected as mobile phase. Selection of wavelength: The more resolving wavelength was found to be 210 nm and selected for determination of racecadotril. Finalised chromatographic conditions: Table 2: Finalised HPLC system specification Optimization of RP-HPLC method with the QbD Paradigm or software aided method optimization 16-20 The RP-HPLC method for Racecadotril analysis was optimized using a Quality by Design (QbD) framework, incorporating software-assisted experimental design. Central Composite Design under (RSM) was employed, requiring a minimal number of experimental runs. A total of nine runs were performed using a 2-factor, 2-level approach suitable for developing quadratic models. The design and analysis were conducted using Design Expert® software. Method validation: When method development and optimization are completed, it is necessary to study method validation. For validation of analytical method, the ICH Guideline has recommended validation characteristic including accuracy, precision, linearity, specificity, robustness was investigated. Accuracy/ recovery: The method's accuracy was evaluated at multiple concentration levels of Racecadotril, including 100% of the target concentration, in the presence of placebo. The recovery percentage was calculated to assess the method's reliability. 80%, 100%, 120% of concentration (0.4 mg/ml) of racecadotril. For 80% and 120% level, six test solution and for 100% three test solution shall be prepared as below with the Standard solution and analysed as per test method. Table 3: multiple concentration levels of Racecadotril Linearity: Linearity was established by analyzing samples at different concentrations across the specified range. The results were mathematically assessed to confirm a direct relationship between concentration and response. Table 4: Linearity for RP-HPLC Method Precision: Six independent test solutions using racecadotril sachet 30 mg, shall be prepared along with standard solution. Specificity: Specificity was determined by injecting blank, standard (400 ppm), and sample solutions into the HPLC system. Chromatograms confirmed no interference from impurities, ensuring the method's ability to selectively measure Racecadotril. Robustness: Robustness was evaluated by intentionally varying key method parameters: Flow rate: ±10% (1.65 mL/min and 1.35 mL/min) Column temperature: ±5°C (25°C and 35°C) Buffer pH: ±0.2 units (pH 2.3 and 2.7) Organic phase composition: ±10% Phosphate buffer (pH 2.5): Acetonitrile 39:61 and 51:49 Forced degradation study 18-22 In order to establish the forced degradation study, the racecadotril granules was subjected in to the various stress conditions as follows: Degradation experiment for granules Crush granules to fine powder: Test preparation for control sample Weigh 20 filled sachets. Empty the granules from all the sachet in a mortar and crush the granules in a mortar to fine powder. Equivalent to 20 mg of racecadotril powder + 30 ml diluent, sonicate for 30 min + 50 ml with diluent + filter through 0.45 m nylon filter. collect the filtrate. Acid Degradation Equivalent to 20 mg of powder of granules + 30 ml diluent, sonicate for 30 min. Add 2ml of 0.1 M HCL, kept at RT for 1 hr. add 2 ml of 0.1 M NaOH + 50 ml diluent. Base Degradation Equivalent to 20 mg of powder of granules + 30 ml diluent, sonicate for 30 min. Add 2ml of 0.1 M NaOH, kept at RT for 1 hr. add 2 ml of 0.1 M HCL + 50 ml diluent. Oxidative Degradation Equivalent to 20 mg of powder of granules + 30 ml diluent, sonicate for 30 min. Add 10ml of 30% Peroxide solution, reflux at 80C for 1 hr + 50 ml diluent. Thermal Degradation Equivalent to 20 mg of exposed powder of granules (60 C for 24 hr + 30 ml diluent, sonicate for 30 min + 50 ml diluent. RESULTS AND DISCUSSION 1. Preliminary Studies Initial evaluations of Racecadotril included organoleptic and solubility assessments. The drug substance was found to be off-white in color and odorless. Solubility analysis demonstrated that Racecadotril is freely soluble in methanol and acetonitrile, but practically insoluble in water. For wavelength selection, a UV scan was conducted using a double-beam UV spectrophotometer within the 200–400 nm range, employing methanol as the blank. The maximum absorbance (λmax) of Racecadotril in methanol was identified at 208 nm. 2. Method Development and Optimization Adjusting the mobile phase to a 60:40 (v/v) ratio eliminated placebo interference, but yielded suboptimal recovery. The optimized mobile phase was found to be a 45:55 (v/v) mixture of buffer (pH 2.5) and acetonitrile, which resulted in excellent recovery and no interference from placebo, blank, or impurities at the retention time of Racecadotril. 