Regulatory Based Method equivalency study to Estimate the Terbutaline sulfate API by RP-HPLC method

Regulatory Based Method equivalency study to Estimate the Terbutaline sulfate API by RP-HPLC method | 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 Regulatory Based Method equivalency study to Estimate the Terbutaline sulfate API by RP-HPLC method Alexandar Sekar, Ravisankar M, Sangeetha Sridharan, Pasupathi Chinnappan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6145442/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 novel, regulatory based HPLC method has been developed and validated for the estimation of Terbutaline sulphate API. Column is X- bridge shield RP-18 150×4.6 mm, 3.5 µm, wave length 223 nm, injection volume 15 µL, column temperature is 30°C,sampler temperature 5°C and flow rate 0.8 mL/min. Retention time of terbutaline sulfate is about 7.226 minutes and Runtime 10 minutes. The developed methods have been validated as per ICH guidelines. The method exhibited satisfactory with precision, specificity, linearity, robustness and ruggedness. The proposed method was found to be equivalent to USP method. HPLC Method validation Method Equivalence ICH Q2R1 USP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION HPLC is a sophisticated separation technique used to Isolation, Elucidation and Quantification of the components. The aim of present study is to analyze the Terbutaline sulfate API. Terbutaline sulfate known as 1,3-Benzenediol, 5-[2-[(1,1-dimethylethyl)amino]− 1-hydroxyethyl]-, sulfate (2:1) (salt). (±)-α-[(tert-Butylamino) methyl]-3,5-dihydroxybenzyl alcohol sulfate (2:1) (salt) by chemically. The classification of drug is short acting beta2–adrenergic agonist. Terbutaline sulfate is prescribed to treat bronchospasm in bronchial asthma, chronic bronchitis, emphysema, chronic obstructive pulmonary disease and prophylaxis of exercise–induced asthma and. Terbutaline sufate is acting on the smooth muscles from the trachea to the end of the bronchioles, thus making breathing easier. Physical appearance is a white or almost white crystalline powder, freely soluble in water, Slightly soluble in ethanol practically insoluble in heptane [ 1 – 4 ]. A Brief literature survey discovered few RP-HPLC methods for routine analysis, related substances and impurity determinations in individual and multi component dosage forms containing ambroxol HCl, guaifenesin and terbutaline sulphate [ 5 – 7 ]. Degradation study of Terbutaline sulfate have been performed [ 8 ].Numerous UV, HPLC, GC and HPTLC based methods have been reported for estimation of these drugs alone as well as in combination with other drugs in pharmaceutical dosage forms [ 9 – 17 ]. Literatures survey revealed there is no method equivalency study related to HPLC method the for Assay of Terbutaline sulfate API. Hence A novel and Regulatory based RP-HPLC In house method has been developed to show the equivalent to USP method for analyse of Terbutaline sulphate API. MATERIALS AND METHODS Drugs and Chemicals Terbutaline sulphate RS, procured from sigma Aldrich. Terbutaline sulphate API obtained from FDC Ltd, India. Impurities standards (Impurity A − 3,5-dihydroxybenzoic acid (α-resorcylic acid), ), (Impurity-B (4RS)-2-tert-butyl-1,2,3,4-tetrahydroisoquinoline-4,6,8- triol,), (Impurity-C- 2-(tert-butylamino)-1-(3,5-dihydroxyphenyl)ethan-1-one), Impurity-D − 2-[benzyl(tert-butyl)amino]-1-(3,5-dihydroxyphenyl) ethan-1-one. purchased from USP and TLC pharmaceutical standards. HPLC grade heptane sulfonic acid, potassium dihydrogen phosphate, acetonitrile, phosphoric acid and Milli-Q water obtained from Merck. Instrumentation A waters HPLC (E-2695 – PDA 2996) instrument controlled with Empower − 3 software. Column utilized X Bridge Shield RP-18 (150x4.6mm, 3.5µ) .Thermo fisher sonicator used to dissolve reference standard and API. A Mettler Toledo electronic balance was used for weighing process. All pH adjustments were made using a Met Rohm pH meter. Mobile Phase Accurately weighed and transferred about 2.87g of heptane sulfonic acid and 2.5 g of potassium dihydrogen phosphate in a suitable container. Added about 1000mL of water into same container. Mixed well and adjust the pH to 3.60 ± 0.05 with dilute phosphoric acid. Filtered through a 0.45µm membrane filter. The mobile phase composed of pH 3.60 Buffer: Acetonitrile (80:20), at flow rate of 0.8 ml/min with run time of 10 min. Detection wavelength at 276 nm. Retention time of Terbutaline sulfate was found to be 7.266 minutes. Injection volume 10 µl, Column oven thermostatically controlled with 30°C, Sampler temperature maintained at 5°C. Diluent Preparation Transferred 900mL of water and 100mL of acetonitrile in a suitable container and mix well. Resolution Solution Preparation Weighed accurately and transferred about 14 mg of Terbutaline sulfate RS and 3 mg of Terbutaline sulfate Related Compound A RS into a 100 mL volumetric flask, added 10 mL of diluent and sonicated to dissolve. Diluted to volume with diluent and mixed well. (Concentration of about 140 µg/mL of Terbutaline sulfate and 30 µg/mL of Terbutaline sulfate Related Compound A) Standard Preparation Standard stock Preparation: Weighed accurately and transferred about 25 mg of Terbutaline sulfate RS into a 25- mL volumetric flask, added 10mL of diluent and sonicated to dissolve. Dilute to volume with diluent. Mixed well. (Concentration of about 1000 µg/mL of Terbutaline Sulfate). Working Standard Preparation: Pipetted out 4.0 mL of stock standard solution into a 50 mL volumetric flask. Diluted to volume with diluent, Mixed well. (Concentration of about 80 µg/mL of Terbutaline Sulfate). Sample Preparation Sample stock Preparation: Weighed accurately and transferred about 50 mg of Terbutaline sulfate sample into a 50mL volumetric flask, added 30mL of diluent and sonicated to dissolve. Diluted to volume with diluent, Mixed well. (Concentration of about 1000 µg/mL of Terbutaline Sulfate). Sample Preparation: Pipetted out 4.0 mL of stock