Center Composite Design Assisted Optimization of Rp-hplc Method for Simultaneous Estimation of Linagliptin and Empagliflozin Using Aqbd Perspective

Center Composite Design Assisted Optimization of Rp-hplc Method for Simultaneous Estimation of Linagliptin and Empagliflozin Using Aqbd Perspective | 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 Method Article Center Composite Design Assisted Optimization of Rp-hplc Method for Simultaneous Estimation of Linagliptin and Empagliflozin Using Aqbd Perspective Khushbu Patel, Zeel Modi, Chhaganbhai. N. Patel This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9035757/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract This study reports the development of a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Linagliptin and Empagliflozin in pharmaceutical dosage forms using a Quality by Design (QbD) approach. In the QbD strategy, a central composite design (CCD) was employed to evaluate the influence of critical method parameters, particularly the composition of the mobile phase and the flow rate. The interaction effects of these parameters on critical responses, including retention time and resolution of Linagliptin and Empagliflozin, were analyzed using three-dimensional response surface plots. These plots helped to establish the optimal chromatographic conditions for the developed method. The proposed method is simple, precise, and accurate for the estimation of Linagliptin and Empagliflozin. Chromatographic separation was achieved using a C18 column (NOVATOZ C18, 250 × 4.6 mm, 5 µm) with gradient elution. The mobile phase consisted of Methanol and Acetonitrile (90:10, v/v) delivered at a flow rate of 1 mL/min. Detection of analytes was performed using a photodiode array (PDA) detector at 225 nm. Linearity was observed over the concentration range of 2.5–12.5 µg/mL for Linagliptin and 5–25 µg/mL for Empagliflozin, with correlation coefficients (r²) of 0.993 and 0.991, respectively. The developed method was successfully applied for the quantitative analysis of Linagliptin and Empagliflozin in pharmaceutical dosage forms. Anti diabetic Dipeptidyl peptidase-4 Sodium-glucose co-transporter 2 Validation Recovery Figures Figure 1 Figure 2 INTRODUCTION Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor that is used either alone or in conjunction with other oral hypoglycemic medications to treat type 2 diabetes, along with diet and exercise. There have been very few reports of clinically noticeable liver damage associated with Linagliptin. Chemically, it is 8-[(3R)-3-aminopiperidin-1-yl]− 7- but- 2- ynyl- 3- methyl1- [(4-methylquinazolin-2-yl) methyl] purine-2,6-dione. [ 1 – 3 ] Empagliflozin belongs to the class of diabetic medications known as sodium-glucose co-transporter 2 (SGLT-2). This exercise explains the use of Empagliflozin as a beneficial treatment for type 2 diabetes mellitus, including its indications, mode of action, and contraindications. The mechanism of action, dosage, and monitoring that are important for interprofessional team members to know when managing patients with type 2 diabetes will be highlighted in this exercise. Chemically, it is 4’-[(1,4’-dimethyl-2’-propyl[2,6’-bi-1Hbenzimidazole]-1’-yl) methyl] [1,1’- biphenyl]-2-carboxylicacid. [ 4 – 5 ] Literature reveals various analytical method have been reported such as UV [ 6 – 11 ], HPLC [ 12 – 21 ], HPTLC [ 22 – 23 ] for estimation of Linagliptin and Empagliflozin. The purpose of the present work is to establish and implement a new RP-HPLC method for determination of Linagliptin and Empagliflozin in bulk and pharmaceutical dosage form adding analytical QbD principles. Application of QbD principle for analytical method development has been practiced quite popular for attaining high robustness and improving method performance. The QbD method was based on the principles and approaches of guideline ICH Q8 pharmaceutical development, ICH Q9 quality risk management and ICH Q10 pharmaceutical quality system [ 24 – 32 ] . MATERIALS AND METHODS: Material: A gift sample of Linagliptin and Empagliflozin was provided by Cubic Analytical Solution, Gujarat. HPLC grade methanol and acetonitrile were bought from Finar life science private limited, Ahmedabad, India. HPLC grade water was ordered from Loba chemise private limited, Mumbai, India. The marketed formulations GLYXAMBI, 5 mg Linagliptin and 10 mg Empagliflozin, are by Boehringer Ingelheim private limited, Gujarat, India. Instruments and Reference Standard: The HPLC study was carried out on Noavatoz C18 (250 x 4.6mm, 5µm) was used at room temperature. Other equipment Shimadzu HPLC instrument (LC_2010 CHT) [software LC solution, equipped with UV detector, Auto-sampler], UV visible spectrophotometer double beam 1800 (Shimadzu), Electronic analytical balance (Acculab ALC210.4, Huntingdon Valley, PA), Ultra sonicator (EN 30 US, Enertech fast clean, Mumbai, India) QbD Software: Design Expert software 13.0 (Trial Version) Preparation of Standard stock solution: Weighed 50 mg of Linagliptin and 100 mg of Empagliflozin. Transferred to a 100 mL flask and diluted with methanol to make 500 μg/mL Linagliptin and 1000 μg/mL Empagliflozin. Diluted 1 mL of this solution to 10 mL for 50 μg/mL Linagliptin and 100 μg/mL Empagliflozin. Preparation of sample stock solution: Twenty tablets of Glyxambi 5 mg / 10 mg were weighed and powdered. The powder equivalent to 10 mg Empagliflozin and 5 mg Linagliptin was mixed with methanol in a volumetric flask. The solution was shaken, sonicated, filtered, and diluted to obtain specific concentrations. Method validation : Developed method was validated according to ICH Q2 (R1) guidelines. Specificity : Comparison of the chromatograms of the standard and test solutions allowed researchers to assess the method’s specificity. Linearity: Aliquots of working standard solutions (0.5, 1, 1.5, 2, 2.5 mL) were transferred into series of 10 mL volumetric flask and diluted up to mark with methanol. These yielded solution of concentration 2.5-12.5 μg/mL of Linagliptin and 5-25 μg/mL of Empagliflozin. An aliquot of 20 μL of each solution was injected under operating chromatographic condition. Precision : Intraday standard solutions of Linagliptin and Empagliflozin at 5 μg/mL, 7. 5 μg/mL, 10 μg/mL and 10 μg/mL, 15 μg/mL, 20 μg/mL were analyzed three times in one day. Interday solutions were analyzed over three different days, measuring absorbance and calculating % RSD. Accuracy: Accuracy was determined by calculating the recovery of Linagliptin and Empagliflozin using the standard addition method. Known amounts of Linagliptin (4 μg/mL, 5 μg/mL, and 6 μg/mL) and Empagliflozin (8 μg/mL, 10 μg/mL, 12 μg/mL) were added to their sample solution in a volumetric flask. Each solution was diluted with methanol, injected three times, and recovery was calculated using a regression equation from the calibration curve by measuring peak areas. Limit of Detection and Limit of Quantification: The LOD and LOQ were calculated based on the standard deviation of the response (y intercepts of regression lines) and the slope using 3 independent analytical curves, as defined by ICH. LOD and LOQ were calculated as 3.3 σ/S and 10 σ/S, respectively, where σ is the standard deviation of Y intercept (ICH guidelines) and S is the slope of the Linagliptin and Empagliflozin calibration curve. Robustness: The robustness study was carried out to assess the impact of subtle but intentional changes to the chromatographic condition. The robustness was tested by making four minor adjustments. Flow rate (1 ± 0.1 mL/min), Mobile phase (90 ± 5 mL), Injection volume (20 ± 5 μL) Analysis of marketed formulation An aliquot of 20 μL from sample solution was injected under a chromatographic condition and peak area was measured and % assay was calculated from regression equation. Three determinations on average were made in response. Method Optimization by QbD Approach Implementation of QbD approach The following experimental study was planned and the QbD technique was used in accordance with ICH Q8 (R2) principles. Method performance requirements: Using a QbD method, it is important to fully document the goals or execution needs of the technique and to consider how the strategy will be used. This involves the Analytical