Eco-Friendly UV Spectroscopic and RP-HPLC Methods for Simultaneous Estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan: A DOE-Optimized Approach with Greenness and Whiteness Assessment

Eco-Friendly UV Spectroscopic and RP-HPLC Methods for Simultaneous Estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan: A DOE-Optimized Approach with Greenness and Whiteness Assessment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Eco-Friendly UV Spectroscopic and RP-HPLC Methods for Simultaneous Estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan: A DOE-Optimized Approach with Greenness and Whiteness Assessment VISHWA VYAS, DR. DILIP MAHESHWARI, Dr. Darshil Shah, Dr. Jignesh Shah This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6433370/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract An accurate, simple and environment friendly Ultraviolet-visible spectroscopic method and Reverse phase High performance liquid chromatography method was established for simultaneous estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan in their pharmaceutical dosage form. Ultraviolet-visible spectroscopic method utilized the simultaneous equation method using 251 nm and 296 nm wavelengths for Efonidipine Hydrochloride Ethanolate and Telmisartan, respectively. Reverse phase high performance liquid chromatography was optimizing with the help of Central Composite Design which is a Design of Experiment approach, to accomplish optimal separation of both drugs with using minimum solvent. Optimal chromatographic separation was obtained by Shimandzu C18 column (250 × 4.6 mm, 5 µm) with Mobile phase which comprising Ethanol: Acetonitrile: Water (70:20:10%v/v) at pH 4, flow rate 1 ml/min at detection wavelength 250 nm. Both methods were validated as per ICH Q2 (R1) guideline, showing linearity, precision, accuracy, limit of detection, limit of quantification. Assessment of greenness and whiteness score, ensures that methods have environmental sustainability and analytical efficiency. Telmisartan Efonidipine Hydrochloride Ethanolate RP-HPLC UV Central Composite Design Greenness and Whiteness Assessment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Hypertension is a chronic condition in which blood pressure in the arteries remains high. On the ranking of the determinants of the disease burden across life, It is placed third in terms of disability-adjusted life years.[ 1 ] Numerous medications in various forms can be used to treat the hypertension. Calcium channels blockers, Diuretics, Angiotensin II receptor blockers, Angiotensin-converting enzyme inhibitors are classes of drugs which are used to treat the hypertension.[ 2 ] Efonidipine Hydrochloride Ethanolate (EFD) chemically referred as 2-( N -benzylanilino)ethyl 5-(5,5-dimethyl-2-oxo-1,3,2λ 5 -dioxaphosphinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylate;ethanol;hydrochloride.[ 3 ] It is a dihydropyridine (DHP) calcium channel blocker. It shows antihypertensive impact through vasodilatation by blocking T-type and L-type calcium channels.[ 4 ] Telmisartan (TEL) chemically referred as 2-[4-[[4-methyl-6-(1-methylbenzimidazol-2-yl)-2-propylbenzimidazol-1-yl]methyl]phenyl]benzoic acid. Angiotensin II produced by a catalytic reaction between Angiotensin I and Angiotensin-converting enzyme (AT1), is the primary presser agent of the renin-angiotensin system, causing vasoconstriction, aldosterone release, cardiac stimulation, and sodium renal reabsorption. Telmisartan selectively blocks its binding to AT1 receptors and exhibit vasodilation.[ 5 ] Choosing High-Performance liquid chromatography (HPLC) and UV-Visible (UV-Vis) Spectroscopy for research provides a robust platform for analysis. UV-Vis Spectroscopy is a cost-effective, simple and less time consuming. It is perfect for routine analysis while HPLC provides precise and accurate analysis of mixtures. It has high-resolution capabilities. Both techniques are ideal for identify the components in pharmaceutical compositions and improves the depth and reliability of research outcomes.