sample into a 50- mL volumetric flask. Diluted to volume with diluent, Mix well. (Concentration of about 80 µg/mL of Terbutaline Sulfate). Chromatographic conditions X Bridge Shield RP-18 (150 x 4.6mm, 3.5µ) was used for chromatographic separation. The mobile phase composed of pH 3.60 Buffer: Acetonitrile (80:20), at flow rate of 0.8 ml/min with run time of 10 min. Detection wavelength at 276 nm. Retention time of Terbutaline sulfate was found to be 7.266 minutes. Injection volume 10 µl, Column oven thermostatically controlled with 30°C, Sampler temperature maintained at 5°C. METHOD VALIDATION The developed method was validated in terms of linearity, specificity, precision, accuracy, limit of detection, limit of quantitation, robustness and system suitability testing as per the ICH guidelines and Validation of compendial procedures USP 40 NF 35 Analytical procedures [ 18 – 19 ] System Precision System precision established by injecting standard solutions and resolution solution. Relative standard deviation of peak area responses for five replicate injections of the standard solution and resolution of impurity mixture solution reported. Method Precision Precision of the assay method was determined by injecting, six individual sample preparations of Terbutaline Sulfate. The samples were prepared as per the method. Reported with Relative standard deviation for assay. Linearity and range Linearity performed by Solutions of Terbutaline sulfate varying concentrations ranging from 40–150% of the standard concentration of (80 µg/mL) were injected into HPLC system. The linearity graph was plotted from 40–150%. Reported with correlation coefficient. Ruggedness Ruggedness of the method performed by analyst-2 on different day from method precision. Standard and sample solutions prepared and injected. Reported with Relative standard deviation for assay and system suitability parameters were calculated. Solution stability Solution stability study was performed at different days against the freshly prepared standard, and sample solution and the results were represented .The standard and sample solutions were prepared and injected. Replicate injections of the standard and sample solution were made at the following time intervals, Initial, 33-hrs, 50-hrs for standard and Initial, 27-hrs, 60-hrsfor sample stored at 5°C cooled temperature. The %difference in % assay from initial and time point for both standard and sample solution were generated. Specificity Specificity of the method can be studied in the presence of excipients, degradation products and impurities. Blank, standard and stressed sample solutions were prepared and injected into the chromatographic system for identification and impurity interference with the Terbutaline sulfate peak. Specificity by Forced Degradation: A forced degradation study is performed in order to prove that the method is stability indicating.The Terbutaline Sulfatedrug substance was stressed under the following conditions. Control Sample Preparation Weighed and transferred about 25mg of Terbutaline sulfatedrug substance in to a 25mL volumetric flask, added 10mL of diluent then sonicated to dissolve and diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well. Acid Degradation Weighed and transferred about 25mg of Terbutaline sulfatedrug substance in to a 25 mL volumetric flask, added 10 mL of diluent and sonicated to dissolve. Added 2.5 mL of 5N HCl and kept in a water bath maintained at a temperature of 80°C for 1 hour. After 1 hour, the sample was allowed to cool to room temperature and then neutralized with 2.5 mL of 5N NaOH. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well. Base Degradation Weighed and transferred about 25mg of Terbutaline sulfate drug substance in to a 25mL volumetric flask,, added 10 mL of diluent and sonicated to dissolve. Added 2.5 mL of 2N NaOH and kept in a water bath maintained at a temperature of 80°C for 1 hour. After 1 hour, the sample was allowed to cool to room temperature and then neutralized with 2.5 mL of 2N HCl. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well. Peroxide Degradation Weighed and transferred about 25mg of Terbutaline sulfate drug substance in to a 25mL volumetric flask, added 10 mL of diluent and sonicated to dissolve. Added 2.5 mL of 3%H2O2and kept in a water bath maintained at a temperature of 80°C for 1 hour. After 1 hour, the sample was allowed to cool to room temperature and then diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well. Heat Degradation Required amount of drug substance transferred in to a Petri dish, heated in an oven for 24 hours at 80°C, then sample taken out and cooled to room temperature. Weighed and transferred about 25mg of this heat exposed sample into a 25mL volumetric flask, 10mL of diluent added, sonicated to dissolve. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well. UV-Light Degradation Required amount of drug substance transferred in to a Petri dish, exposed to 254nm UV light at room temperature for 24 hours. After 24 hours of exposure time, about 25mg of this UV light exposed sample weighed and transferred into a 25mL volumetric flask, 10mL of diluent added, sonicated to dissolve. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well. Preparation of Individual Impurity Stock Solutions Accurately Weighed and transferred each about 0.5 mg of Impurity-A, Impurity-B, Impurity C and Impurity-D into individual 10mL volumetric flasks. Added diluent about 3/4th volume of the flask and sonicated to dissolve. Made up to volume with diluent and mixed well. Impurity Mixture Solution Preparation Pipetted about 2.5mL of Impurity-A, Impurity-B, Impurity-C and Impurity-D stock solutions into 25mL volumetric flask. Diluted to volume with diluent and mixed well. Preparation of Spiked Sample Solution Weighed and transferred about 25 mg of Terbutaline sulfate drug substance in to a 25 mL volumetric flask. Transferred 2.5mL of Impurity mixture solution preparation into the same volumetric flask and sonicated to dissolve. Diluted to volume with diluent and mixed well. All the control, stress samples and spiked samples were injected for determination of % assay and peak purity. Robustness Robustness of the method evaluated by varying chromatographic method parameters such as column temperature ± 5°C and Mobile phase pH ± 0.2 from the optimized conditions. Injected Standard solution into the chromatographic system. System suitability results were reported. METHOD EQUIVALENCY Method equivalency study was determined by injecting, Standard, Resolution solution and six individual sample solutions of Terbutaline Sulfate. The samples were prepared and analyzed as per both USP method and In-house method and results were compared. RESULTS AND DISCUSSION Table.1 Parameter Terbutaline sulfate System precision % RSD = 0.1 Resolution = 24.6 Method precision % RSD = 0.3 Method precision assay (Average of six preparations) 101.2% Linearity and Range (Correlation co-efficient) 0.9999 Ruggedness (System precision) % RSD = 0.2 Resolution = 37.2 Ruggedness (Average of six preparations) 98.5% Solution stability Standard stability − 50 hours at 5°C Sample stability – 60 hours at 5°C Method precision assay (Average of six preparations) USP Method 99.5% System precision parameters such % RSD and Resolution between Main compound and impurity’s found within limit .Blank, standard and System suitability chromatograms shown from Fig. 1 to Fig. 3 . Method precision result shows repeatability of the method. Linear over the concentration range of 40–150% of assay concentration. Ruggedness study exhibited reproducibility of the developed method. Solution Stability data demonstrated the sample and standard solutions stable up to 60 h and 50 h respectively at 5°C.Method precision study performed by USP method revealed that the developed inhouse method equivalence to USP method. Above all the results reported in Table 1. Table.2 S.no Sample name % Assay Purity angle Purity Threshold 01 Control API 105.0 0.255 0.461 02 Acid 5N HCl 180°C 1Hour 100.7 0.261 0.467 03 Base 2N NaOH 80° 1 Hour 97.3 0.257 0.460 04 3% H 2 O 2 80°C 1Hour 90.2 0.282 0.500 05 UV 24 hours 103.7 0.219 0.433 06 Heat 24 hours 105.1 0.243 0.443 07 Spiked Sample 0.160 0.294 In forced degradation study Terbutaline sulfate treated with different stress conditions such as Acid stress, base stress, peroxide stress, UV and Heat. Purity plots presented from Fig. 4 to Fig. 8 .In all conditions Purity threshold found more than the purity angle. It’s highy showing the specificity of the method mentioned in Table 2 . Table.3 Method Parameters Retention Time (1st injection) %RSD USP Resolution Normal Condition Column Temp: 30°C Mobile phase pH: 3.60 7.301 0.1 30.7 Column Temp. Minus 25°C 7.610 0.1 30.3 Column Temp. Plus 35°C 6.984 0.1 30.4 Mobile phase pH Minus 3.40 7.213 0.2 32.1 Mobile phase pH Plus 3.80 7.245 0.2 32.1 Robustness study results showed the developed method not affected by the small changes. Method had undergone with Column compartment temperature change and mobile phase pH changes. Results reported in Table 3. CONCLUSION In the present study, the new In-house HPLC analytical method was developed for the estimation of Terbutaline sulfate API .Based on the results of the above studies, it is concluded that the method for Assay of Terbutaline sulfate API is specific, precise, rugged, robust and linear over the concentration range. Standard and sample solutions are stable up to 50 hours and 60 hours respectively when stored at a temperature of 5°C (Refrigerator Condition). Results of In-house method comparable with USP method with respect to validation parameters, method equivalency study protocols and regulatory requirements. Declarations CONFLICT OF INTEREST None Author Contribution Ravisankar.M - Writing – original draftS.Alexandar -Project administration S.Sangeetha - VisualizationC.Pasupathi - Resource ContributionM.Kumar - Formal analysis ACKNOWLEDGEMENT The authors are thankful to Srinivasan college of pharmaceutical sciences, Trichy and P&A pharma and neutraceuticals testing lab,Chennai providing necessary facilities to carry out the research work. References https://www.rxlist.com/terbutaline-sulfate-drug.htm https:// pubchem.ncbi.nlm.nih.gov/compound/Terbutaline-sulphate European Pharmacopoeia Page No:3979 USP 40 NF 35–Terbutaline sulfate N Itagimatha, D H Manjunatha. RP-HPLC-UV method development and validation for simultaneous determination of terbutaline sulphate, ambroxol HCl and guaifenesin in pure and dosage forms. Annales Pharmaceutiques Françaises.2019:7(4):295–301. K.Lakshmi Narasimha Rao,Ch.Krishnaiah,K.Sudheer Babu,K.Padmaja Reddy. Development and validation of a stability-indicating LC method for simultaneous determination of related compounds of guaifenesin, terbutaline sulfate and ambroxol HCl in cough syrup formulation.Journal of Saudi Chemical Society.2014:18(5):593–600 Ghosh A, Mandal S. K.,Ghosh S.and Deb S. Development And Validation of RP-HPLC Method For Simultaneous Determination of Terbutaline Sulphate, Guaiphenesin And Ambroxol Hydrochloride From An Oral Liquid.India drugs.2016:53(5):53–56. N Daraghmeh,M.MAl-Omari,ZSara, A.ABadwan,A.M.YJaber. Determination of terbutaline sulfate and its degradation products in pharmaceutical formulations using LC. Journal of Pharmaceutical and Biomedical Analysis.2002:29(5):927–937. Maulik Oza1, Jagdish Kakadiya1, Chirag Oza. Development and Validation of Solvent Extraction Spectrophotometric Method for Simultaneous Estimation of Doxofylline and Terbutaline sulphate In their Combined Dosage Form.American journal of pharm tech research.2012:2(4):593–604 B. 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Thirupathi Dongala,Lakshmi Narasimha Rao Katakam,Ashok Kumar Palakurthi,Naresh Kumar Katari.RP-HPLC Stability Indicating Method Development and Validation of Pseudoephedrine Sulfate and Related Organic Impurities in Tablet Dosage Forms, Robustness by QbD Approach.2019:697–710. A.Susmitha,B.Satya Narayana, E.Masthani, S.Bindu Bhargavi,Dr.C.Gopinath. Development And Validation of UV Spectrophotometric Method For The Estimation Of Terbutaline Sulphate In Its Syrup Dosage form. International Journal of Advances in Pharmaceutical Research.2015:6(8):221–227. Gananadhamu Samanthula,Krishnaveni Yadiki,shantikumar saladi ,Sreekanth Gutala and K.V.Surendranath. Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Doxofylline and Terbutalinesulphate in Pharmaceutical Formulations.Scientia pharmaceutica.2013.969-982. Gajanan b kasawar,Mazahar farooqui. Development and validation of a stability indicating RP- HPLC method for the simultaneous determination of related substances of Terbutaline sulfateand ipratropium bromide in nasal solution. Journal Of Analytical And Bio Analytical Techniques.,2010;52(1):19–29. Validation of analytical procedures ICH Q2R1 Validation of compendial procedures USP 40 NF 35 Analytical procedures 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. 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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-6145442","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":425996538,"identity":"2b753545-43db-45aa-a4cb-a92259b29af6","order_by":0,"name":"Alexandar 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5","display":"","copyAsset":false,"role":"figure","size":104199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurity plot for Base Stress\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6145442/v1/7151fd11e69d48ebedac2043.png"},{"id":78121548,"identity":"dd3589b4-02d7-4d9a-b9b8-189d3c1b5d70","added_by":"auto","created_at":"2025-03-10 07:10:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":98511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurity plot for Peroxide Stress\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6145442/v1/4a82f01308a972771e0708a0.png"},{"id":78121546,"identity":"70645f33-8272-43d8-99cf-6977519378a1","added_by":"auto","created_at":"2025-03-10 07:10:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":102419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurity plot for Heat Stress\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6145442/v1/0a333a5035f6fa5e82e65c77.png"},{"id":78121571,"identity":"9a0f54f5-f0bc-4db3-9e0d-96481e467473","added_by":"auto","created_at":"2025-03-10 07:10:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":87459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePurity plot for UV Stress\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6145442/v1/b149966b5661edbd6ec9e780.png"},{"id":78760068,"identity":"baca6dbc-48b1-43e9-b3c9-610c510f13b1","added_by":"auto","created_at":"2025-03-18 13:46:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1209729,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6145442/v1/ba5e7e77-7ffb-420a-8bbe-a4b6f44e126f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulatory Based Method equivalency study to Estimate the Terbutaline sulfate API by RP-HPLC method","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHPLC is a sophisticated separation technique used to Isolation, Elucidation and Quantification of the components. The aim of present study is to analyze the Terbutaline sulfate API. Terbutaline sulfate known as 1,3-Benzenediol, 5-[2-[(1,1-dimethylethyl)amino]\u0026minus;\u0026thinsp;1-hydroxyethyl]-, sulfate (2:1) (salt). (\u0026plusmn;)-α-[(tert-Butylamino) methyl]-3,5-dihydroxybenzyl alcohol sulfate (2:1) (salt) by chemically. The classification of drug is short acting beta2\u0026ndash;adrenergic agonist. Terbutaline sulfate is prescribed to treat bronchospasm in bronchial asthma, chronic bronchitis, emphysema, chronic obstructive pulmonary disease and prophylaxis of exercise\u0026ndash;induced asthma and. Terbutaline sufate is acting on the smooth muscles from the trachea to the end of the bronchioles, thus making breathing easier. Physical appearance is a white or almost white crystalline powder, freely soluble in water, Slightly soluble in ethanol practically insoluble in heptane [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A Brief literature survey discovered few RP-HPLC methods for routine analysis, related substances and impurity determinations in individual and multi component dosage forms containing ambroxol HCl, guaifenesin and terbutaline sulphate [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Degradation study of Terbutaline sulfate have been performed [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].Numerous UV, HPLC, GC and HPTLC based methods have been reported for estimation of these drugs alone as well as in combination with other drugs in pharmaceutical dosage forms [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Literatures survey revealed there is no method equivalency study related to HPLC method the for Assay of Terbutaline sulfate API. Hence A novel and Regulatory based RP-HPLC In house method has been developed to show the equivalent to USP method for analyse of Terbutaline sulphate API.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eDrugs and Chemicals\u003c/p\u003e \u003cp\u003eTerbutaline sulphate RS, procured from sigma Aldrich. Terbutaline sulphate API obtained from FDC Ltd, India. Impurities standards (Impurity A \u0026minus;\u0026thinsp;3,5-dihydroxybenzoic acid (α-resorcylic acid), ), (Impurity-B (4RS)-2-tert-butyl-1,2,3,4-tetrahydroisoquinoline-4,6,8-\u003c/p\u003e \u003cp\u003etriol,), (Impurity-C- 2-(tert-butylamino)-1-(3,5-dihydroxyphenyl)ethan-1-one), Impurity-D \u0026minus;\u0026thinsp;2-[benzyl(tert-butyl)amino]-1-(3,5-dihydroxyphenyl) ethan-1-one. purchased from USP and TLC pharmaceutical standards. HPLC grade heptane sulfonic acid, potassium dihydrogen phosphate, acetonitrile, phosphoric acid and Milli-Q water obtained from Merck.\u003c/p\u003e \u003cp\u003eInstrumentation\u003c/p\u003e \u003cp\u003eA waters HPLC (E-2695 \u0026ndash; PDA 2996) instrument controlled with Empower \u0026minus;\u0026thinsp;3 software. Column utilized X Bridge Shield RP-18 (150x4.6mm, 3.5\u0026micro;) .Thermo fisher sonicator used to dissolve reference standard and API. A Mettler Toledo electronic balance was used for weighing process. All pH adjustments were made using a Met Rohm pH meter.