3. HPLC Method Optimization Using QbD Approach A QbD approach was employed to enhance method robustness and reproducibility. The Quality Target Product Profile included key chromatographic performance indicators. To identify CQAs, the influence of mobile phase composition and flow rate was studied. These factors were considered critical due to their impact on the resolution and efficiency of the chromatographic method. 4. Experimental Design and Optimization A Central Composite Design based on (RSM), was utilized for method optimization. This design enabled efficient exploration of experimental variables with minimal runs. A 2-factor, 2-level model was selected to evaluate the relationship between the independent variables—flow rate (Factor A) and mobile phase composition (Factor B)—and the dependent responses—retention time and theoretical plates. The Design Expert® software was used to generate and analyze the experimental matrix. Design Space A Central Composite Design (CCD) with a quadratic model was employed using a response surface methodology involving 9 experimental runs. The study focused on optimizing two key parameters mobile phase composition and flow rate against two critical responses: retention time and theoretical plate count. Figure 2 illustrates the contour plot showing the influence of flow rate and mobile phase ratio on the retention time. Figure 3 presents a similar contour plot depicting their effect on the theoretical plates. Figures 4 and 5 display predicted vs. actual plots from multiple linear regression analysis, highlighting the model's accuracy and identifying any outliers or deviations from predicted trends. Fig. 6 & 7 shows the 3D surface plot of desirability for obtaining optimized formulations. The selected batch on which the further validation was performed for the more robust method shown in table no. 6 and figure 8. Shows overlay plot for selected factors. Analysis of marketed formulation The optimized RP-HPLC method for Racecadotril demonstrated a well-resolved peak with a retention time of 8.56 minutes when analyzed from the sachet formulation. The assay of the marketed product Torsec showed a drug content of 99.23%, confirming the method’s suitability for commercial formulations. Method validation: Linearity: The method showed excellent linearity over the concentration range of 25–150 µg/mL. The correlation coefficient (r²) was 0.9999, and the regression equation was y = 296743308.0966x + 1271544.9233, indicating strong linear correlation between concentration and response. Precision: Six independent test solutions, using racecadotril sachet 30 mg, were prepared along with standard solution. Method precision and intermediate precisions were shown in Table 8. The % difference was found to be 0.0 which indicated that the developed method was found to be precise. Accuracy The accuracy was done by recovery study. Sample solutions were prepared of 80%, 100%, and 120% concentration of racecadotril. For 80% and 120% level, six test solution and for 100% three test solution were prepared. The % recovery data obtained by the proposed Recovery studies confirmed the accuracy of the method, with individual recovery values ranging from 97% to 103%, and overall recovery between 98% and 102%, meeting standard validation criteria. Specificity: For the specificity study, solutions of blank, sample and standard solution were used; the standard and sample solutions of 400 ppm were used and each solution were injected into the system and chromatograms were recorded. It was found that the no interferences are observed between blank, standard solution and test solution. Robustness: Robustness of the method was studied by changing flow rate of mobile phase±10% (i.e.1.65 ml/min and 1.35 ml/min), Change in column oven temperature, ±5°C (i.e. 35°C to 25°C), Change in pH of buffer solution in mobile phase±0.2 (pH2.7and pH2.3), Change in composition of organic component of organic component in mobile phase, ±10%. [Phosphate buffer solution pH 2.5: ACN (39:61), Phosphate buffer solution pH 2.5: ACN (51:49)]. Variation in above parameter no significant effect on chromatographic response, indicating that the method was robust. The results are shown in table 10. Forced degradation study The percent degradation of Racecadotril after acidic (0.1M HCl), basic (0.1M NaOH) & oxidative (30% H2O2), stress was found to be 7.3%, 13.4% &5.2%respectively. While the product remain unchanged after thermal stress. The results for forced degradation study are shown in table 11. CONCLUSION The application of the QbD approach in developing a stability-indicating HPLC method significantly enhanced the understanding of critical method parameters, reducing the risk of failure during validation and transfer processes. Utilizing Design Expert® software streamlined the optimization process, making it faster and more efficient compared to conventional manual techniques. The optimized method successfully separated the parent compound from its degradation products, demonstrating its specificity. Overall, the developed method proved to be simple, accurate, sensitive, precise, and robust, with no observed interference. Hence, it is well-suited for routine quality control analysis in the pharmaceutical industry. Declarations Author Contribution Priya and Akshata - wrote the manuscript under the guidance of Dr. bhushan and Dr. bhushan review the all manuscript. all figure are Prepared by Akshata. References Doltade M, Saudagar R. 