RP-HPLC Photodiode Array Based Method for Simultaneous Estimation of Aspirin and Pantoprazole Sodium

RP-HPLC Photodiode Array Based Method for Simultaneous Estimation of Aspirin and Pantoprazole Sodium | 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 RP-HPLC Photodiode Array Based Method for Simultaneous Estimation of Aspirin and Pantoprazole Sodium Amitkumar J. Vyas, Bharat S. Pithiya, Sinchan M. Patel, Ashvin V. Dudhrejiya, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5879425/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 A specific, precise, accurate, robust, cost-effective and reproducible diode array detector based RP-HPLC method was developed and validated for quantitative analysis of Pantoprazole sodium and Aspirin in synthetic mixture. In RP-HPLC method drugs were separated by Inertsil C18 ODS (250 × 4.7mm, 5µm), utilizing a mobile phase buffer(0.05 m potassium dihydrogen phosphate pH 3 Adjusted by using 10% Orthophosphoric acid) : Acetonitrile (60 : 40%) at temperature(TEMP) 30º C, flow rate of 1 mL/min, injection volume 10 µL with UV detection at 230 nm. Aspirin and pantoprazole sodium were eluted with the retention time of 5.330 and 7.201 min respectively. The calibration plots were established over the concentration range of 4.05–121.5 µg/ml and 1–30 µg/ml for Aspirin and Pantoprazole Sodium with correlation coefficient 0.9997 and 0.9995 respectively. The method was found to comply all the validation parameter as per the ICH guideline, such as accuracy, precision, and linearity, Robustness, specificity. This method can be effectively employed for the day to day analysis of both drug. Aspirin Pantoprazole Sodium RP-HPLC Photodiode Array Method validation ICH guideline (Q2R2) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Aspirin (ASP) is used as an Anticoagulant, antipyretic, analgesic and non-steroidal anti-inflammatory agent. It acts as non selective cyclo-oxygenase Inhibitors. It is officials in Indian pharmacopoeia, US pharmacopoeia, British pharmacopoeia and European pharmacopoeia. The chemical structure of the ASP is shown in Fig. 1 [ 1 ]. Pantoprazole Sodium (PPZ) is used as an Anti-ulcerative agent. It falls under the category of Proton pump inhibitors. Its chemical structure is shown in Fig. 2 [ 2 ]. When combined with PPZ, ASP works in concert to lower total and LDL cholesterol levels. Additionally, lower the ASP dose needed. PPZ has modest HMG-CoA reductase activity and may potentially reduce the likelihood of ASP side effects such myopathy [ 3 ]. UV spectrophotometry, HPLC, ICP‒MS and LC‒MS/MS are essential for quality control in pharmaceuticals, ensuring substance stability and integrity for safety and effectiveness[ 4 – 6 ]. Impurities significantly impact the Pharmacological activity and stability of pharmaceutical APIs and pharmaceuticals [ 7 – 9 ]. For the development of RP-HPLC techniques, stress testing, and stability-indicating techniques, AQbD and CCD support regulatory compliance[ 10 – 13 ]. A literature survey discloses that there is single UV method is available for the simultaneous estimation of ASP and PPZ the ratio of (1:1) [ 14 ]. Many more analytical methods are published for the estimation for single drug and in combine with other drugs. It includes HPTLC [ 15 ], Stability-indicating HPLC [ 16 – 19 ], UV [ 20 – 24 ], RP-HPLC [ 25 – 30 ], UPLC [ 31 ], LC-MS/MS [ 32 ]. The method is developed in a ratio of (4.05:1) ASP and PPZ. This study aims to developed and validate a RP-HPLC method which can be utilize for simultaneous estimation of ASP and PPZ by industry and analytical laboratory. The devised approach may be effectively used to quality control decisions and was verified in accordance with ICH guidelines. 2. MATERIAL AND METHOD Chemicals and Reagents : ASP and PPZ standards were kindly provided by Sidmak laboratories India Pvt. Ltd.– Valsad and Umedica Pvt. Ltd – Vapi respectively. HPLC grade Methanol, Acetonitrile(ACN), and Milli Q water, ACN, Potassium dihydrogen phosphate and Orthophosphoric acid were used. Instrumentation : Chromatographic analysis was carried out using a Shimadzu LC-10 HPLC, PDA detector, and Lab Solution software for data acquisition. RP-HPLC method Table 1 Chromatographic Condition: Sr. No. Chromatographic condition Optimized condition 1 Column Inertsil C18 ODS (250 × 4.7mm, 5µm) 2 Flow rate (FR) 1.0 ml/min 3 Run time 10 min 4 Column Temp. 30 ºC 5 Sample cooler Temp. 4 ºC 6 Detection 230 nm 7 Injection volume 10 µL 8 Diluent (ACN 9 Mobile phase(MP) 0.05M KH 2 PO 4 Bufferr (pH 3):ACN (60:40%) Preparation of Standard stock solution (SSS) : Weigh accurately about 4 mg of PPZ and 16.2 mg of ASP into 100 ml volumetric flask. Dilute up to the mark with diluents and mix well. The solution formed will have concentration of 40 µg/ml of PPZ and 162 µg/ml of ASP. Synthetic mixture preparation Synthetic mixture was prepared by mixing ASP (81.0 mg) and PPZ (20.0 mg) with sucrose (30.mg), starch (80.0 mg) polyethylene glycol (27.0 mg), micro-crystalline cellulose (50.0 mg), magnesium stearate (7.0 mg) and talc (5.0 mg). 