[ 6 ] Green Analytical Chemistry (GAC) enhance analytical methods through reducing the amount of waste, Improving the efficiency of energy using, using green solvent which are not harmful to humans and environment and making a method eco-friendly. [ 7 , 8 ] White Analytical Chemistry (WAC) builds on the idea of GAC. It is a cost-effective, efficient and reproducible analytical approach. It contains three main aspects: Red (analytical efficiency), Green (environmental impact), Blue (Practicality).[ 9 ] Design of Experiments (DOE) is a methodical approach that searches into the corelation between numerous input factors and its effects on intended output responses. Central Composite Design (CCD) is the significant method in DOE. It is a valuable in developing analytical method, like improving sustainability and stability of HPLC method for determining pharmaceutical compounds.[ 10 ] According to literature survey, a various analytical methods have been reported for the analysis of EFD and TEL. [ 3 , 5 , 11 – 23 ] Nevertheless, there are no UV-vis Spectroscopic and HPLC techniques have been reported with greenness and whiteness assessment of EFD and TEL. The main objective of this research is to develop and validate a precise and eco-friendly UV-vis Spectroscopic and DOE assisted RP-HPLC which can replace the hazardous and costly method for determination of EFD and TEL in their pharmaceutical dosage form. Materials and Reagents A gift sample of EFD was provided by Zuventus healthcare ltd., Pune. TEL gift sample was provided by Zydus lifesciences, Ahmedabad. Combination product of EFD and TEL (Label claim: 40 mg EFD and 40 mg TEL) EFNOCAR-T Manufactured by Zuventus healthcare ltd was bought by local pharmacy. Acetonitrile, Ethanol, methanol and water (HPLC grade) acquired from Thermo Fisher Scientific India Pvt. Ltd. The supplier of Orthophosphoric acid was ACS Chemicals in India. Apparatus and Software: The study was carried out by UV- Visible spectrophotometer (Shimadzu model 1800 (Japan)) with software UV Probe, HPLC was completed with a Shimandzu HPLC system with model LC-2010 CHT fitted with the UV detector and serial dual plugger pump with software Labsolution version 5.52. Chromatographic separations were done by Shimadzu C 18 (250 × 4.6 mm, 5 µm) column. For Design of experiments (Central Composite Design) Design-Expert v13.0.12.0 by Stat Ease was used. For weighing purpose, analytical weight balance (Wensar electronic balance) was used. Prior to use, every piece of glasswares were calibrated. Preparation of Mobile phase: 70 ml of ethanol, 20 ml of acetonitrile and 10 ml of water were taken and mixed with the use of sonicator. pH 4 adjusted with the 1% Orthophosphoric acid. Preparation of 1% Orthophosphoric acid: 1 ml of orthophophoric acid dissolved in 100 ml of water. Preparation of Standard Stock Solution: For UV-vis Spectroscopic technique, 40 mg of TEL and EFD dissolved in 100 ml of Methanol to make standard stock solution of TEL (400 µg/ml) and EFD (400 µg/ml), respectively. After that, Methanol was added in the standard solution to make a various concentrations 10–60 µg/ml for TEL and 10–60 µg/ml for EFD. For HPLC technique, 40 mg of TEL and EFD dissolved in 100 ml of Ethanol to make standard stock solution of TEL (400 µg/ml) and EFD (400 µg/ml), respectively. After that, the solution of standard was diluted with Mobile phase to make a various concentrations 10–60 µg/ml for TEL and 10–60 µg/ml for EFD. UV-Visible Spectroscopic method: Simultaneous equation approach: TEL and EFD mixture is present in ratio of 1:1, respectively. Using methanol as a solvent, spectra of absorption of API and their mixture were measured in range 200 to 400 nm. Absorbance of TEL and EFD at 296 nm and 251 nm respectively. This method is evaluated by below formula:[ 3 ] $$\:Cx=\left(\frac{{A}_{2}a{y}_{1}-{A}_{1}a{y}_{2}}{a{x}_{2}a{y}_{1}-a{x}_{1}a{y}_{2}}\right)$$ $$\:{C}_{y}=\left(\frac{{A}_{1}a{x}_{2}-{A}_{2}a{x}_{1}}{a{y}_{1}a{x}_{2}-a{y}_{2}a{x}_{1}}\right)$$ where, Cx = Concentration of TEL, Cy = Concentration of EFD, A 1 = Absorbance of test at λ 1 (λ max of TEL), A 2 = Absorbance of test at λ 2 (λ max of EFD), ax1 = Absorptivity of x drug (TEL) at λ1, ax2 = Absorptivity of x drug (TEL) at λ2, ay1 = Absorptivity of y drug (EFD) at λ1, ay2 = Absorptivity of y drug (EFD) at λ2. Optimized Chromatographic condition for HPLC: The Reverse phase technique was used for developing RP-HPLC method with a Shimadzu C 18 (250 × 4.6 mm, 5 µm) column. Mobile phase comprised of Ethanol: Acetonitrile: Water (70:20:10%v/v) with pH 4 adjusted with orthophosphoric acid. Detection was carried out at 250nm wavelength with a flow rate 1 ml/min. Run time was 10 min and injection volume was 10 µl. Experimental Design: Faced central composite design (FCCD) was used for the optimization of pH, % ethanol and flow rate for the effective separation. FCCD included five replicates including axial and center points. Using Derringer’s desirability, we evaluated the R2 coefficient for determining polynomial models to access the optimize condition. Results and Discussion Method development and optimization: RP-HPLC method was developed by application of DOE to explore the three independent variables: pH, Flow rate and %ethanol in the mobile phase. 250 nm of wavelength was selected by the analysis of UV–vis overlay spectra of EFD and TEL drugs. Chromatographic separation of compounds was done by using a composite ratio of ethanol (70%) at a pH 4 and flow rate 1 ml/min. The present study emphasizes on studying tailing factor and retention time in 15 experiments. The effective range for flow rate between 0.8 to 1.2 ml/min, pH between 3.5 to 4.5 and % ratio of ethanol is 65 to 75%v/v. Applying CCD finds optimal conditions and gives model’s suitability. Table 3 shows indicate the regression model and statistical parameters obtained by ANOVA which shows that factors affecting the tailing factor and retention time for TEL and EFD respectively. P-value is less than 0.5 which indicates that models were significant. Adjusted R 2 for both drugs was greater than 0.8 which shows that drugs both drugs provide linear response. Table 1 HPLC independent variables Factor Name Level (-) Level (0) Level (+) A Flow rate(ml/min) 0.8 1 1.2 B pH 3.5 4 4.5 C Ethanol 65 70 75 Table 2 Conditions and responses of HPLC Design Points Factor Level Responses Factor 1 Flow rate(ml/min) Factor2 pH Factor 3 Concof Ethanol Tailing Factor of TEL Retention time of EFD 1 1 4 75 1.301 5.56 2 0.8 4.5 75 1.234 5.671 3 1 4 65 1.38 6.74 4 1.2 4.5 65 1.24 6.59 5 1.2 4.5 75 1.18 5.78 6 1 4 70 1.322 6.264 7 1 4.5 70 1.201 6.31 8 0.8 3.5 65 1.427 6.93 9 1.2 3.5 65 1.43 6.67 10 1 3.5 70 1.41 6.19 11 0.8 3.5 75 1.39 5.76 12 1.2 4 70 1.319 6.024 13 0.8 4 70 1.31 6.29 14 0.8 4.5 65 1.25 6.87 15 1.2 3.5 75 1.383 5.42 Table 3 Statistical paramters and regression model which are based on ANOVA Res pons e Regression Model Adjusted R2 Model P-Value % C.V Adequa te Precisio n Predicte d R2 F valu e TF + 1.32-0.059A-0.0935B-0.0239C-0.0075AB-0.0068AC + 0.010BC 0.9513 ˂0.0001 1.39 20.9782 0.9035 46.56 RT + 6.20-0.1037A + 0.0251B-0.5609C + 0.0536AB + 0.0386AC + 0.0514BC 0.9723 ˂0.0001 1.32 27.6001 0.8596 82.93 Derringer’s Desirability Function (D): It is a technique which optimize multiple objectives with multiple targets and responses. Design Expert 13 was used to optimize standards which focuses on retention time while method development. By utilizing a pH 4, flow rate 1 ml/min and %ethanol concentration (v/v), Total desirability value found to be 1 which indicates that these coordinates are the most appropriate parameters for recommended method. System suitability studies: The system suitability was assessed by six replicate analyses of EFD and TEL at concentration of 40 µg/ml. The column resolution, tailing factor, theoretical plates were analysed for standard solutions. Peak area