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Patel YS, Sen AK, Shah B, Seth AK. Development and validation of new analytical method for quantitative estimation of racecadotril as an active pharmaceutical ingredient by RP-HPLC. Pharma Sci Monit. 2012;3(4). Basniwal PK, Srivastava PK, Jain SK, Jain D. RP-LC analysis and hydrolytic degradation profile of racecadotril. Chromatographia. 2008;68(7–8):641. National Center for Biotechnology Information. PubChem Database. Racecadotril; CID=107751. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/racecadotril (accessed August 27, 2019). ChemicalBook. Racecadotril—Chemical Product Info. Available from: https://www.chemicalbook.com/chemicalproductproperty_en_cb3378722.htm Medicine India. Pharmacology of Racecadotril. Available from: https://www.medicineindia.org/pharmacology-for-generic/1475/racecadotril Tables Tables 1 to 11 are available in the Supplementary Files section Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":13489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSturcture of Racecadotril\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/e24f9b455f3b78e5a423ffed.png"},{"id":97341788,"identity":"912a5a69-0070-4bd5-9966-a935ad02999a","added_by":"auto","created_at":"2025-12-03 11:16:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecounter graph for retention time R1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/9d655e050dd8583ebc0420f0.png"},{"id":97370494,"identity":"ddd3d69d-d8d4-4c2f-879a-836db9708bad","added_by":"auto","created_at":"2025-12-03 16:27:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecounter graph for retention time R2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/78b4b80827374eedcaedd03d.png"},{"id":97341794,"identity":"f9c6d892-b046-4836-934e-28322e4e41fc","added_by":"auto","created_at":"2025-12-03 11:16:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epredicted vs actual graph of racecadotril R1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/cff5e5f6aeae0180fdb867ac.png"},{"id":97341795,"identity":"99e6995a-36d0-4d05-89d9-2b7992e314d5","added_by":"auto","created_at":"2025-12-03 11:16:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epredicted vs actual graph of racecadotril R2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/8c88d974199ac9604f22f544.png"},{"id":97341801,"identity":"e9396ef0-ecfc-4324-b427-f3278bd30cd8","added_by":"auto","created_at":"2025-12-03 11:16:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":141123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D surface plot by central composite R1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/248ce8a03ee976d7890149ea.png"},{"id":97370036,"identity":"457859b0-e55e-4b50-90dc-4a2cfcfae653","added_by":"auto","created_at":"2025-12-03 16:26:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":155093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D surface plot by central composite R2\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/111547abcaeaa4207623be41.png"},{"id":97370430,"identity":"ce7dfbd2-9b6a-4eb9-b143-d788879b0ba6","added_by":"auto","created_at":"2025-12-03 16:27:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":25839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverlay plot of the factors\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/6dd206f0049573ebe672fde0.png"},{"id":97370731,"identity":"83c8f837-48a3-45ec-8f06-aa69395cd911","added_by":"auto","created_at":"2025-12-03 16:27:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":61005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStandard chromatogram for racecadotril\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/5be806c584c2fe251a222e8f.png"},{"id":97371012,"identity":"3c179716-9820-4f99-87aa-ade463acc64b","added_by":"auto","created_at":"2025-12-03 16:28:15","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":105562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStandard Calibration curve for racecadotril\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/76124769fcfbc32d4909b2ae.png"},{"id":98626773,"identity":"d6b7d178-fd3e-492c-87bb-2d094ae5cc43","added_by":"auto","created_at":"2025-12-19 17:09:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1689401,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/128e312b-19e1-4d78-a2a6-6f80ce5b967f.pdf"},{"id":97341787,"identity":"04888951-15a6-455e-98d4-82cc6a8d2284","added_by":"auto","created_at":"2025-12-03 11:16:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36342,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8172934/v1/b822e123a0ae7aea7dd543af.