\u003c/p\u003e \u003cp\u003eMobile Phase\u003c/p\u003e \u003cp\u003eAccurately weighed and transferred about 2.87g of heptane sulfonic acid and 2.5 g of potassium dihydrogen phosphate in a suitable container. Added about 1000mL of water into same container. Mixed well and adjust the pH to 3.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 with dilute phosphoric acid. Filtered through a 0.45\u0026micro;m membrane filter. The mobile phase composed of pH 3.60 Buffer: Acetonitrile (80:20), at flow rate of 0.8 ml/min with run time of 10 min. Detection wavelength at 276 nm. Retention time of Terbutaline sulfate was found to be 7.266 minutes. Injection volume 10 \u0026micro;l, Column oven thermostatically controlled with 30\u0026deg;C, Sampler temperature maintained at 5\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDiluent Preparation\u003c/h2\u003e \u003cp\u003eTransferred 900mL of water and 100mL of acetonitrile in a suitable container and mix well.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eResolution Solution Preparation\u003c/h3\u003e\n\u003cp\u003eWeighed accurately and transferred about 14 mg of Terbutaline sulfate RS and 3 mg of Terbutaline sulfate Related Compound A RS into a 100 mL volumetric flask, added 10 mL of diluent and sonicated to dissolve. Diluted to volume with diluent and mixed well. (Concentration of about 140 \u0026micro;g/mL of Terbutaline sulfate and 30 \u0026micro;g/mL of Terbutaline sulfate Related Compound A)\u003c/p\u003e\n\u003ch3\u003eStandard Preparation\u003c/h3\u003e\n\u003cp\u003eStandard stock Preparation: Weighed accurately and transferred about 25 mg of Terbutaline sulfate RS into a 25- mL volumetric flask, added 10mL of diluent and sonicated to dissolve. Dilute to volume with diluent. Mixed well. (Concentration of about 1000 \u0026micro;g/mL of Terbutaline Sulfate).\u003c/p\u003e \u003cp\u003eWorking Standard Preparation: Pipetted out 4.0 mL of stock standard solution into a 50 mL volumetric flask. Diluted to volume with diluent, Mixed well. (Concentration of about 80 \u0026micro;g/mL of Terbutaline Sulfate).\u003c/p\u003e\n\u003ch3\u003eSample Preparation\u003c/h3\u003e\n\u003cp\u003eSample stock Preparation: Weighed accurately and transferred about 50 mg of Terbutaline sulfate sample into a 50mL volumetric flask, added 30mL of diluent and sonicated to dissolve. Diluted to volume with diluent, Mixed well. (Concentration of about 1000 \u0026micro;g/mL of Terbutaline Sulfate).\u003c/p\u003e \u003cp\u003eSample Preparation: Pipetted out 4.0 mL of stock sample into a 50- mL volumetric flask. Diluted to volume with diluent, Mix well. (Concentration of about 80 \u0026micro;g/mL of Terbutaline Sulfate).\u003c/p\u003e\n\u003ch3\u003eChromatographic conditions\u003c/h3\u003e\n\u003cp\u003eX Bridge Shield RP-18 (150 x 4.6mm, 3.5\u0026micro;) was used for chromatographic separation. The mobile phase composed of pH 3.60 Buffer: Acetonitrile (80:20), at flow rate of 0.8 ml/min with run time of 10 min. Detection wavelength at 276 nm. Retention time of Terbutaline sulfate was found to be 7.266 minutes. Injection volume 10 \u0026micro;l, Column oven thermostatically controlled with 30\u0026deg;C, Sampler temperature maintained at 5\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMETHOD VALIDATION\u003c/h2\u003e \u003cp\u003eThe developed method was validated in terms of linearity, specificity, precision, accuracy, limit of detection, limit of quantitation, robustness and system suitability testing as per the\u003c/p\u003e \u003cp\u003eICH guidelines and Validation of compendial procedures USP 40 NF 35 Analytical procedures [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eSystem Precision\u003c/p\u003e \u003cp\u003eSystem precision established by injecting standard solutions and resolution solution. Relative standard deviation of peak area responses for five replicate injections of the standard solution and resolution of impurity mixture solution reported.\u003c/p\u003e \u003cp\u003eMethod Precision\u003c/p\u003e \u003cp\u003ePrecision of the assay method was determined by injecting, six individual sample preparations of Terbutaline Sulfate. The samples were prepared as per the method. Reported with Relative standard deviation for assay.\u003c/p\u003e \u003cp\u003eLinearity and range\u003c/p\u003e \u003cp\u003eLinearity performed by Solutions of Terbutaline sulfate varying concentrations ranging from 40\u0026ndash;150% of the standard concentration of (80 \u0026micro;g/mL) were injected into HPLC system. The linearity graph was plotted from 40\u0026ndash;150%. Reported with correlation coefficient.\u003c/p\u003e \u003cp\u003eRuggedness\u003c/p\u003e \u003cp\u003eRuggedness of the method performed by analyst-2 on different day from method precision. Standard and sample solutions prepared and injected. Reported with Relative standard deviation for assay and system suitability parameters were calculated.\u003c/p\u003e \u003cp\u003eSolution stability\u003c/p\u003e \u003cp\u003eSolution stability study was performed at different days against the freshly prepared standard, and sample solution and the results were represented .The standard and sample solutions were prepared and injected. Replicate injections of the standard and sample solution were made at the following time intervals, Initial, 33-hrs, 50-hrs for standard and Initial, 27-hrs, 60-hrsfor sample stored at 5\u0026deg;C cooled temperature. The %difference in % assay from initial and time point for both standard and sample solution were generated.\u003c/p\u003e \u003cp\u003eSpecificity\u003c/p\u003e \u003cp\u003eSpecificity of the method can be studied in the presence of excipients, degradation products and impurities. Blank, standard and stressed sample solutions were prepared and injected into the chromatographic system for identification and impurity interference with the Terbutaline sulfate peak.