Target Profile (ATP), which evaluates the active pharmaceutical ingredient (API) using chromatographic and/or spectrophotometric methods. ATP is similar to the Quality Target Product/Process Profile (QTPP) for the pharmaceutical process. ATP consists of models outlining what will be measured, the grid used, the concentration range, and the required method performance metrics, known as Critical Quality Attributes. Method development: Following the characterization of the ATP, an appropriate protocol and technique condition should be selected in order to satisfy all of the ATP's requirements. Method understanding: Based on a risk assessment, an exercise focused on comprehending the approach can be conducted to better grasp the potential impact of important information components on the presentation qualities of the technique. This allows for the recognition of certain functional technique controls. Risk assessment: To understand the practical relationship between method input components and all of the attributes of strategy execution, investigations might be hurried. Data gathered during events and the start of the strategy's application provides input into a risk assessment (using tools like the Fishbone chart, FMEA, and Ishikawa Diagram), which may determine which factors need consideration and which require controls. Design of experiments: Design of Experiment (DoE) is used for power testing of parametric factors to achieve the best setup while reducing the number of tests. It evaluates proxy measures, like precision, based on the method type. It explains critical parameters by showing how influential limits affect strategy implementation after a risk assessment. Method design output: To meet the ATP, several procedure conditions will have been developed and improved based on their impact on the plan. Assessing a scientific strategy using QbD requires examining key characteristics affecting selectivity. Continuous variable testing is impractical, so some authors use statistical methods like Placket-Burman to reduce exploratory costs. Mechanized strength testing is used in some methods. RESULTS AND DISCUSSION Optimization phase: It is necessary to enhance peak symmetry and reduce tailing from the chromatographic conditions by adopting an appropriate experiment design. Selection of factors: Variables affecting separation procedures include mobile phase composition, flow rate, temperature, stationary phase, and injection volume. For the study, mobile phase composition, flow rate, and injection volume were significant for retention and peak shape. The mobile phase used was Methanol: Acetonitrile (90:10 %v/v). The upper and lower levels of the mobile phase were chosen to adjust polarity for optimal retention time. Flow rate and mobile phase also needed optimization to improve system suitability parameters like the number of theoretical plates. Selection of level: In most cases, components were examined at least twice (-1, +1) during optimization designs. Factor levels for method optimization are chosen from existing knowledge and literature, often covering the broadest range for the factor. Selection of optimization design Using the response-surface design process, key components were examined. Central composite design can determine the best conditions with fewer runs, saving time and money. Below is a list of tests for two factors and two levels for various experimental designs. Formula for CCD: 2 (K) + 2K + C, where K = No. of Factors, C = No. of Center points. Selection of response Response can be chosen for peak tailing, retention time, and chromatographic separations, among other things. Actually, it is important to select optimization conditions so that the Rt values of the components do not conflict with those of impurities or degradation products. The peak tailing must be less than 1.5 at that moment. The optimization of both response variables must have more theoretical plates and better suitability parameters (Table1). Table 1: Software-aided Method Optimization Standard Experiment No. Run Factor 1 A: Mobile phase composition (mL) Factor 2 B: Flow rate (mL/min) Response 1 Retention time (min) Empagliflozin Response 2 Retention time (min) Linagliptin Response 3 Resolution 11 1 90 1 2.921 4.27 6.541 12 2 90 1 2.99 4.202 6.454 13 3 90 1 2.93 4.2 6.468 2 4 95 0.9 3.211 4.47 6.681 6 5 97.07 1 2.958 3.715 7.399 3 6 85 1.1 2.621 3.8 4.88 8 7 90 1.1 2.697 3.51 4.636 1 8 85 0.9 3.15 4.3 4.789 5 9 82.92 1 2.715 3.87 4.651 9 10 90 1 2.93 4.21 6.457 7 11 95 1.1 2.9 3.42 5.248 4 12 90 0.85 3.446 4.782 5.49 10 13 90 1 2.92 4.209 6.459 Method performance can be improved throughout the method development. The analyst has opportunities to placed inventive approaches to improve quality. Process performance can be monitored to make sure consistency in quality. The QbD approach avails the continuous improvement throughout the method life cycle (Fig. 1A, 1B, and 1C). The Anova response of reduced model were shown in table 2. Table 2: ANOVA responses for Model ANOVA for response Retention time of Empagliflozin Source Sum of Squares df Mean Square F-value p-value Model 0.5782 5 0.1156 80.95 < 0.0001 significant A-Mobile Phase 0.0584 1 0.0584 40.90 0.0004 B-Flow Rate 0.4509 1 0.4509 315.64 < 0.0001 ANOVA for response Retention time of Linagliptin Model 1.80 5 0.3590 196.68 < 0.0001 significant A-Mobile Phase 0.0230 1 0.0230 12.61 0.0093 B-Flow Rate 1.40 1 1.40 767.96 < 0.0001 ANOVA for reduced model for response resolution Model 13.19 5 2.64 38.69 < 0.0001 Significant A-Mobile Phase 6.45 1 6.45 94.61 < 0.0001 B-Flow Rate 1.81 1 1.81 26.53 0.0013 Continuous Improvement throughout Product Life Cycle The Linagliptin and Empagliflozin standard solution of 5 μg/mL and 10 μg/mL were injected in six replicates. The Standard and sample chromatogram of Linagliptin and Empagliflozin was shown in the figure 2a and 2b. The retention time of Linagliptin was 4.160 min and 2.889 min for Empagliflozin. Linearity The working concentration 2.5-12.5 µg/mL for Linagliptin and 5-25 µg/mL for Empagliflozin. The calibration curve was plotted for the response (area) and concentration (amount) and the correlation coefficient (r 2 ) and y-intercept were calculated. Precision Intraday precision (repeatability) of a standard solution with (5,7. 5,10µg/mL) Linagliptin and (10,15,20 µg/mL) Empagliflozin was tested three times in one day. Interday precision of the same standard solution was analyzed over three different days. Accuracy The % recovery studies involved spiking Linagliptin and Empagliflozin at 80%, 100%, and 120% concentrations in triplicates. Chromatograms were taken for each spiked sample, and the total drug amount was calculated to find % recovery. (Table 2). Table 2: Accuracy data of developed method validation Linagliptin level Amount of test solution (µg/mL) Amount of standard added (µg/mL) Amount found* ± SD % Recovery*± SD % RSD 80% 5 4 8.95±0.03 99.48±0.39 0.39 100% 5 5 9.97±0.02 99.70±0.2 0.20 120% 5 6 10.97±0.01 99.72±0.15 0.15 Empagliflozin 80% 10 8 17.95 ± 0.02 99.78 ± 0.17 0.11 100% 10 10 19.96 ± 0.02 99.80 ± 0.1 0.10 120% 10 12 21.95 ± 0.02 99.76 ± 0.12 0.12 Limit of Detection (LOD) and Limit of Quantification (LOQ) The LOD and LOQ were obtained by successively decreasing the concentration of Linagliptin and Empagliflozin as long as a signal to noise ratio of not less than 3:1 and 10:1 is maintained, respectively. Robustness The flow rate, mobile phase composition, and injection volume are the usual variations investigated under these parameters; the findings are displayed in table 3. The approach was proven to be robust because the variation was within an acceptable range with regard to peak asymmetry and theoretical plates. Table 3: Robustness data of developed method validation Condition Variation Peak area Retention time Tailing factor Linagliptin Mobile phase (90 ± 5 mL) 95:5 mL 468209 3.473 1.308 100 mL 468100 3.470 1.302 Flow rate (0.9 ± 0.1 mL/min) 0.9mL/min 642001 4.801 1.318 1.1mL/min 471463 3.507 1.314 Injection volume (20 ± 5 µL) 15 µL 498410 4.172 1.320 25 µL 498408 4.170 1.315 Applicability of developed method to Marketed formulation By analysing the GLYXAMBI tablet, which is used in industry, the proposed method's applicability was evaluated. The results are shown in