3. METHOD VALIDATION Analytical validation parameters for the analysis of the proposed method were detected as stated in ICH(Q2R1) guideline [ 21 ]. Linearity The SSS was diluted appropriately to obtain a concentration of 4.05–121.5 µg/ml of ASP and 1–30 µg/ml of PPZ with diluent (ACN). Specificity Specificity was performed by injecting diluent, placebo and sample solution to check the interference of excipients. LOD and LOQ The limit of detection (LOD) and limit of quantification (LOQ) were calculated by formula as given in ICH (Q2R1) [ 21 ]. LOD = 3.3 x Standard deviation/ Slope LOQ = 10 x Standard deviation/ Slope Accuracy The accuracy of the method was performed by placebo spiking in triplicate at three different concentration levels of 80%, 100% and 120% of the targeted concentration of drugs. Precision Repeatability was performed at the assay concentration with 6 replicates of ASP and PPZ with concentration of 81 µg/mL and 20 µg/mL. Intra-day and Inter-day variations of ASP and PPZ were evaluated in triplicate at three contrasting concentration levels 80%, 100% and 180% at 64.8, 81 and 97.2 µg/mL and 16, 20 and 24 µg/mL for ASP and PPZ respectively for three different days and 3 different timings within a day. Robustness The robustness of the proposed method was determined by analysis of the sample, during which Change in wavelength (230 nm ± 1), Temperature (30 ̊C ± 1 ̊C), MP composition (± 1% for Buffer : ACN) and FR (1 ml/min ± 0.1). Assay of Synthetic mixture : For determination of the concentration of ASP and PPZ in a synthetic mixture. Weight Equivalent to 8.1 mg ASP and 2.0 mg PPZ of synthetic mixture (30 mg) and transferred into a 100 ml volumetric flask and add excipients. Add 70% of ACN and sonicate. Dilute up to the mark with the same and mix it well. Take out 2ml of filtrate solution to 10ml volumetric flask make up to the mark. The final concentration is 16.2 µg/mL of ASP and 4.0 µg/mL of PPZ is assessed for assay. 4. RESULTS AND DISCUSSIONS 1. System suitability test: Drug concentration prepared for system suitability test were ASP (81 μg/mL) and PPZ (20 μg/mL). The system suitability parameters for ASP and PPZ were acceptable shown in Table 2. Table : 2 Results of System Suitability Test Parameters Observation Specification ASP PPZ Repeatability 0.23 0.18 RSD ≤ 1% Retention time 5.33 7.021 - Resolution (RS) - 6.83 RS > 2 Tailing Factor (T) 1.02 1.01 T ≤ 2 Theoretical Plates (N) 8116 7201 ≥2000 Area 2007476 416550 - The repeatability of ASP and PPZ was found to be less than 1% RSD, indicating acceptable precision. The resolution between the two peaks was satisfactory, and both tailing factor and theoretical plate count were within the acceptable range. 2. Linearity: Linearity was checked at five concentration level. The calibration curve obtained for ASP and PPZ in the range of 4.05-121.5 µg/ml and 1-30 µg/ml and the correlation coefficient was found to be 0.9997 and 0.9995 respectively. Linearity graph is revealed in Figure 3 and 4. 3. Specificity: The % interference was found to be ASP (0.13 %) and PPZ (0.01 %), which is less than 0.5 %, thus making specificity to be acceptable. It was found by comparing chromatograms of blank, placebo, sample solution and standard solution allow that there was no interference of excipients. Chromatograph showed with ASP and PPZ peak in Fig.6. 4. Accuracy: The percentage recovery for ASP was found to be within 98.34 %-99.76 % and that for PPZ was found to be in range of 98.23 %-99.60 %. Thus, the % recover of both drug is acceptable, as shown in Table 3. 5. Precision: Precision found to be less than 2 % RSD, Thus acceptable. Data of RSD result are shown in table 3. Table 3: Precision and Accuracy data for ASP and PPZ Intraday precision Interday precision Mean % Recovery (n = 3) Drug % Level (n=3) RSD RSD ASP 80 1.58 0.24 99.76 100 0.38 0.39 99.57 120 1.26 1.34 98.37 PPZ 80 0.55 0.20 99.60 100 0.27 0.22 99.77 120 0.57 0.99 98.23 6. Robustness: Relative standard deviation of peak area were found to be < 2, indicating that the proposed method is robust. Results remained unaffected by small changes of these parameters. 7. LOD and LOQ: LOD and LOQ were found to be 0.091 μg/ml & 0.273 μg/ml for ASP and 0.012 μg/ml and 0.038 μg/ml for PPZ respectively, thus acceptable. 8. Assay: The assay of synthetic mixture results falls within the official limits of both drug which is 99.5 -101 % for ASP and 99-101 % for PPZ. The data is given in Table 4. Table 4 : Assay of synthetic mixture Drugs Label claim (mg) Conc. ( μg/mL) % Assay PPZ 20 20 100.42 ASP 81 81 100.59 Validation Parameter summary is tabulated in table 5. Table 5 : Regression analysis data and summary of validation parameter for the current method RP-HPLC Photo-diode array method Parameters ASP PPZ Wavelength (nm) 230 Retention time (min) 5.330 7.201 Beer's law limit (μg/ml) 4.05-121.5 µg/ml 1-30 µg/ml Regresssion equation y = 25364x + 2008.6 y = 21034x + 32.699 Correlation coefficient(r2) 0.9999 0.9999 % recovery 98.34-99.76 98.23-99.60 Intraday (n=3)(%RSD) Interday (n=3)(%RSD) LOD (μg/ml) 0.091 0.012 LOQ (μg/ml) 0.273 0.038 5. CONCLUSION Based on the research mentioned above, it can be said that the suggested approach is straightforward, sensitive, repeatable, robust, specific, and has excellent accuracy and precision. The major advantage of this technique is that it is developed for combination in actual dose ratio (4.05:1). So that method can be implemented for quantification by the pharmaceutical industry and analytical laboratories. Declarations Consent to Participate declaration: Not