\u003c/p\u003e \u003cp\u003eSpecificity by Forced Degradation:\u003c/p\u003e \u003cp\u003eA forced degradation study is performed in order to prove that the method is stability indicating.The Terbutaline Sulfatedrug substance was stressed under the following conditions.\u003c/p\u003e \u003cp\u003eControl Sample Preparation\u003c/p\u003e \u003cp\u003eWeighed and transferred about 25mg of Terbutaline sulfatedrug substance in to a 25mL volumetric flask, added 10mL of diluent then sonicated to dissolve and diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003eAcid Degradation\u003c/p\u003e \u003cp\u003eWeighed and transferred about 25mg of Terbutaline sulfatedrug substance in to a 25 mL volumetric flask, added 10 mL of diluent and sonicated to dissolve. Added 2.5 mL of 5N HCl and kept in a water bath maintained at a temperature of 80\u0026deg;C for 1 hour. After 1 hour, the sample was allowed to cool to room temperature and then neutralized with 2.5 mL of 5N NaOH. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003eBase Degradation\u003c/p\u003e \u003cp\u003eWeighed and transferred about 25mg of Terbutaline sulfate drug substance in to a 25mL volumetric flask,, added 10 mL of diluent and sonicated to dissolve. Added 2.5 mL of 2N NaOH and kept in a water bath maintained at a temperature of 80\u0026deg;C for 1 hour. After 1 hour, the sample was allowed to cool to room temperature and then neutralized with 2.5 mL of 2N HCl. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003ePeroxide Degradation\u003c/p\u003e \u003cp\u003eWeighed and transferred about 25mg of Terbutaline sulfate drug substance in to a 25mL volumetric flask, added 10 mL of diluent and sonicated to dissolve. Added 2.5 mL of 3%H2O2and kept in a water bath maintained at a temperature of 80\u0026deg;C for 1 hour. After 1 hour, the sample was allowed to cool to room temperature and then diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003eHeat Degradation\u003c/p\u003e \u003cp\u003eRequired amount of drug substance transferred in to a Petri dish, heated in an oven for 24 hours at 80\u0026deg;C, then sample taken out and cooled to room temperature. Weighed and transferred about 25mg of this heat exposed sample into a 25mL volumetric flask, 10mL of diluent added, sonicated to dissolve. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003eUV-Light Degradation\u003c/p\u003e \u003cp\u003eRequired amount of drug substance transferred in to a Petri dish, exposed to 254nm UV light at room temperature for 24 hours. After 24 hours of exposure time, about 25mg of this UV light exposed sample weighed and transferred into a 25mL volumetric flask, 10mL of diluent added, sonicated to dissolve. Diluted to volume with diluent and mixed well. Pipetted out 2.0 mL of stock solution into 25mL volumetric flask. Diluted to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003ePreparation of Individual Impurity Stock Solutions\u003c/p\u003e \u003cp\u003eAccurately Weighed and transferred each about 0.5 mg of Impurity-A, Impurity-B, Impurity C and Impurity-D into individual 10mL volumetric flasks. Added diluent about 3/4th volume of the flask and sonicated to dissolve. Made up to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003eImpurity Mixture Solution Preparation\u003c/p\u003e \u003cp\u003ePipetted about 2.5mL of Impurity-A, Impurity-B, Impurity-C and Impurity-D stock solutions into 25mL volumetric flask. Diluted to volume with diluent and mixed well.\u003c/p\u003e \u003cp\u003ePreparation of Spiked Sample Solution\u003c/p\u003e \u003cp\u003eWeighed and transferred about 25 mg of Terbutaline sulfate drug substance in to a 25 mL volumetric flask. Transferred 2.5mL of Impurity mixture solution preparation into the same volumetric flask and sonicated to dissolve. Diluted to volume with diluent and mixed well. All the control, stress samples and spiked samples were injected for determination of % assay and peak purity.\u003c/p\u003e \u003cp\u003eRobustness\u003c/p\u003e \u003cp\u003eRobustness of the method evaluated by varying chromatographic method parameters such as column temperature\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C and Mobile phase pH\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 from the optimized conditions. Injected Standard solution into the chromatographic system. System suitability results were reported.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMETHOD EQUIVALENCY\u003c/h3\u003e\n\u003cp\u003eMethod equivalency study was determined by injecting, Standard, Resolution solution and six individual sample solutions of Terbutaline Sulfate. The samples were prepared and analyzed as per both USP method and In-house method and results were compared.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e \u003cb\u003eTable.1\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTerbutaline sulfate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSystem precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% RSD\u0026thinsp;=\u0026thinsp;0.1\u003c/p\u003e \u003cp\u003eResolution\u0026thinsp;=\u0026thinsp;24.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethod precision\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% RSD\u0026thinsp;=\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethod precision assay\u003c/p\u003e \u003cp\u003e(Average of six preparations)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinearity and Range\u003c/p\u003e \u003cp\u003e(Correlation co-efficient)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRuggedness\u003c/p\u003e \u003cp\u003e(System precision)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% RSD\u0026thinsp;=\u0026thinsp;0.2\u003c/p\u003e \u003cp\u003eResolution\u0026thinsp;=\u0026thinsp;37.