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eQBD base stability indicating method development and validation of rocecadotril and its formulation\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRacecadotril, commonly referred to as acetorphan, is an antidiarrheal agent that exerts its effect by peripherally inhibiting the enzyme enkephalinase. As a prodrug, it is rapidly converted into its active metabolite, thiorphan, which specifically binds to the active site of the enzyme. Unlike conventional opioid antidiarrheal drugs that primarily slow down intestinal motility, racecadotril reduces intestinal secretion of water and electrolytes, thereby exhibiting an antisecretory mechanism.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalytical method development involves selecting a suitable procedure to accurately assess the composition of a pharmaceutical formulation. Among various analytical techniques, HPLC stands out as a robust and reliable method for the qualitative and quantitative evaluation of compounds, particularly when the analyte is soluble in a liquid medium. RP-HPLC utilizes a non-polar stationary phase and a polar or moderately polar mobile phase. \u003csup\u003e4\u0026ndash;5\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eForced degradation studies are an integral part of stability testing, involving the deliberate exposure of drug substances and formulations to stress conditions.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e These tests help identify potential degradation products, determine the molecule\u0026rsquo;s stability profile, and confirm that the method is stability-indicating..\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAnalytical QbD applies QbD concepts to analytical method development. A central concept in AQbD is the \u0026ldquo;Design Space,\u0026rdquo; which refers to the multidimensional range of input variables that have been demonstrated to produce a product of consistent quality. The design space is established through risk assessment and links CQAs with their associated CPPs, ultimately ensuring method reliability and robustness.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eInstruments and\u0026nbsp;chemicals:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApparatus and Equipment\u0026rsquo;s \u0026ndash; HPLC System specification:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Instrument and chemical\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRacecadotril (API) was obtained from Athena Drug Delivery Solutions Pvt. Ltd., Mumbai. The marketed formulation used was \u003cem\u003eTorsec\u003c/em\u003e 30 mg. All chemicals and reagents, including acetonitrile and water, methanol, potassium dihydrogen phosphate, and phosphoric acid, were of AR or HPLC grade and used as received.\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod Development:\u003csup\u003e9-13\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of solvent:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003edepend upon solubility of the drug solvent methanol use for preparation of solutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of chromatographic mode:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRP-HPLC method use for the separation of the drug as it gives more resolve peak than other liquid chromatography at specific solvent and pH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRP-HPLC method development and optimization:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStandard solution of racecadotril was used for HPLC method development trails and then optimized method used to determine racecadotril.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColumn selection:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Water reliant C18, 250 mm x 4.6mm, 5 \u0026mu;m. Column is versatile column and gives good separation used for the method development with UV detector.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of mobile phase:\u003csup\u003e14-15\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are various factors to be related with the selection of MP such as best separation, peak index, peak summary, theoretical plates etc. MP gives symmetrical and well separate peak in less time. After trying various MP, Buffer: ACN (45:55) was selected as mobile phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of wavelength:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe more resolving wavelength was found to be 210 nm and selected for determination of racecadotril.