applicable Ethics statement: Not applicable Competing interests The authors declared that they have no competing interests Clinical trial number Not applicable Consent to Publish All the authors agree to publish Data Availability Not applicable Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Acknowledgment The authors are thankful to Sidmak laboratory (India) PVT. LTD. Valsad for providing necessary facilities to carry out the research work and provide ASP drug and Umedica PVT. LTD. Vapi for providing the PPZ drug. Author Contribution Amitkumar J. Vyas guided the research. Bharat S. Pithiya and Sinchan M. Patel Performed entire research. Ajay I. Patel and Nilesh K. assisted with HPLC. Ashvin V. Dudhrejiya helped procure API. Sunny R. Shah and Hetal B. gavit advised on the manuscript. References US Pharmacopoeia. (2016). NF 41, The United State Pharmacopoeials Convention Inc, Rockville, pp 391–395. Indian Pharmacopoeia. (2018). Government of India, Ministry of Health & Family Welfare; 8th Edn; Indian Pharmacopoeia Commission, Ghaziabad, pp 1604–1606. CDSCO – clinical trial phaseIII approval, october, 2020, https://cdsco.gov.in/opencms/opencms/system/modules/CDSCO.WEB/elements/download_file_division.jsp?num_id=NTQyNw Vyas AJ, Jha SA, Patel AB, Patel AI, Shah SR, Sheth DB. Review on simultaneous equation method (Vierodt’s method). 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Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC–MS/MS: Application to bioequivalence study. 2010; 878: 1499–1505. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5879425","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":422305569,"identity":"d8d29679-e5e4-4e60-a6ef-372d6c4bf892","order_by":0,"name":"Amitkumar J. Vyas","email":"","orcid":"","institution":"B. K. Mody Government Pharmacy College","correspondingAuthor":false,"prefix":"","firstName":"Amitkumar","middleName":"J.","lastName":"Vyas","suffix":""},{"id":422305570,"identity":"b24f9aa2-fc19-402a-abbc-e7ac0df84fa4","order_by":1,"name":"Bharat S. 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Mody Government Pharmacy College","correspondingAuthor":false,"prefix":"","firstName":"Sinchan","middleName":"M.","lastName":"Patel","suffix":""},{"id":422305572,"identity":"920c6408-18f0-4565-b81d-a1d0e1fad589","order_by":3,"name":"Ashvin V. Dudhrejiya","email":"","orcid":"","institution":"B. K. Mody Government Pharmacy College","correspondingAuthor":false,"prefix":"","firstName":"Ashvin","middleName":"V.","lastName":"Dudhrejiya","suffix":""},{"id":422305574,"identity":"35f9bdc0-5dc1-408b-9b31-e7e408e21407","order_by":4,"name":"Nilesh K. Patel","email":"","orcid":"","institution":"B. K. Mody Government Pharmacy College","correspondingAuthor":false,"prefix":"","firstName":"Nilesh","middleName":"K.","lastName":"Patel","suffix":""},{"id":422305577,"identity":"ff8a5d42-c7fc-4e5e-bd0b-685a20ce52f6","order_by":5,"name":"Ajay I. Patel","email":"","orcid":"","institution":"B. K. Mody Government Pharmacy College","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"I.","lastName":"Patel","suffix":""},{"id":422305578,"identity":"2bad5753-270b-40c6-aaff-f6d15e1efab4","order_by":6,"name":"Sunny R. Shah","email":"","orcid":"","institution":"Government pharmacy college","correspondingAuthor":false,"prefix":"","firstName":"Sunny","middleName":"R.","lastName":"Shah","suffix":""},{"id":422305579,"identity":"dd9ab53f-c3fc-4141-9120-9c60174e0bcc","order_by":7,"name":"Hetal B. Gavit","email":"","orcid":"","institution":"Government pharmacy college","correspondingAuthor":false,"prefix":"","firstName":"Hetal","middleName":"B.","lastName":"Gavit","suffix":""}],"badges":[],"createdAt":"2025-01-22 09:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5879425/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5879425/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77623550,"identity":"48400b89-ec7a-4d50-b70e-9f6cd12f359f","added_by":"auto","created_at":"2025-03-03 16:11:49","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAspirin\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5879425/v1/793ff8e18b8bef72e4d306df.jpeg"},{"id":77623485,"identity":"01465596-9e7e-48ce-a740-7fbaa5c51aef","added_by":"auto","created_at":"2025-03-03 16:11:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePantoprazole Sodium\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5879425/v1/aa58ceb830de598ebdd1454d.jpeg"},{"id":77623561,"identity":"eb88ccc4-7d17-4527-8539-be16b1bc6158","added_by":"auto","created_at":"2025-03-03 16:11:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLinearity of PPZ\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5879425/v1/d0a9a23256895acd8eca97b8.png"},{"id":77623503,"identity":"95d879e8-bd08-4c39-98a4-77cd9ee6a6e3","added_by":"auto","created_at":"2025-03-03 16:11:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLinearity of PPZ\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5879425/v1/2595452f5771c0940aefdac9.png"},{"id":77623546,"identity":"556358a4-8389-4a9c-8252-135f2f496917","added_by":"auto","created_at":"2025-03-03 16:11:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOverlay chromatogram of ASP and PPZ\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5879425/v1/bc7d735043a89b7ac583c790.png"},{"id":77623602,"identity":"91d791db-f8b7-40cf-8de3-70898b824f23","added_by":"auto","created_at":"2025-03-03 16:11:51","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":814295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChromatograms of (a) placebo, (b) diluent, (c) PPZ (d) APZ\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5879425/v1/8a27fcc2d89ea5047fde6ce2.jpeg"},{"id":82184188,"identity":"307d0387-a82a-49e0-85c5-d4ab5dc666df","added_by":"auto","created_at":"2025-05-07 12:38:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1980137,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5879425/v1/1a5b1e7f-2d02-411f-88d2-3afc5760e229.