table 4. Table 4: Analysis data of marketed formulation Tablet mg/tablet(Label claim) Amount recovered (mg) ± SD %Assay* (% of label claim) ± SD %RSD Lina Empa Lina Empa %Lina %Empa Lina Empa Glyxambi 5 10 5.02± 0.01 10.03± 0.01 100.03± 0.01 100.04 ±0.03 0.22 0.15 The assay results were comparable to labelled value of each drug in tablet dosage form. These results indicate that the developed method is accurate, precise, simple and rapid. DISCUSSION The centre composite design was applied for three factors at three levels with use of the Design Expert software trial version 13.0. Design of experimental results, including ANOVA, diagnostic graphs and model graphs, were examined for each factor. The effect of each factor on the response result was investigated in this results part. The solvent system (Methanol: Acetonitrile 90: 10%v/v), flow rate (1 mL/min), and UV wavelength at 225 nm was found to be the optimized chromatographic condition for Linagliptin and Empagliflozin. Linearity data shows that the developed method was linear in the range of 2.5–12 µg/mL of Linagliptin and 5–25 µg/mL of Empagliflozin with 0.993 and 0.991 correlation coefficient of Linagliptin and Empagliflozin respectively. The % RSD was less than 2% in intraday and interday precision study of proposed method and % recovery was found to be 99.48–99.72 and 99.76–99.80 for Linagliptin and Empagliflozin respectively. Limit of detection and limit of Empagliflozin was determined to be 0.281 µg/mL and 0.873 µg/mL for Linagliptin and 0.186 µg/mL and 0.566 µg/mL for Empagliflozin. Applicability of developed method for quantification of Linagliptin (tR = 4.174 min) and Empagliflozin (tR = 2.900 min) in tablet dosage form was shown the absence of any wanted peak and no change in retention time, resolution and peak symmetry. CONCLUSION An analytical quality by design (Central Composite Design) approach was successfully employed for the optimization of the RP-HPLC method. The developed RP-HPLC was specific, accurate and precise method as per ICH guidelines. Hence, the method can be successfully used for the simultaneous estimation of Linagliptin and Empagliflozin in pharmaceutical dosage form in routine quality control testing. Abbreviations ANOVA : Analysis of variance, AQbD: analytical quality by design, CMA : Critical material attributes, CCD: Centre composite design, ICH : International Conference on Harmonization, LOD: Limit of Detection, LOQ : Limit of Quantitation, RSM: Response surface method, RP-HPLC: reverse phase high performance liquid chromatography. Declarations CONFLICTS OF INTEREST : The authors declare no conflicts of interest. ETHICS DECLARATION Not applicable FUNDING STATEMENT No Funding was received to assist with the preparation of this manuscript DATA AVAILABILITY STATEMENT The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. ACKNOWLEDGEMENT Authors express their sincere gratitude to shri Sarvajanik pharmacy college, Mehsana, Gujarat, India for continuous motivation, support and guidance for research activity and for providing all required facilities to accomplish the entitled work. AUTHORS' CONTRIBUTION STATEMENT: Conceptualization: Khushbu Patel, Zeel modi Methodology: Zeel modi, Chhaganbhai. N. Patel Investigation: Khushbu Patel, Chhaganbhai. N. Patel Manuscript writing: Khushbu Patel, Zeel modi Visulization: Khushbu Patel, Chhaganbhai. N. Patel Supervision: Khushbu Patel Project Administration: Khushbu Patel References Drugs.com, Linagliptin Dosage, November 2023, https://www.drugs.com/dosage/linagliptin.html Thakkar Kinjal K, Patel UB, Patel DCN, Patel. Evogliptin tartrate a new drug of DPP-4 inhibitor: an overview. World J Pharm Res. 2021;10(5):1921–9. Pubchem L. November 2023, https://pubchem.ncbi.nlm.nih.gov/compound/Linagliptin Empagliflozin Dcom. 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Pharmaceutical Development Q8, Q9 and Q10 International Conference on harmonization, Geneva, Switzerland,2003:1–20. Additional Declarations No competing interests reported. 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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-9035757","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":607940010,"identity":"a12d7a0b-3f91-4ad0-a0bc-92541d143942","order_by":0,"name":"Khushbu Patel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACCR4wZSEH5TODEWEtBxgkjEnXktgA10IISPacPfj5Q4VE+vz+swc/MFRYJzaw8x7Aq0Waty9Z4sAZidwNN/KSJRjOpCc2MPMl4NUix89jIHGwDahFgseMgbHtMFALjwEhLcY/Dv6TSJfvPwPU8o8ILdK8PWYSBxskEhgO5AC1NBChRbLnXJrFmWMShmC/JBxLN24jpEXiTO7hGxU1NvLyoBD7UGMt289/Br8WBgYeJEYCkGIjoB5NyygYBaNgFIwCbAAAxho/ou3jcEQAAAAASUVORK5CYII=","orcid":"","institution":"Shri Sarvajanik Pharmacy College, Gujarat Technology University","correspondingAuthor":true,"prefix":"","firstName":"Khushbu","middleName":"","lastName":"Patel","suffix":""},{"id":607940012,"identity":"dcc76608-9570-41c2-a92f-45eb53b866f2","order_by":1,"name":"Zeel Modi","email":"","orcid":"","institution":"Shri Sarvajanik Pharmacy College, Gujarat Technology University","correspondingAuthor":false,"prefix":"","firstName":"Zeel","middleName":"","lastName":"Modi","suffix":""},{"id":607940017,"identity":"75698be9-400c-4687-8bc6-c92ed7959bb0","order_by":2,"name":"Chhaganbhai. N. Patel","email":"","orcid":"","institution":"Shri Sarvajanik Pharmacy College, Gujarat Technology University","correspondingAuthor":false,"prefix":"","firstName":"Chhaganbhai.","middleName":"N.","lastName":"Patel","suffix":""}],"badges":[],"createdAt":"2026-03-05 04:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9035757/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9035757/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105000837,"identity":"adb8a0ce-61b7-4c6e-9c44-9d16ed6dd165","added_by":"auto","created_at":"2026-03-19 16:50:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":190344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Effect of interaction term Mobile phase (A) and Flow rate (B) on response variable retention time (3D plot) of Empagliflozin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB)\u003c/strong\u003e Effect of interaction term Mobile phase (A) and Flow rate (B) on response variable retention time (3D plot) of linagliptin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC)\u003c/strong\u003eEffect of interaction term Mobile phase (A) and Flow rate (B) on response variable resolution (3D plot)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9035757/v1/971414260e3027a5da07b076.png"},{"id":105000838,"identity":"f7e98029-7120-4048-bae3-f2315043feb8","added_by":"auto","created_at":"2026-03-19 16:50:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2a) \u003c/strong\u003eChromatogram of standard solution Linagliptin (5 μg/mL) and Empagliflozin (10 μg/mL)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2b) \u003c/strong\u003eChromatogram of sample solution Linagliptin (5 μg/mL) and Empagliflozin (10 μg/mL)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9035757/v1/c32b098a389649911df19d11.png"},{"id":105035810,"identity":"dc18c4c9-02e3-492c-8ca9-9ac6cdafb8f2","added_by":"auto","created_at":"2026-03-20 07:26:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1774940,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9035757/v1/af8941b0-c0f6-4525-becb-00a2488bc945.