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRuggedness\u003c/p\u003e \u003cp\u003e(Average of six preparations)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolution stability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStandard stability \u0026minus;\u0026thinsp;50 hours at 5\u0026deg;C\u003c/p\u003e \u003cp\u003eSample stability \u0026ndash; 60 hours at 5\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethod precision assay\u003c/p\u003e \u003cp\u003e(Average of six preparations) USP Method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.5%\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\u003eSystem precision parameters such % RSD and Resolution between Main compound and impurity\u0026rsquo;s found within limit .Blank, standard and System suitability chromatograms shown from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eto\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Method precision result shows repeatability of the method. Linear over the concentration range of 40\u0026ndash;150% of assay concentration. Ruggedness study exhibited reproducibility of the developed method. Solution Stability data demonstrated the sample and standard solutions stable up to 60 h and 50 h respectively at 5\u0026deg;C.Method precision study performed by USP method revealed that the developed inhouse method equivalence to USP method. Above all the results reported in \u003cb\u003eTable\u0026nbsp;1.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.2\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS.no\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% Assay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePurity angle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePurity Threshold\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl API\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e105.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.461\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcid 5N HCl 180\u0026deg;C 1Hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.467\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBase 2N NaOH 80\u0026deg; 1 Hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.460\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 80\u0026deg;C 1Hour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUV 24 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e103.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.433\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeat 24 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e105.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.443\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpiked Sample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.294\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\u003eIn forced degradation study Terbutaline sulfate treated with different stress conditions such as Acid stress, base stress, peroxide stress, UV and Heat. Purity plots presented from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eto\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.In all conditions Purity threshold found more than the purity angle. It\u0026rsquo;s highy showing the specificity of the method mentioned in \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.3\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eMethod Parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRetention Time\u003c/p\u003e \u003cp\u003e(1st injection)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUSP Resolution\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal Condition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColumn Temp: 30\u0026deg;C\u003c/p\u003e \u003cp\u003eMobile phase\u003c/p\u003e \u003cp\u003epH: 3.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColumn Temp. Minus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColumn Temp. Plus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.984\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMobile phase pH Minus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMobile phase pH Plus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32.1\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\u003eRobustness study results showed the developed method not affected by the small changes. Method had undergone with Column compartment temperature change and mobile phase pH changes. Results reported in \u003cb\u003eTable\u0026nbsp;3.\u003c/b\u003e\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn the present study, the new In-house HPLC analytical method was developed for the estimation of Terbutaline sulfate API .Based on the results of the above studies, it is concluded that the method for Assay of Terbutaline sulfate API is specific, precise, rugged, robust and linear over the concentration range. Standard and sample solutions are stable up to 50 hours and 60 hours respectively when stored at a temperature of 5\u0026deg;C (Refrigerator Condition). Results of In-house method comparable with USP method with respect to validation parameters, method equivalency study protocols and regulatory requirements.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRavisankar.M - Writing \u0026ndash; original draftS.Alexandar -Project administration S.Sangeetha - VisualizationC.Pasupathi - Resource ContributionM.Kumar - Formal analysis\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThe authors are thankful to Srinivasan college of pharmaceutical sciences, Trichy and P\u0026amp;A pharma and neutraceuticals testing lab,Chennai providing necessary facilities to carry out the research work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rxlist.com/terbutaline-sulfate-drug.htm\u003c/span\u003e\u003cspan address=\"https://www.rxlist.com/terbutaline-sulfate-drug.