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"RP-HPLC Photodiode Array Based Method for Simultaneous Estimation of Aspirin and Pantoprazole Sodium","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eAspirin (ASP) is used as an Anticoagulant, antipyretic, analgesic and non-steroidal anti-inflammatory agent. It acts as non selective cyclo-oxygenase Inhibitors. It is officials in Indian pharmacopoeia, US pharmacopoeia, British pharmacopoeia and European pharmacopoeia. The chemical structure of the ASP is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePantoprazole Sodium (PPZ) is used as an Anti-ulcerative agent. It falls under the category of Proton pump inhibitors. Its chemical structure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen combined with PPZ, ASP works in concert to lower total and LDL cholesterol levels. Additionally, lower the ASP dose needed. PPZ has modest HMG-CoA reductase activity and may potentially reduce the likelihood of ASP side effects such myopathy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUV spectrophotometry, HPLC, ICP‒MS and LC‒MS/MS are essential for quality control in pharmaceuticals, ensuring substance stability and integrity for safety and effectiveness[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Impurities significantly impact the Pharmacological activity and stability of pharmaceutical APIs and pharmaceuticals [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. For the development of RP-HPLC techniques, stress testing, and stability-indicating techniques, AQbD and CCD support regulatory compliance[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A literature survey discloses that there is single UV method is available for the simultaneous estimation of ASP and PPZ the ratio of (1:1) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Many more analytical methods are published for the estimation for single drug and in combine with other drugs. It includes HPTLC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], Stability-indicating HPLC [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], UV [\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], RP-HPLC [\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], UPLC [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], LC-MS/MS [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe method is developed in a ratio of (4.05:1) ASP and PPZ. This study aims to developed and validate a RP-HPLC method which can be utilize for simultaneous estimation of ASP and PPZ by industry and analytical laboratory. The devised approach may be effectively used to quality control decisions and was verified in accordance with ICH guidelines.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHOD","content":"\u003cp\u003e \u003cb\u003eChemicals and Reagents\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eASP and PPZ standards were kindly provided by Sidmak laboratories India Pvt. Ltd.\u0026ndash; Valsad and Umedica Pvt. Ltd \u0026ndash; Vapi respectively. HPLC grade Methanol, Acetonitrile(ACN), and Milli Q water, ACN, Potassium dihydrogen phosphate and Orthophosphoric acid were used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInstrumentation\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eChromatographic analysis was carried out using a Shimadzu LC-10 HPLC, PDA detector, and Lab Solution software for data acquisition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRP-HPLC method\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChromatographic Condition:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChromatographic condition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOptimized condition\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColumn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInertsil C18 ODS (250 \u0026times; 4.7mm, 5\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlow rate (FR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0 ml/min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRun time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColumn Temp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 \u0026ordm;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample cooler Temp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 \u0026ordm;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e230 nm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInjection volume\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 \u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiluent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(ACN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMobile phase(MP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003eBufferr (pH 3):ACN (60:40%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of Standard stock solution (SSS)\u003c/b\u003e:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWeigh accurately about 4 mg of PPZ and 16.2 mg of ASP into 100 ml volumetric flask. Dilute up to the mark with diluents and mix well. The solution formed will have concentration of 40 \u0026micro;g/ml of PPZ and 162 \u0026micro;g/ml of ASP.