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCenter Composite Design Assisted Optimization of Rp-hplc Method for Simultaneous Estimation of Linagliptin and Empagliflozin Using Aqbd Perspective\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLinagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor that is used either alone or in conjunction with other oral hypoglycemic medications to treat type 2 diabetes, along with diet and exercise. There have been very few reports of clinically noticeable liver damage associated with Linagliptin. Chemically, it is 8-[(3R)-3-aminopiperidin-1-yl]\u0026minus;\u0026thinsp;7- but- 2- ynyl- 3- methyl1- [(4-methylquinazolin-2-yl) methyl] purine-2,6-dione. \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEmpagliflozin belongs to the class of diabetic medications known as sodium-glucose co-transporter 2 (SGLT-2). This exercise explains the use of Empagliflozin as a beneficial treatment for type 2 diabetes mellitus, including its indications, mode of action, and contraindications. The mechanism of action, dosage, and monitoring that are important for interprofessional team members to know when managing patients with type 2 diabetes will be highlighted in this exercise. Chemically, it is 4\u0026rsquo;-[(1,4\u0026rsquo;-dimethyl-2\u0026rsquo;-propyl[2,6\u0026rsquo;-bi-1Hbenzimidazole]-1\u0026rsquo;-yl) methyl] [1,1\u0026rsquo;- biphenyl]-2-carboxylicacid. \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eLiterature reveals various analytical method have been reported such as UV [\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e], HPLC [\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e], HPTLC [\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e] for estimation of Linagliptin and Empagliflozin. The purpose of the present work is to establish and implement a new RP-HPLC method for determination of Linagliptin and Empagliflozin in bulk and pharmaceutical dosage form adding analytical QbD principles. Application of QbD principle for analytical method development has been practiced quite popular for attaining high robustness and improving method performance. The QbD method was based on the principles and approaches of guideline ICH Q8 pharmaceutical development, ICH Q9 quality risk management and ICH Q10 pharmaceutical quality system \u003csup\u003e[\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29 CR30 CR31\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS:","content":"\u003cp\u003e\u003cstrong\u003eMaterial:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA gift sample of Linagliptin and Empagliflozin was provided by Cubic Analytical Solution, Gujarat.\u0026nbsp;HPLC grade methanol and acetonitrile were bought from Finar life science private limited, Ahmedabad, India.\u0026nbsp;HPLC grade water was ordered from Loba chemise private limited, Mumbai, India.\u0026nbsp;The marketed formulations GLYXAMBI, 5 mg Linagliptin and 10 mg Empagliflozin, are by Boehringer Ingelheim private limited, Gujarat, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstruments and Reference Standard:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HPLC study was carried out on Noavatoz C18 (250 x 4.6mm, 5\u0026micro;m) was used at room temperature. Other equipment Shimadzu HPLC instrument (LC_2010 CHT) [software LC solution, equipped with UV detector, Auto-sampler], UV visible spectrophotometer double beam 1800 (Shimadzu), Electronic analytical balance (Acculab ALC210.4, Huntingdon Valley, PA), Ultra sonicator (EN 30 US, Enertech fast clean, Mumbai, India) QbD Software: Design Expert software 13.0 (Trial Version)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Standard stock solution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeighed 50 mg of Linagliptin and 100 mg of Empagliflozin.\u0026nbsp;Transferred to a 100 mL flask and diluted with methanol to make 500 \u0026mu;g/mL Linagliptin and 1000 \u0026mu;g/mL Empagliflozin.\u0026nbsp;Diluted 1 mL of this solution to 10 mL for 50 \u0026mu;g/mL Linagliptin and 100 \u0026mu;g/mL Empagliflozin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of sample stock solution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty tablets of Glyxambi 5 mg / 10 mg were weighed and powdered. The powder equivalent to 10 mg Empagliflozin and 5 mg Linagliptin was mixed with methanol in a volumetric flask. The solution was shaken, sonicated, filtered, and diluted to obtain specific concentrations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod validation\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eDeveloped method was validated according to ICH Q2 (R1) guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eComparison\u0026nbsp;of\u0026nbsp;the\u0026nbsp;chromatograms\u0026nbsp;of\u0026nbsp;the\u0026nbsp;standard\u0026nbsp;and\u0026nbsp;test\u0026nbsp;solutions\u0026nbsp;allowed\u0026nbsp;researchers to assess the method\u0026rsquo;s specificity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAliquots of working standard solutions (0.5, 1, 1.5, 2, 2.5 mL) were transferred into series of 10 mL volumetric flask and diluted up to mark with methanol. These yielded solution of concentration 2.5-12.5 \u0026mu;g/mL of Linagliptin and 5-25 \u0026mu;g/mL of Empagliflozin. An aliquot of 20 \u0026mu;L of each solution was injected under operating chromatographic condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIntraday standard solutions of Linagliptin and Empagliflozin at 5 \u0026mu;g/mL, 7.\u0026nbsp;5 \u0026mu;g/mL, 10 \u0026mu;g/mL and 10 \u0026mu;g/mL, 15 \u0026mu;g/mL, 20 \u0026mu;g/mL were analyzed three times in one day.\u0026nbsp;Interday solutions were analyzed over three different days, measuring absorbance and calculating % RSD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy: \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccuracy was determined by calculating the recovery of Linagliptin and Empagliflozin using the standard addition method.\u0026nbsp;Known amounts of Linagliptin (4 \u0026mu;g/mL, 5 \u0026mu;g/mL, and 6 \u0026mu;g/mL) and Empagliflozin (8 \u0026mu;g/mL, 10 \u0026mu;g/mL, 12 \u0026mu;g/mL) were added to their sample solution in a volumetric flask.\u0026nbsp;Each solution was diluted with methanol, injected three times, and recovery was calculated using a regression equation from the calibration curve by measuring peak areas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimit of Detection and Limit of Quantification:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LOD and LOQ were calculated based on the standard deviation of the response (y intercepts of regression lines) and the slope using 3 independent analytical curves, as defined by ICH.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLOD \u0026nbsp;and \u0026nbsp;LOQ \u0026nbsp; were calculated \u0026nbsp;as \u0026nbsp;3.3 \u0026nbsp; \u0026sigma;/S \u0026nbsp;and \u0026nbsp;10 \u0026nbsp; \u0026sigma;/S, \u0026nbsp;respectively, where \u0026sigma; is the standard deviation \u0026nbsp;of Y intercept (ICH \u0026nbsp;guidelines) \u0026nbsp;and \u0026nbsp; S \u0026nbsp;is \u0026nbsp;the \u0026nbsp; slope \u0026nbsp;of \u0026nbsp;the Linagliptin and Empagliflozin calibration curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe robustness study was carried out to assess the impact of subtle but intentional\u0026nbsp;changes to the chromatographic condition. The robustness was tested by making four\u0026nbsp;minor\u0026nbsp;adjustments.\u0026nbsp;Flow rate (1 \u0026plusmn; 0.1 mL/min), Mobile phase (90 \u0026plusmn; 5 mL), Injection volume (20 \u0026plusmn; 5 \u0026mu;L)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of marketed formulation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn aliquot of 20 \u0026mu;L from sample solution was injected under a chromatographic condition and peak area was measured and % assay was calculated from regression equation. Three determinations\u0026nbsp;on\u0026nbsp;average were\u0026nbsp;made\u0026nbsp;in\u0026nbsp;response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod Optimization by QbD Approach\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImplementation of QbD approach\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following experimental study was planned and the QbD technique was used in accordance with ICH Q8 (R2) principles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod performance requirements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing a QbD method, it is important to fully document the goals or execution needs of the technique and to consider how the strategy will be used.\u0026nbsp;This involves the Analytical Target Profile (ATP), which evaluates the active pharmaceutical ingredient (API) using chromatographic and/or spectrophotometric methods.\u0026nbsp;ATP is similar to the Quality Target Product/Process Profile (QTPP) for the pharmaceutical process.