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinalised chromatographic conditions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Table 2: Finalised HPLC system specification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of RP-HPLC method with the QbD Paradigm or software aided method optimization\u003csup\u003e16-20\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RP-HPLC method for Racecadotril analysis was optimized using a Quality by Design (QbD) framework, incorporating software-assisted experimental design. Central Composite Design under (RSM) was employed, requiring a minimal number of experimental runs. A total of nine runs were performed using a 2-factor, 2-level approach suitable for developing quadratic models. The design and analysis were conducted using Design Expert\u0026reg; software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod validation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen method development and optimization are completed, it is necessary to study method validation. For validation of analytical method, the ICH Guideline has recommended validation characteristic including accuracy, precision, linearity, specificity, robustness was investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy/\u0026nbsp;recovery:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method\u0026apos;s accuracy was evaluated at multiple concentration levels of Racecadotril, including 100% of the target concentration, in the presence of placebo. The recovery percentage was calculated to assess the method\u0026apos;s reliability. 80%, 100%, 120% of concentration (0.4 mg/ml) of racecadotril. For 80% and 120% level, six test solution and for 100% three test solution shall be prepared as below with the Standard solution and analysed as per test method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emultiple concentration levels of Racecadotril\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLinearity was established by analyzing samples at different concentrations across the specified range. The results were mathematically assessed to confirm a direct relationship between concentration and response.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Linearity for RP-HPLC Method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix independent test solutions using racecadotril sachet 30 mg, shall be prepared along with standard solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecificity was determined by injecting blank, standard (400 ppm), and sample solutions into the HPLC system. Chromatograms confirmed no interference from impurities, ensuring the method\u0026apos;s ability to selectively measure Racecadotril.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRobustness was evaluated by intentionally varying key method parameters:\u003c/p\u003e\n\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003eFlow rate: \u0026plusmn;10% (1.65 mL/min and 1.35 mL/min)\u003c/li\u003e\n \u003cli\u003eColumn temperature: \u0026plusmn;5\u0026deg;C (25\u0026deg;C and 35\u0026deg;C)\u003c/li\u003e\n \u003cli\u003eBuffer pH: \u0026plusmn;0.2 units (pH 2.3 and 2.7)\u003c/li\u003e\n \u003cli\u003eOrganic phase composition: \u0026plusmn;10%\u003c/li\u003e\n \u003cli\u003ePhosphate buffer (pH 2.5): Acetonitrile 39:61 and 51:49\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eForced degradation study\u003csup\u003e18-22\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to establish the forced degradation study, the racecadotril granules was subjected in to the various stress conditions as follows:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDegradation experiment for granules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrush granules to fine powder: Test preparation for control sample\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeigh 20 filled sachets. Empty the granules from all the sachet in a mortar and crush the granules in a mortar to fine powder. Equivalent to 20 mg of racecadotril powder + 30 ml diluent, sonicate for 30 min + 50 ml with diluent + filter through 0.45 m nylon filter. collect the filtrate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcid Degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEquivalent to 20 mg of powder of granules + 30 ml diluent, sonicate for 30 min. Add 2ml of 0.1 M HCL, kept at RT for 1 hr. add 2 ml of 0.1 M NaOH + 50 ml diluent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBase Degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEquivalent to 20 mg of powder of granules + 30 ml diluent, sonicate for 30 min. Add 2ml of 0.1 M NaOH, kept at RT for 1 hr. add 2 ml of 0.1 M HCL + 50 ml diluent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative Degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEquivalent to 20 mg of powder of granules + 30 ml diluent, sonicate for 30 min. Add 10ml of 30% Peroxide solution, reflux at 80C for 1 hr + 50 ml diluent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThermal Degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEquivalent to 20 mg of exposed powder of granules (60 C for 24 hr + 30 ml diluent, sonicate for 30 min + 50 ml diluent.