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthetic mixture preparation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSynthetic mixture was prepared by mixing ASP (81.0 mg) and PPZ (20.0 mg) with sucrose (30.mg), starch (80.0 mg) polyethylene glycol (27.0 mg), micro-crystalline cellulose (50.0 mg), magnesium stearate (7.0 mg) and talc (5.0 mg).\u003c/p\u003e"},{"header":"3. METHOD VALIDATION","content":"\u003cp\u003eAnalytical validation parameters for the analysis of the proposed method were detected as stated in ICH(Q2R1) guideline [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eLinearity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe SSS was diluted appropriately to obtain a concentration of 4.05\u0026ndash;121.5 \u0026micro;g/ml of ASP and 1\u0026ndash;30 \u0026micro;g/ml of PPZ with diluent (ACN).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpecificity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSpecificity was performed by injecting diluent, placebo and sample solution to check the interference of excipients.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLOD and LOQ\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe limit of detection (LOD) and limit of quantification (LOQ) were calculated by formula as given in ICH (Q2R1) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLOD\u0026thinsp;=\u0026thinsp;3.3 x Standard deviation/ Slope\u003c/p\u003e \u003cp\u003eLOQ\u0026thinsp;=\u0026thinsp;10 x Standard deviation/ Slope\u003c/p\u003e \u003cp\u003e \u003cb\u003eAccuracy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe accuracy of the method was performed by placebo spiking in triplicate at three different concentration levels of 80%, 100% and 120% of the targeted concentration of drugs.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrecision\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRepeatability was performed at the assay concentration with 6 replicates of ASP and PPZ with concentration of 81 \u0026micro;g/mL and 20 \u0026micro;g/mL. Intra-day and Inter-day variations of ASP and PPZ were evaluated in triplicate at three contrasting concentration levels 80%, 100% and 180% at 64.8, 81 and 97.2 \u0026micro;g/mL and 16, 20 and 24 \u0026micro;g/mL for ASP and PPZ respectively for three different days and 3 different timings within a day.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRobustness\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe robustness of the proposed method was determined by analysis of the sample, during which Change in wavelength (230 nm\u0026thinsp;\u0026plusmn;\u0026thinsp;1), Temperature (30 ̊C\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ̊C), MP composition (\u0026plusmn;\u0026thinsp;1% for Buffer : ACN) and FR (1 ml/min\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssay of Synthetic mixture\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eFor determination of the concentration of ASP and PPZ in a synthetic mixture. Weight Equivalent to 8.1 mg ASP and 2.0 mg PPZ of synthetic mixture (30 mg) and transferred into a 100 ml volumetric flask and add excipients. Add 70% of ACN and sonicate. Dilute up to the mark with the same and mix it well. Take out 2ml of filtrate solution to 10ml volumetric flask make up to the mark. The final concentration is 16.2 \u0026micro;g/mL of ASP and 4.0 \u0026micro;g/mL of PPZ is assessed for assay.\u003c/p\u003e"},{"header":"4. RESULTS AND DISCUSSIONS","content":"\u003cp\u003e\u003cstrong\u003e1. \u0026nbsp; System suitability test:\u0026nbsp;\u003c/strong\u003eDrug concentration prepared for system suitability test were ASP (81 \u0026mu;g/mL) and PPZ (20 \u0026mu;g/mL). The system suitability parameters for ASP and PPZ were acceptable shown in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable : 2 Results of System Suitability Test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 251px;\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 170px;\"\u003e\n \u003cp\u003eObservation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 183px;\"\u003e\n \u003cp\u003eSpecification\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eASP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ePPZ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 251px;\"\u003e\n \u003cp\u003eRepeatability\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003eRSD \u0026le; 1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 251px;\"\u003e\n \u003cp\u003eRetention time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e5.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e7.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 251px;\"\u003e\n \u003cp\u003eResolution (RS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e6.