\u0026nbsp;ATP consists of models outlining what will be measured, the grid used, the concentration range, and the required method performance metrics, known as Critical Quality Attributes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod development:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the characterization of the ATP, an appropriate protocol and technique condition should be selected in order to satisfy all of the ATP\u0026apos;s requirements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod understanding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on a risk assessment, an exercise focused on comprehending the approach can be conducted to better grasp the potential impact of important information components on the presentation qualities of the technique. This allows for the recognition of certain functional technique controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Risk assessment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the practical relationship between method input components and all of the attributes of strategy execution, investigations might be hurried.\u0026nbsp;Data gathered during events and the start of the strategy\u0026apos;s application provides input into a risk assessment (using tools like the Fishbone chart, FMEA, and Ishikawa Diagram), which may determine which factors need consideration and which require controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesign of experiments:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesign of Experiment (DoE) is used for power testing of parametric factors to achieve the best setup while reducing the number of tests.\u0026nbsp;It evaluates proxy measures, like precision, based on the method type.\u0026nbsp;It explains critical parameters by showing how influential limits affect strategy implementation after a risk assessment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod design output:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo meet the ATP, several procedure conditions will have been developed and improved based on their impact on the plan.\u0026nbsp;Assessing a scientific strategy using QbD requires examining key characteristics affecting selectivity.\u0026nbsp;Continuous variable testing is impractical, so some authors use statistical methods like Placket-Burman to reduce exploratory costs.\u0026nbsp;Mechanized strength testing is used in some methods.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eOptimization phase:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is necessary to enhance peak symmetry and reduce tailing from the chromatographic conditions by adopting an appropriate experiment design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of factors:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVariables affecting separation procedures include mobile phase composition, flow rate, temperature, stationary phase, and injection volume.\u0026nbsp;For the study, mobile phase composition, flow rate, and injection volume were significant for retention and peak shape.\u0026nbsp;The mobile phase used was Methanol: Acetonitrile (90:10 %v/v).\u0026nbsp;The upper and lower levels of the mobile phase were chosen to adjust polarity for optimal retention time.\u0026nbsp;Flow rate and mobile phase also needed optimization to improve system suitability parameters like the number of theoretical plates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of level:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn most cases, components were examined at least twice (-1, +1) during optimization designs.\u0026nbsp;Factor levels for method optimization are chosen from existing knowledge and literature, often covering the broadest range for the factor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of optimization design\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the response-surface design process, key components were examined.\u0026nbsp;Central composite design can determine the best conditions with fewer runs, saving time and money.\u0026nbsp;Below is a list of tests for two factors and two levels for various experimental designs.\u0026nbsp;Formula for CCD: 2 (K) + 2K + C, where K = No.\u0026nbsp;of Factors, C = No.\u0026nbsp;of Center points.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSelection of response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResponse can be chosen for peak tailing, retention time, and chromatographic separations, among other things. Actually, it is important to select optimization conditions so that the Rt values of the components do not conflict with those of impurities or degradation products. The peak tailing must be less than 1.5 at that moment. The optimization of both response variables must have more theoretical plates and better suitability parameters (Table1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Software-aided Method Optimization\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.5e+2pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eStandard Experiment No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRun\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFactor 1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA: Mobile phase composition (mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFactor 2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB: Flow rate (mL/min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResponse 1 Retention time\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(min) Empagliflozin\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResponse 2 Retention time\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(min) Linagliptin\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResponse 3\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eResolution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.921\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.541\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.454\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.468\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.681\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.958\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.715\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.399\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.697\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.636\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.789\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e82.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.715\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.651\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.457\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.248\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.446\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.782\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.459\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMethod performance can be improved throughout the method development. The analyst has opportunities to placed inventive approaches to improve quality. Process performance can be monitored to make sure consistency in quality. The QbD approach avails the continuous improvement throughout the method life cycle (Fig. 1A, 1B, and 1C). The Anova response of reduced model were shown in table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: ANOVA responses for Model\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.3e+2pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eANOVA for response Retention time of Empagliflozin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSum of Squares\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003edf\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean Square\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eF-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\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\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.5782\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.1156\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e80.95\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003esignificant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA-Mobile Phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0584\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0584\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e40.90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0004\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\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\u003e\n \u003cp\u003e\u003cstrong\u003eB-Flow Rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.4509\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.4509\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e315.64\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\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 colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eANOVA for response Retention time of Linagliptin \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.80\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.3590\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e196.68\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003esignificant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA-Mobile Phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0230\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0230\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e12.