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003e1. Preliminary Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitial evaluations of Racecadotril included organoleptic and solubility assessments. The drug substance was found to be off-white in color and odorless. Solubility analysis demonstrated that Racecadotril is freely soluble in methanol and acetonitrile, but practically insoluble in water. For wavelength selection, a UV scan was conducted using a double-beam UV spectrophotometer within the 200\u0026ndash;400 nm range, employing methanol as the blank. The maximum absorbance (\u0026lambda;max) of Racecadotril in methanol was identified at 208 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Method Development and Optimization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdjusting the mobile phase to a 60:40 (v/v) ratio eliminated placebo interference, but yielded suboptimal recovery. The optimized mobile phase was found to be a 45:55 (v/v) mixture of buffer (pH 2.5) and acetonitrile, which resulted in excellent recovery and no interference from placebo, blank, or impurities at the retention time of Racecadotril.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. HPLC Method Optimization Using QbD Approach\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA QbD approach was employed to enhance method robustness and reproducibility. The Quality Target Product Profile included key chromatographic performance indicators. To identify CQAs, the influence of mobile phase composition and flow rate was studied. These factors were considered critical due to their impact on the resolution and efficiency of the chromatographic method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Experimental Design and Optimization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Central Composite Design based on (RSM), was utilized for method optimization. This design enabled efficient exploration of experimental variables with minimal runs. A 2-factor, 2-level model was selected to evaluate the relationship between the independent variables\u0026mdash;flow rate (Factor A) and mobile phase composition (Factor B)\u0026mdash;and the dependent responses\u0026mdash;retention time and theoretical plates. The Design Expert\u0026reg; software was used to generate and analyze the experimental matrix.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesign Space\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Central Composite Design (CCD) with a quadratic model was employed using a response surface methodology involving 9 experimental runs. The study focused on optimizing two key parameters mobile phase composition and flow rate against two critical responses: retention time and theoretical plate count. Figure 2 illustrates the contour plot showing the influence of flow rate and mobile phase ratio on the retention time. Figure 3 presents a similar contour plot depicting their effect on the theoretical plates. Figures 4 and 5 display predicted vs. actual plots from multiple linear regression analysis, highlighting the model\u0026apos;s accuracy and identifying any outliers or deviations from predicted trends. Fig. 6 \u0026amp; 7 shows the 3D surface plot of desirability for obtaining optimized formulations.\u003c/p\u003e\n\u003cp\u003eThe selected batch on which the further validation was performed for the more robust method shown in table no. 6 and figure 8. Shows overlay plot for selected factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of marketed formulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimized RP-HPLC method for Racecadotril demonstrated a well-resolved peak with a retention time of 8.56 minutes when analyzed from the sachet formulation. The assay of the marketed product \u003cem\u003eTorsec\u003c/em\u003e showed a drug content of 99.23%, confirming the method\u0026rsquo;s suitability for commercial formulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod validation:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method showed excellent linearity over the concentration range of 25\u0026ndash;150 \u0026micro;g/mL. The correlation coefficient (r\u0026sup2;) was 0.9999, and the regression equation was y = 296743308.0966x + 1271544.9233, indicating strong linear correlation between concentration and response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix independent test solutions, using racecadotril sachet 30 mg, were prepared along with standard solution. Method precision and intermediate precisions were shown in Table 8. The % difference was found to be 0.0 which indicated that the developed method was found to be