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003eRS \u0026nbsp;\u0026gt; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 251px;\"\u003e\n \u003cp\u003eTailing Factor (T)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003eT \u0026le; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 251px;\"\u003e\n \u003cp\u003eTheoretical Plates (N)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e8116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e7201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e\u0026ge;2000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 251px;\"\u003e\n \u003cp\u003eArea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2007476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e416550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 183px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cbr\u003e\n\u003cp\u003eThe repeatability of ASP and PPZ was found to be less than 1% RSD, indicating acceptable precision. The resolution between the two peaks was satisfactory, and both tailing factor and theoretical plate count were within the acceptable range.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. \u0026nbsp; Linearity:\u0026nbsp;\u003c/strong\u003eLinearity was checked at five concentration level.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe calibration curve obtained for ASP and PPZ\u003csub\u003e\u0026nbsp;\u003c/sub\u003ein the range of 4.05-121.5 \u0026micro;g/ml and 1-30 \u0026micro;g/ml and the correlation coefficient was found to be 0.9997 and 0.9995 respectively. Linearity graph is revealed in Figure 3 and 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. \u0026nbsp; Specificity:\u0026nbsp;\u003c/strong\u003eThe % interference was found to be ASP (0.13 %) and PPZ (0.01 %), which is less than 0.5 %, thus making specificity to be acceptable. It was found by comparing chromatograms of blank, placebo, sample solution and standard solution allow that there was no interference of excipients. Chromatograph showed with ASP and PPZ peak in Fig.6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Accuracy:\u003c/strong\u003e The percentage recovery for ASP was found to be within 98.34 %-99.76 % and that for PPZ was found to be in range of 98.23 %-99.60 %. Thus, the % recover of both drug is acceptable, as shown in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. \u0026nbsp; Precision:\u0026nbsp;\u003c/strong\u003ePrecision found to be less than 2 % RSD, Thus acceptable. Data of RSD result are shown in table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eTable 3: Precision and Accuracy data for ASP and PPZ\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"606\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntraday precision\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterday precision\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean % Recovery\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrug\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Level\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eASP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e1.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e99.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e99.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e98.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePPZ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e99.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e99.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003e98.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cbr\u003e\n\u003cp\u003e\u003cstrong\u003e6. \u0026nbsp; Robustness:\u0026nbsp;\u003c/strong\u003eRelative standard deviation of peak area were found to be \u0026lt; 2, indicating that the \u0026nbsp;proposed method is robust. Results remained unaffected by small changes of these parameters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. \u0026nbsp; LOD and LOQ:\u003c/strong\u003e LOD and LOQ were found to be 0.091 \u0026mu;g/ml \u0026amp; 0.273 \u0026mu;g/ml for ASP and 0.012 \u0026mu;g/ml and 0.038 \u0026mu;g/ml for PPZ\u003csub\u003e\u0026nbsp;\u003c/sub\u003erespectively, thus acceptable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. \u0026nbsp; Assay:\u003c/strong\u003e The assay of synthetic mixture results falls within the official limits of both drug which is 99.5 -101 % for ASP and 99-101 % for PPZ. The data is given in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 4 : Assay\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eof synthetic mixture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrugs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLabel claim (mg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003e\u0026mu;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Assay\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePPZ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e100.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eASP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e100.