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0093\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\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\u003e\n \u003cp\u003e\u003cstrong\u003eB-Flow Rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.40\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e767.96\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\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 colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eANOVA for reduced model for response resolution \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e13.19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2.64\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e38.69\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSignificant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA-Mobile Phase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e6.45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e6.45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e94.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\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\u003e\n \u003cp\u003e\u003cstrong\u003eB-Flow Rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.81\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1.81\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e26.53\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;0.0013\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eContinuous Improvement throughout Product Life Cycle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Linagliptin and Empagliflozin standard solution of 5 \u0026mu;g/mL and 10 \u0026mu;g/mL were injected in six replicates. The Standard and sample chromatogram of Linagliptin and Empagliflozin was shown in the figure 2a and 2b. The retention time of Linagliptin was 4.160 min and 2.889 min for Empagliflozin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe working concentration 2.5-12.5 \u0026micro;g/mL for Linagliptin and 5-25 \u0026micro;g/mL for Empagliflozin. The calibration curve was plotted for the response (area) and concentration (amount) and the correlation coefficient (r\u003csup\u003e2\u003c/sup\u003e) and y-intercept were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntraday precision (repeatability) of a standard solution with (5,7.\u0026nbsp;5,10\u0026micro;g/mL) Linagliptin and (10,15,20 \u0026micro;g/mL) Empagliflozin was tested three times in one day.\u0026nbsp;Interday precision of the same standard solution was analyzed over three different days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe % recovery studies involved spiking Linagliptin and Empagliflozin at 80%, 100%, and 120% concentrations in triplicates.\u0026nbsp;Chromatograms were taken for each spiked sample, and the total drug amount was calculated to find % recovery. (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Accuracy data of developed method validation\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.2e+2pt;\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eLinagliptin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003elevel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmount of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003etest solution (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmount of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003estandard added \u0026nbsp; \u0026nbsp;(\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmount\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003efound* \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% Recovery*\u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.95\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.48\u0026plusmn;0.39\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9.97\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.70\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e120%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.97\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.72\u0026plusmn;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eEmpagliflozin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17.95 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.78 \u0026plusmn; 0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19.96 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.80 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e120%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21.95 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.76 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eLimit of Detection (LOD) and Limit of Quantification (LOQ)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LOD and LOQ were obtained by successively decreasing the concentration of Linagliptin and Empagliflozin as long as a signal to noise ratio of not less than 3:1 and 10:1 is maintained, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe flow rate, mobile phase composition, and injection volume are the usual variations investigated under these parameters; the findings are displayed in table 3. The approach was proven to be robust because the variation was within an acceptable range with regard to peak asymmetry and theoretical plates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Robustness data of developed method validation\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.1e+2pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCondition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVariation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePeak area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRetention time\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTailing factor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\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; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Linagliptin\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMobile \u0026nbsp; \u0026nbsp; phase (90 \u0026plusmn; 5 mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e95:5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e468209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.473\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.308\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e100 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e468100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.302\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlow rate (0.9 \u0026plusmn; 0.1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003emL/min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9mL/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e642001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.318\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1.1mL/min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e471463\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.314\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eInjection volume (20 \u0026plusmn; 5 \u0026micro;L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e498410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e498408\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.315\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eApplicability of developed method to Marketed formulation \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy analysing the GLYXAMBI tablet, which is used in industry, the