precise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accuracy was done by recovery study. Sample solutions were prepared of 80%, 100%, and 120% concentration of racecadotril. For 80% and 120% level, six test solution and for 100% three test solution were prepared. The % recovery data obtained by the proposed Recovery studies confirmed the accuracy of the method, with individual recovery values ranging from 97% to 103%, and overall recovery between 98% and 102%, meeting standard validation criteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the specificity study, solutions of blank, sample and standard solution were used; the standard and sample solutions of 400 ppm were used and each solution were injected into the system and chromatograms were recorded. It was found that the no interferences are observed between blank, standard solution and test solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRobustness of the method was studied by changing flow rate of mobile phase\u0026plusmn;10% (i.e.1.65 ml/min and 1.35 ml/min), Change in column oven temperature, \u0026plusmn;5\u0026deg;C (i.e. 35\u0026deg;C to 25\u0026deg;C), Change in pH of buffer solution in mobile phase\u0026plusmn;0.2 (pH2.7and pH2.3), Change in composition of organic component of organic component in mobile phase, \u0026plusmn;10%. [Phosphate buffer solution pH 2.5: ACN (39:61), Phosphate buffer solution pH 2.5: ACN (51:49)]. Variation in above parameter no significant effect on chromatographic response, indicating that the method was robust. The results are shown in table 10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eForced degradation study\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percent degradation of Racecadotril after acidic (0.1M HCl), basic (0.1M NaOH) \u0026amp; oxidative (30% H2O2), stress was found to be 7.3%, 13.4% \u0026amp;5.2%respectively. While the product remain unchanged after thermal stress. The results for forced degradation study are shown in table 11.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe application of the QbD approach in developing a stability-indicating HPLC method significantly enhanced the understanding of critical method parameters, reducing the risk of failure during validation and transfer processes. Utilizing Design Expert\u0026reg; software streamlined the optimization process, making it faster and more efficient compared to conventional manual techniques. The optimized method successfully separated the parent compound from its degradation products, demonstrating its specificity. Overall, the developed method proved to be simple, accurate, sensitive, precise, and robust, with no observed interference. Hence, it is well-suited for routine quality control analysis in the pharmaceutical industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePriya and Akshata - wrote the manuscript under the guidance of Dr. bhushan and Dr. bhushan review the all manuscript. all figure are Prepared by Akshata.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDoltade M, Saudagar R. The analytical method development and validation: a review. J Drug Deliv Ther. 2019;9(3):563\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003eVidushi Y, Meenakshi B. A review on HPLC method development and validation. Res J Life Sci Bioinform Pharm Chem Sci. 2017;2(6):178.\u003c/li\u003e\n\u003cli\u003eChauhan A, Mittu B, Chauhan P. Analytical method development and validation: a concise review. J Anal Bioanal Tech. 2015;6(1):1.\u003c/li\u003e\n\u003cli\u003eChaudhary A, Choudhary AN, Dutta KK. QbD approach for development of stability indicating RP-HPLC method and its validation: a review. Int J Pharm Sci Rev Res. 2018; Available from: [Exact journal name or link needed].\u003c/li\u003e\n\u003cli\u003eSharma S, Goyal S, Chauhan K. A review on analytical method development and validation. Int J Appl Pharm. 2018;10(6):8\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eVenkataraman S, Manasa M. Forced degradation studies: regulatory guidance, characterization of drugs and their degradation products \u0026ndash; a review. Drug Invent Today. 2018;10(2):137\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eHossain M, Bhadra S, Kumar U, Rouf A. The ICH guidance in practice: stress degradation studies on aceclofenac and development of a validated stability-indicating RP-HPLC assay in tablet dosage form. Der Pharm Chem. 2013;5(4):131\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003ePatil AS, Pethe AM. Quality by design (QbD): a new concept for development of quality pharmaceuticals. Int J Pharm Qual Assur. 2013;4(2):13\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eNadpara NP, Thumar RV, Kalola VN, Patel PB. Quality by design (QbD): a complete review. Int J Pharm Sci Rev Res. 2012;17(2):20\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003ePramod K, Tahir MA, Charoo NA, Ansari SH, Ali J. Pharmaceutical product development: a quality by design approach. Int J Pharm Investig. 