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cbr\u003e\n\u003cp\u003eValidation Parameter summary is tabulated in table 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 5 : Regression analysis data and summary of validation parameter for the current method\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"558\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 351px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRP-HPLC Photo-diode array method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eASP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePPZ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eWavelength (nm)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 351px;\"\u003e\n \u003cp\u003e230\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eRetention time (min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e5.330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e7.201\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eBeer\u0026apos;s law limit (\u0026mu;g/ml)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e4.05-121.5 \u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e1-30 \u0026micro;g/ml\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eRegresssion equation\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003ey = 25364x + 2008.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003ey = 21034x + 32.699\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eCorrelation coefficient(r2)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e% recovery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e98.34-99.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e98.23-99.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eIntraday (n=3)(%RSD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eInterday (n=3)(%RSD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eLOD (\u0026mu;g/ml)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e0.091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003eLOQ (\u0026mu;g/ml)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 153px;\"\u003e\n \u003cp\u003e0.273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eBased on the research mentioned above, it can be said that the suggested approach is straightforward, sensitive, repeatable, robust, specific, and has excellent accuracy and precision. The major advantage of this technique is that it is developed for combination in actual dose ratio (4.05:1). So that method can be implemented for quantification by the pharmaceutical industry and analytical laboratories. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent to Participate declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors agree to publish\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Sidmak laboratory (India) PVT. LTD. Valsad for providing necessary facilities to carry out the research work and provide ASP drug and Umedica PVT. LTD. Vapi for providing the PPZ drug.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAmitkumar J. Vyas guided the research. Bharat S. Pithiya and Sinchan M. Patel Performed entire research. Ajay I. Patel and Nilesh K. assisted with HPLC. Ashvin V. Dudhrejiya helped procure API. Sunny R. Shah and Hetal B. gavit advised on the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eUS Pharmacopoeia. (2016). NF 41, The United State Pharmacopoeials Convention Inc, Rockville, pp 391\u0026ndash;395.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIndian Pharmacopoeia. (2018). Government of India, Ministry of Health \u0026amp; Family Welfare; 8th Edn; Indian Pharmacopoeia Commission, Ghaziabad, pp 1604\u0026ndash;1606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCDSCO \u0026ndash; clinical trial phaseIII approval, october, 2020, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cdsco.gov.in/opencms/opencms/system/modules/CDSCO.WEB/elements/download_file_division.jsp?num_id=NTQyNw\u003c/span\u003e\u003cspan address=\"https://cdsco.gov.in/opencms/opencms/system/modules/CDSCO.WEB/elements/download_file_division.jsp?num_id=NTQyNw\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVyas AJ, Jha SA, Patel AB, Patel AI, Shah SR, Sheth DB. Review on simultaneous equation method (Vierodt\u0026rsquo;s method). 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Development and validation of HPTLC method for simultaneous estimation of Aspirin and Pantoprazole Sodium in Pharmaceutical dosage form. World J. Pharm. Sci. 2014; 2(11): 1511\u0026ndash;1519.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlessy, M. Patel, R.D. Prajapati, P.N. and Agrawal, Y.K. Development of forced degradation and stability indicating studies of drugs \u0026ndash; A review. J. Phar. Anal. 2013; 4: 159\u0026ndash;165.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaheer, Z. Farooqui, M.N. Mangle, A.A. and Nikalje, A.G. Stability indicating high performance liquid chromatographic determination of Aspirin in pharmaceutical dosage form. Afr. J. Pharm. Pharmacol. 2008; 2(10): 204\u0026ndash;210.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTemova, Z. and Roskar, R. Stability-indicating HPLC-UV method for Pantoprazole Sodium determination in Solutions, Nutritional Supplements and Pharmaceuticals. J. Chromatogr. Sci. 2016; 54(7): 1180\u0026ndash;1186.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRudwan, A.H. Hussein, A.B. and Saheed, E.M. Development and validation of stability indicating high perfermance liquid chromatography method for determination of Aspirin in the presence of its degradation products. Der. Pharm. Sinica.2015; 6(11): 19\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrajapati, K.P. and Bhanderi, A. Spectroscopic method for estimation of Aspirin in Tablet dosage form. Indo glob. J. Pharm. Sci. 2011; 1(4): 294\u0026ndash;299.