proposed method\u0026apos;s applicability was evaluated. The results are shown in table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Analysis data of marketed formulation\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.2e+2pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTablet\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003emg/tablet(Label claim)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmount recovered (mg) \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e%Assay* (% of label claim) \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLina\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEmpa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLina\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEmpa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e%Lina\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e%Empa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLina\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eEmpa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGlyxambi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.02\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10.03\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.03\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.04\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe assay results were comparable to labelled value of each drug in tablet dosage form. These results indicate that the developed method is accurate, precise, simple and rapid.\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe centre composite design was applied for three factors at three levels with use of the Design Expert software trial version 13.0. Design of experimental results, including ANOVA, diagnostic graphs and model graphs, were examined for each factor. The effect of each factor on the response result was investigated in this results part. The solvent system (Methanol: Acetonitrile 90: 10%v/v), flow rate (1 mL/min), and UV wavelength at 225 nm was found to be the optimized chromatographic condition for Linagliptin and Empagliflozin. Linearity data shows that the developed method was linear in the range of 2.5\u0026ndash;12 \u0026micro;g/mL of Linagliptin and 5\u0026ndash;25 \u0026micro;g/mL of Empagliflozin with 0.993 and 0.991 correlation coefficient of Linagliptin and Empagliflozin respectively. The % RSD was less than 2% in intraday and interday precision study of proposed method and % recovery was found to be 99.48\u0026ndash;99.72 and 99.76\u0026ndash;99.80 for Linagliptin and Empagliflozin respectively. Limit of detection and limit of Empagliflozin was determined to be 0.281 \u0026micro;g/mL and 0.873 \u0026micro;g/mL for Linagliptin and 0.186 \u0026micro;g/mL and 0.566 \u0026micro;g/mL for Empagliflozin. Applicability of developed method for quantification of Linagliptin (tR\u0026thinsp;=\u0026thinsp;4.174 min) and Empagliflozin (tR\u0026thinsp;=\u0026thinsp;2.900 min) in tablet dosage form was shown the absence of any wanted peak and no change in retention time, resolution and peak symmetry.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eAn analytical quality by design (Central Composite Design) approach was successfully employed for the optimization of the RP-HPLC method. The developed RP-HPLC was specific, accurate and precise method as per ICH guidelines. Hence, the method can be successfully used for the simultaneous estimation of Linagliptin and Empagliflozin in pharmaceutical dosage form in routine quality control testing.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eANOVA\u003c/strong\u003e: Analysis of variance, \u003cstrong\u003eAQbD:\u003c/strong\u003e analytical quality by design, \u003cstrong\u003eCMA\u003c/strong\u003e: Critical material attributes, \u003cstrong\u003eCCD:\u003c/strong\u003e Centre composite design, \u003cstrong\u003eICH\u003c/strong\u003e: International Conference on Harmonization, \u003cstrong\u003eLOD:\u0026nbsp;\u003c/strong\u003eLimit of Detection, \u003cstrong\u003eLOQ\u003c/strong\u003e: Limit of Quantitation, \u003cstrong\u003eRSM:\u0026nbsp;\u003c/strong\u003eResponse surface method, \u003cstrong\u003eRP-HPLC:\u003c/strong\u003e reverse phase high performance liquid chromatography.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS DECLARATION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING STATEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo Funding was received to assist with the preparation of this manuscript \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors express their sincere gratitude to shri Sarvajanik pharmacy college, Mehsana, Gujarat, India for continuous motivation, support and guidance for research activity and for providing all required facilities to accomplish the entitled work.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026apos; CONTRIBUTION STATEMENT:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u0026nbsp;\u003c/strong\u003eKhushbu Patel, Zeel modi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e Zeel modi, Chhaganbhai. N. Patel\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u0026nbsp;\u003c/strong\u003eKhushbu Patel, Chhaganbhai. N. Patel\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManuscript writing:\u0026nbsp;\u003c/strong\u003eKhushbu Patel, Zeel modi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisulization:\u0026nbsp;\u003c/strong\u003eKhushbu Patel, Chhaganbhai. N. Patel\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u0026nbsp;\u003c/strong\u003eKhushbu Patel\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject Administration:\u003c/strong\u003e Khushbu Patel\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDrugs.com, Linagliptin Dosage, November 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.drugs.com/dosage/linagliptin.html\u003c/span\u003e\u003cspan address=\"https://www.drugs.com/dosage/linagliptin.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThakkar Kinjal K, Patel UB, Patel DCN, Patel. Evogliptin tartrate a new drug of DPP-4 inhibitor: an overview. World J Pharm Res. 2021;10(5):1921\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePubchem L. November 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/Linagliptin\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/compound/Linagliptin\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmpagliflozin Dcom. November 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.drugs.com/mtm/empagliflozin.html\u003c/span\u003e\u003cspan address=\"https://www.drugs.com/mtm/empagliflozin.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmpagliflozin Dbank. November 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/Empagliflozin#section=DrugIndication\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/compound/Empagliflozin#section=DrugIndication\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSri K, Vijaya, Anusha A. UV-Spectrophotometry method for the estimation of Linagliptin in bulk and pharmaceutical formulations. Asian J Res Chem. 2016;9:47\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmruta Dalal and Rajashri Waghmode. Development and validation of a simple and rapid Uv Spectrophotometeric method for Linagliptin in bulk and marketed dosage form. Res - Der Pharm Chem. 2022;14:23\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeepika Joshi and Nidhi Semwal. Analytical method development and validation of UV-Visible Spectrophotometric method for the estimation of Linagliptin. Glob J Nano. 2021;5:30\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManish Mishra and Govinda Verma. Analytical method development and Validation for determination of Linagliptin in bulk and dosage form by UV Spectroscopy. J Emerg Tech Innovative Res. 2018;5:908\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatil Sushil D, Chaure Sayali K. Development and Validation of Simple UV-Spectrophotometric Method for the Determination of Empagliflozin. Asian J Pharm Anal. 2017;7:18\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenazir Shaik Bima and Archana Jorige. Method development and validation of Empagliflozin in bulk and pharmaceutical dosage form using UV spectroscopy. Asian J Pharm Anal. 2021;11:123\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLakshman Raju Badugu. A