2016;6(3):129\u0026ndash;38. https://doi.org/10.4103/2230-973x.187350\u003c/li\u003e\n\u003cli\u003eSangshetti JN, Deshpande M, Zaheer Z, Shinde DB, Arote R. Quality by design approach: regulatory need. Arab J Chem. 2017;10(Suppl):S3412\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eGandhi A, Roy C. Quality by design (QbD) in pharmaceutical industry: tools, perspectives and challenges. PharmaTutor. 2016;4(11):12\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eMogal V, Dusane J, Borase P, Thakare P, Kshirsagar S. A review on quality by design. Pharm Biol Eval. 2016;3:313\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eSankar MS, Arulantony S. New spectrophotometric method for the determination of racecadotril in pharmaceutical dosage forms. Int J Drug Res Technol. 2017;3(3):6.\u003c/li\u003e\n\u003cli\u003eAnton SA, Madhusudhana RI, Manavalan R, Varaprasad K. Development and validation of a rapid RP-HPLC method for the determination of racecadotril in formulation. Int J ChemTech Res. 2009;1(4):1090\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003ePrabu SL, Singh T, Joseph A, Dinesh Kumar C, Shirwaikar A. Determination of racecadotril by HPLC in capsules. Indian J Pharm Sci. 2007;69(6):819.\u003c/li\u003e\n\u003cli\u003eAnnapurna MM, Narendra A, Sahu A. Development and validation of a stability-indicating RP-HPLC method for analysis of racecadotril in pharmaceutical dosage forms. Chem Sci Trans. 2014;3(2):518\u0026ndash;29.\u003c/li\u003e\n\u003cli\u003ePrabu SL, Sivagurunathan N, Dinesh Kumar C, Vasantharaju SG, Meenashi Vanathi B. Stability indicating HPLC method for determination of racecadotril in solid dosage form. J Pharm Res. 2009;8(1):39\u0026ndash;41.\u003c/li\u003e\n\u003cli\u003ePatel YS, Sen AK, Shah B, Seth AK. Development and validation of new analytical method for quantitative estimation of racecadotril as an active pharmaceutical ingredient by RP-HPLC. Pharma Sci Monit. 2012;3(4).\u003c/li\u003e\n\u003cli\u003eBasniwal PK, Srivastava PK, Jain SK, Jain D. RP-LC analysis and hydrolytic degradation profile of racecadotril. Chromatographia. 2008;68(7\u0026ndash;8):641.\u003c/li\u003e\n\u003cli\u003eNational Center for Biotechnology Information. PubChem Database. Racecadotril; CID=107751. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/racecadotril (accessed August 27, 2019).\u003c/li\u003e\n\u003cli\u003eChemicalBook. Racecadotril\u0026mdash;Chemical Product Info. Available from: https://www.chemicalbook.com/chemicalproductproperty_en_cb3378722.htm \u003c/li\u003e\n\u003cli\u003eMedicine India. Pharmacology of Racecadotril. Available from: https://www.medicineindia.org/pharmacology-for-generic/1475/racecadotril\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 11 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Reverse- Phase High Performance Liquid Chromatography, Design of Experiments (DoE), Forced degradation study, Racecadotril","lastPublishedDoi":"10.21203/rs.3.rs-8172934/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8172934/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study aims to develop and validate a QbD-based stability-indicating RP-HPLC method for the estimation of Racecadotril and its pharmaceutical formulation. Using a systematic QbD approach, critical method parameters such as mobile phase composition and flow rate were optimized through Design of Experiments. Chromatographic separation was achieved on a Waters Reliant C18 column (4.6 \u0026times; 250 mm, 5 \u0026micro;m) using a mobile phase of phosphate buffer and acetonitrile (45:55 v/v), with a flow rate of 1.5 mL/min and detection at 210 nm. The method demonstrated a retention time of approximately 8.5 minutes for Racecadotril. Stress degradation studies under acidic, basic, oxidative, and thermal conditions confirmed the stability-indicating nature of the method. Validation performed in accordance with ICH guidelines established the method\u0026rsquo;s specificity, accuracy, precision, sensitivity, and robustness. The developed method is suitable for routine quality control analysis of Racecadotril in both bulk drug and finished dosage forms.\u003c/p\u003e","manuscriptTitle":"QBD base stability indicating method development and validation of rocecadotril and its formulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 11:16:29","doi":"10.21203/rs.3.rs-8172934/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f46769ae-a103-4c34-ad00-0020119020db","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-19T03:23:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 11:16:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8172934","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8172934","identity":"rs-8172934","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}