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInda, S.S. Sharma, S. Das, V. Kumar, B. and Bhandari, A. First derivative spectrophotometric method for the estimation of Aspirin as bulk and in Pharmaceutical dosage form. Int. J. Pharm. Sci. 2013; 5(3): 530\u0026ndash;533.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShinde V, Sarode V, Kshirsgar S, Jadhav A. Development and validation of uv spectrophotometric method for estimation of pantoprazole sodium in bulk and tablet dosage form. J. CIBTech journal of pharmaceutical science. 2016; 5(4): 22\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar R, Singh H, Singh P. Development of UV Spectrophotometric method for estimation of Pantoprazole in pharmaceutical dosage forms. J. Journal of Chemical and Pharmaceutical Research. 2011; 3(2): 113\u0026ndash;117.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatil S, Dhable P, Kuchekar B, Spectrophotometric estimation of pantoprazole in tablet dosage form\u0026rdquo; J. International Journal Chemstry Science. 2008; 6(4): 1984\u0026ndash;1990.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlam, S. Saleem, S. Naveed, S. Dilshad, H. Qamar, F. Alam, T. Sadia, H. Khan, S. Karim, M. and Khan, A. HPLC method development and validation of Aspirin in bulk and tablet dosage form. Pharm. Sci. 2018; 6(1): 83\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed, A.W. Mohammed, S.E. Boshra, H.S. Abdelmula, M.H. Mohammed, M.O. Mohammed, A. and Hammad, A. Development and validation method for the determination of Aspirin tablet drugs by using High Performance Liquid Chromatography (HPLC) in Pharmaceutical formulation. Inter. J. of Inno. Sci. Res.Tech. 2019; 4(4): 1009\u0026ndash;1015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohan, T. Prabhakata, S. Anbazhagan, K. Krishnan, P. Bhandare, B. and Sahjanand, H. Development and validation of new RP-HPLC method for quantitative estimation of Vitamin-PPZ in bulk drug and Pharmaceutical Dosage Form. Ind. Drugs 2016; 53(6): 40\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQadi, A. Battah, A. and Hadidi, H. Development of High Performance liquid chromatographic method for Pantoprazole Sodium analysis in pharmaceutical preparation. Jordan J. Pharm. Sci. 2010; 3(2): 78\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNassar M, Attia AM, Mohamed AA, Shahin M. HPLC Method for the Simultaneous Estimation of Aspirin and Omeprazole in their new combination. J. Analytical Chemistry Letters 2017; 7(3): 438\u0026ndash;444.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimionato, L.D. Ferello, L. Stamer, S. Repetto, M.F. Zubata, P.D. and Segal, A.L. A validated reversed phase HPLC method for the determination of Aspirin in Tablets. Aus. Chrom. 2014; 1(1): 1\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, K.K. Rao, C.K. Maddala, V.L. and Mukkanti, K. A validated stability indicating RP-UPLC method for Aspirin. Am. J. Analyt. Chem. 2012; 3: 392\u0026ndash;399.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChallaa B, Boddub S, Awenc Z. Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC\u0026ndash;MS/MS: Application to bioequivalence study. 2010; 878: 1499\u0026ndash;1505.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Aspirin, Pantoprazole Sodium, RP-HPLC, Photodiode Array, Method validation, ICH guideline (Q2R2)","lastPublishedDoi":"10.21203/rs.3.rs-5879425/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5879425/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA specific, precise, accurate, robust, cost-effective and reproducible diode array detector based RP-HPLC method was developed and validated for quantitative analysis of Pantoprazole sodium and Aspirin in synthetic mixture. In RP-HPLC method drugs were separated by Inertsil C18 ODS (250 \u0026times; 4.7mm, 5\u0026micro;m), utilizing a mobile phase buffer(0.05 m potassium dihydrogen phosphate pH 3 Adjusted by using 10% Orthophosphoric acid) : Acetonitrile (60 : 40%) at temperature(TEMP) 30\u0026ordm; C, flow rate of 1 mL/min, injection volume 10 \u0026micro;L with UV detection at 230 nm. Aspirin and pantoprazole sodium were eluted with the retention time of 5.330 and 7.201 min respectively. The calibration plots were established over the concentration range of 4.05\u0026ndash;121.5 \u0026micro;g/ml and 1\u0026ndash;30 \u0026micro;g/ml for Aspirin and Pantoprazole Sodium with correlation coefficient 0.9997 and 0.9995 respectively. The method was found to comply all the validation parameter as per the ICH guideline, such as accuracy, precision, and linearity, Robustness, specificity. This method can be effectively employed for the day to day analysis of both drug.\u003c/p\u003e","manuscriptTitle":"RP-HPLC Photodiode Array Based Method for Simultaneous Estimation of Aspirin and Pantoprazole Sodium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 16:08:10","doi":"10.21203/rs.3.rs-5879425/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":"a6370574-b4f0-4c14-a373-54916786848a","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-07T12:38:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 16:08:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5879425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5879425","identity":"rs-5879425","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}