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ehttps://\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003epubchem.ncbi.nlm.nih.gov/compound/Terbutaline-sulphate\u003c/span\u003e\u003cspan address=\"http://pubchem.ncbi.nlm.nih.gov/compound/Terbutaline-sulphate\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Pharmacopoeia Page No:3979\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUSP 40 NF 35\u0026ndash;Terbutaline sulfate\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN Itagimatha, D H Manjunatha. RP-HPLC-UV method development and validation for simultaneous determination of terbutaline sulphate, ambroxol HCl and guaifenesin in pure and dosage forms. Annales Pharmaceutiques Fran\u0026ccedil;aises.2019:7(4):295\u0026ndash;301.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.Lakshmi Narasimha Rao,Ch.Krishnaiah,K.Sudheer Babu,K.Padmaja Reddy. Development and validation of a stability-indicating LC method for simultaneous determination of related compounds of guaifenesin, terbutaline sulfate and ambroxol HCl in cough syrup formulation.Journal of Saudi Chemical Society.2014:18(5):593\u0026ndash;600\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh A, Mandal S. K.,Ghosh S.and Deb S. Development And Validation of RP-HPLC Method For Simultaneous Determination of Terbutaline Sulphate, Guaiphenesin And Ambroxol Hydrochloride From An Oral Liquid.India drugs.2016:53(5):53\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN Daraghmeh,M.MAl-Omari,ZSara, A.ABadwan,A.M.YJaber. Determination of terbutaline sulfate and its degradation products in pharmaceutical formulations using LC. Journal of Pharmaceutical and Biomedical Analysis.2002:29(5):927\u0026ndash;937.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaulik Oza1, Jagdish Kakadiya1, Chirag Oza. Development and Validation of Solvent Extraction Spectrophotometric Method for Simultaneous Estimation of Doxofylline and Terbutaline sulphate In their Combined Dosage Form.American journal of pharm tech research.2012:2(4):593\u0026ndash;604\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Sunandana, K. Sushmitha, Buchi N. Nalluri. RP-HPLC-PDA Method for the Analysis of Terbutaline sulphate in Bulk, Dosage forms and in Dissolution samples. Journal of Applied Pharmaceutical Science.2013:3(03).126\u0026ndash;132.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel Juhee J,Mrs Chorawala Henal,Dr.Dedania zama R,Dr.Vijendraswamy S.M.Development and Validation of UV Spectroscopic method for simultaneous estimation of doxofylline and Terbutaline sulphate in combined dosage form. Asian Journal of 2015:5(2):74\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeerthisikha Palur, Bharathi Koganti, Sreenivasa Charan Archakam. Chemometric-assisted RP-HPLC method for the simultaneous determination of ambroxol hydrochloride, terbutaline sulfate, and guaiphenesin in combined dosage form. Journal of Applied Pharmaceutical Science.2019:09(09):92\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePorel, Sanjukta Haty, and A. Kundu..Stability-indicating HPLC Method for Simultaneous Determination of Terbutaline Sulphate, Bromhexine Hydrochloride and Guaifenesin.Indian Journal of pharmaceutical sciences.2011:73(1):46\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThirupathi Dongala,Lakshmi Narasimha Rao Katakam,Ashok Kumar Palakurthi,Naresh Kumar Katari.RP-HPLC Stability Indicating Method Development and Validation of Pseudoephedrine Sulfate and Related Organic Impurities in Tablet Dosage Forms, Robustness by QbD Approach.2019:697\u0026ndash;710.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA.Susmitha,B.Satya Narayana, E.Masthani, S.Bindu Bhargavi,Dr.C.Gopinath. Development And Validation of UV Spectrophotometric Method For The Estimation Of Terbutaline Sulphate In Its Syrup Dosage form. International Journal of Advances in Pharmaceutical Research.2015:6(8):221\u0026ndash;227.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGananadhamu Samanthula,Krishnaveni Yadiki,shantikumar saladi ,Sreekanth Gutala and K.V.Surendranath. Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Doxofylline and Terbutalinesulphate in Pharmaceutical Formulations.Scientia pharmaceutica.2013.969-982.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGajanan b kasawar,Mazahar farooqui. Development and validation of a stability indicating RP- HPLC method for the simultaneous determination of related substances of Terbutaline sulfateand ipratropium bromide in nasal solution. Journal Of Analytical And Bio Analytical Techniques.,2010;52(1):19\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValidation of analytical procedures ICH Q2R1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValidation of compendial procedures USP 40 NF 35 Analytical procedures\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":"HPLC, Method validation, Method Equivalence, ICH Q2R1, USP","lastPublishedDoi":"10.21203/rs.3.rs-6145442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6145442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel, regulatory based HPLC method has been developed and validated for the estimation of Terbutaline sulphate API. Column is X- bridge shield RP-18 150\u0026times;4.6 mm, 3.5 \u0026micro;m, wave length 223 nm, injection volume 15 \u0026micro;L, column temperature is 30\u0026deg;C,sampler temperature 5\u0026deg;C and flow rate 0.8 mL/min. Retention time of terbutaline sulfate is about 7.226 minutes and Runtime 10 minutes. The developed methods have been validated as per ICH guidelines. The method exhibited satisfactory with precision, specificity, linearity, robustness and ruggedness. The proposed method was found to be equivalent to USP method.\u003c/p\u003e","manuscriptTitle":"Regulatory Based Method equivalency study to Estimate the Terbutaline sulfate API by RP-HPLC method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-10 07:10:34","doi":"10.21203/rs.3.rs-6145442/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":"7641f3d7-5dfb-4ca6-9f40-e90d065896bf","owner":[],"postedDate":"March 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-18T13:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-10 07:10:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6145442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6145442","identity":"rs-6145442","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}