validated RP-HPLC method for the determination of Linagliptin. Am J Pharm Tech Res. 2012;2:463\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel K, Shah UA, Joshi HV, Patel JK, Patel CN. QbD Stressed development and validation of stability-indicating RP-HPLC method for the simultaneous estimation of linagliptin and metformin HCl in pharmaceutical dosage form. Res J Pharm Tech. 2022;15(5):1917\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoy Chandra Rajbangshi and Md. Mahbubul Alam. Development and Validation of a RP-HPLC Method for Quantitative Analysis of Linagliptin in Bulk and Dosage Forms. Dhaka Univ J Pharm Sci. 2018;17:175\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbeer Hanafy and Hoda Mahgoub. A Validated HPLC Method for the Determination of Linagliptin in Rat Plasma. Application to a Pharmacokinetic Study. J Chrom Scie. 2016;54:1573\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiridevi Mounika P, Kumar Hemant. T.RP-HPLC Method for Quantification of Empagliflozin in Pharmaceutical Formulation. Asian J Pharm Tech. 2019;9:208\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatil Sushil D, Dr. Amurutkar Sunil V. Development and Validation of Stability Indicating RP-HPLC Method for Empagliflozin. Asian J Pharm Tech. 2016;6:201\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnas M. Hanif and Rabia Bushra. Empagliflozin: HPLC based analytical method development and application to pharmaceutical raw material and dosage form. Pak J Pharm Sci. 2021;3:1081\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVankalapati KR, Alegete P, Boodida S. Stability-indicating ultra performance liquid chromatography method development and validation for simultaneous estimation of metformin, linagliptin, and empagliflozin in bulk and pharmaceutical dosage form. Biomed Chromatogr. 2021, 35e5019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRizk M, Attia AK, Mohamed HY, Elshahed MS. Validated Voltammetric Method for the Simultaneous Determination of Anti-diabetic Drugs, Linagliptin and Empagliflozin in Bulk. Pharm Dosage Forms Biol Fluids Electroanalysis. 2020;32:1737.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao Knv, Shirisha V. A New Simple Method Development, Validation and Forced Degradation Studies of Empagliflozin by Using RP-HPLC. Int J Pharm sci. 2019;9:25\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhole RP, Wankhede SB. High performance thin layer chromatographic determination of linagliptin in pharmaceutical formulations and in biological samples. Int J Pharm Chem Anal. 2017;4:13\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManojkumar Munde and Nilesh Kulkarni. A novel validated stability indicating method for quantification of Empagliflozin in bulk and marketed formulation by hptlc applying experimental design approach. Indian Drugs. 2023;60:66\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhatt DA, Rane SI. QbD approach to analytical RP-HPLC method development and its Validation. Int J Pharma Pharma Sci. 2011;3(10):179\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJadhav ML, Tambe SR. Implementation of QbD approach to analytical method development and validation for the estimation of Propafenone Hydrochloride in tablet dosage form. Chrsom Res Int. 2013;4(11):120\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePriya B. Shah Khushbu Patel. Qbd \u0026ndash; A novel setup for the analytical method development. World J Pharm Pharm Sci. 2021;10(5):1645\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJinal A, Goswami ZM, Modi K, Patel, Dr CN, Patel. Review on Implementation of Quality by Design. Int J Innovative Sci Res Technol. 2024;9(2):544\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDivyesh Sharma K, Patel, Patel DCN. QbD-A new era of Pharmaceutical drug development European. J Pharm Med Res. 2021;8(5):688\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoy S. Quality by design: A holistic concept of building quality in pharmaceuticals. Int J Pharma Bio Res. 2012;3(2):100\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel VP, Shihora HD. Experimental Design \u0026amp; Patent; 1st Edn; Akshat publication, 2011, 1\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInternational Conference on Harmonization (ICH) Validation of Analytical Procedure: Text and Methodology Q2 (R1). 2005, 780\u0026ndash;798.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eICH Harmonized Tripartite Guideline. Pharmaceutical Development Q8, Q9 and Q10 International Conference on harmonization, Geneva, Switzerland,2003:1\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Anti diabetic, Dipeptidyl peptidase-4, Sodium-glucose co-transporter 2, Validation, Recovery","lastPublishedDoi":"10.21203/rs.3.rs-9035757/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9035757/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study reports the development of a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Linagliptin and Empagliflozin in pharmaceutical dosage forms using a Quality by Design (QbD) approach. In the QbD strategy, a central composite design (CCD) was employed to evaluate the influence of critical method parameters, particularly the composition of the mobile phase and the flow rate. The interaction effects of these parameters on critical responses, including retention time and resolution of Linagliptin and Empagliflozin, were analyzed using three-dimensional response surface plots. These plots helped to establish the optimal chromatographic conditions for the developed method. The proposed method is simple, precise, and accurate for the estimation of Linagliptin and Empagliflozin. Chromatographic separation was achieved using a C18 column (NOVATOZ C18, 250 \u0026times; 4.6 mm, 5 \u0026micro;m) with gradient elution. The mobile phase consisted of Methanol and Acetonitrile (90:10, v/v) delivered at a flow rate of 1 mL/min. Detection of analytes was performed using a photodiode array (PDA) detector at 225 nm. Linearity was observed over the concentration range of 2.5\u0026ndash;12.5 \u0026micro;g/mL for Linagliptin and 5\u0026ndash;25 \u0026micro;g/mL for Empagliflozin, with correlation coefficients (r\u0026sup2;) of 0.993 and 0.991, respectively. The developed method was successfully applied for the quantitative analysis of Linagliptin and Empagliflozin in pharmaceutical dosage forms.\u003c/p\u003e","manuscriptTitle":"Center Composite Design Assisted Optimization of Rp-hplc Method for Simultaneous Estimation of Linagliptin and Empagliflozin Using Aqbd Perspective","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 16:50:34","doi":"10.21203/rs.3.rs-9035757/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-08T08:25:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-04T11:11:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T13:31:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80885681492242817117352441941123025658","date":"2026-03-30T05:59:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-28T05:11:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21512551814252895524992997490469254417","date":"2026-03-26T17:20:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-26T11:07:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160844743020900340827934149472722897143","date":"2026-03-25T03:33:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50217590133173841252769923817956252061","date":"2026-03-24T20:36:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21763177665663998691438116577064808439","date":"2026-03-18T02:42:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T12:32:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-15T06:37:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-14T09:49:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-14T06:50:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2026-03-14T06:47:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd474689-d0f7-48f7-9f26-3097805d007b","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T12:25:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 16:50:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9035757","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9035757","identity":"rs-9035757","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}