for TEL was found to be 2435985 and Peak area for EFD was found to be 1106705. Retention time for TEL and EFD was found to be 2.288 and 6.264, respectively. Plate count for TEL and EFD was found to be 2094 and 8827 and tailing factor TEL and EFD was found to be 1.322 and 1.643, respectively. Resolution between both drugs were found to be 17.039. Method Validation: The suggested method was validated with regards to linearity, accuracy, limit of detection, limit of quantification, repeatability, intermediate precision based on ICH Q2(R1) guideline. Linearity: The linearity of TEL and EFD was established by scanning different concentrations at six levels. The solution of concentration in range of 10–60 µg/ml for both drugs for both UV-vis Spectroscopic and HPLC method. A graph of Concentration vs Absorbance and Concentration vs Peak area was plotted for UV-vis Spectroscopic and HPLC method respectively and regression equation was acquired. Results of TEL and EFD for both methods was displayed in Table 4 . UV-vis Spectroscopic overlay chromatograph was displayed in Fig. 5 (A) and Fig. 5 (B) of both drugs and it was found to be linear over 10–60 µg/ml with regression line equation y = 0.0361x + 0.0362 with r2 = 0.998 for EFD and y = 0.0437x + 0.2356 with r2 = 0.998 for TEL. (Fig. 6 (A) and 6(B)). HPLC overlay chromatogram was displayed in Fig. 5 (C)10–60 µg/ml with retention time 2.28 min and 6.264 min for TEL and EFD, respectively. Regression line equation y = 63083x-157627 with r2 = 0.9981 for TEL and y = 29313x-80692 with r2 = 0.9979 for EFD(Fig. 6 (C) and 6(D)).. Table 4 Linearity and Precision data of EFD and TEL by UV-vis Spectroscopy and HPLC Linearity Simultaneous equation UV-vis Spectroscopic Method TEL EFD Concentration (µg/ml) Absorbance ± SD %RSD Concentration (µg/ml) Absorbance ± SD %RSD 10 0.672 ± 0.003 0.553 10 0.394 ± 0.003 0.762 20 1.082 ± 0.004 0.412 20 0.771 ± 0.005 0.772 30 1.540 ± 0.006 0.409 30 1.114 ± 0.005 0.520 40 2.021 ± 0.014 0.729 40 1.467 ± 0.006 0.465 50 2.463 ± 0.013 0.554 50 1.842 ± 0.016 0.872 60 2.805 ± 0.018 0.666 60 2.208 ± 0.011 0.539 HPLC TEL EFD Concentration (µg/ml) Peak area ± SD %RSD Concentration (µg/ml) Peak area ± SD %RSD 10 492981 ± 3388 0.764 10 193544 ± 1084 0.560 20 1154859 ± 10758 0.931 20 525975 ± 3189 0.606 30 1718879 ± 11703 0.680 30 803897 ± 7516 0.935 40 2404176 ± 23026 0.957 40 1111288 ± 5390 0.485 50 2915219 ± 15452 0.530 50 1343184 ± 12496 0.930 60 3565496 ± 28028 0.764 60 1693629 ± 13287 0.784 Precision UV-vis Spectroscopic method HPLC method Drug Concentration (µg/ml) Mean absorbance ± SD %RSD Mean peak area ± SD %RSD Intraday precision (n = 3) TEL 10 0.673 ± 0.0051 0.762 497429 ± 2182 0.487 20 1.084 ± 0.0073 0.679 1165358 ± 6929 0.594 30 1.55 ± 0.0091 0.591 1729854 ± 9950 0.575 EFD 10 0.393 ± 0.0032 0.817 193079 ± 1044 0.540 20 0.775 ± 0.0056 0.733 526882 ± 3288 0.624 30 1.122 ± 0.0068 0.606 802822 ± 5568 0.693 Interday precision (n = 3) TEL 10 0.676 ± 0.0049 0.728 493564 ± 2986 0.673 20 1.086 ± 0.0070 0.652 1157278 ± 9669 0.835 30 1.552 ± 0.0117 0.759 1716061 ± 15861 0.924 EFD 10 0.394 ± 0.0032 0.814 193247 ± 1537 0.795 20 0.776 ± 0.0060 0.783 523821 ± 3413 0.651 30 1.128 ± 0.0105 0.934 802646 ± 6098 0.759 Repeatability (n = 6) TEL 20 1.081 ± 0.0044 0.412 1160386 ± 8463 0.729 EFD 20 0.771 ± 0.0059 0.772 525233 ± 3843 0.731 Precision: Intraday precision and intermediate precision (interday precision) were performed by estimating SD and %RSD for three times determinations of 3 concentrations within the range of linearity. Repeatability was performed by calculating SD and %RSD for the six times determinations of 1 concentration. Results of both methods for EFD and TEL were displayed in Table 4 . %RSD value of EFD and TEL for both methods were found to be less than 2 which indicates that method was precise. Accuracy: Accuracy was calculated by determining the % recovery of TEL and EFD at 80%, 100%, 120% in placebo through spiking API. Accuracy results for both methods were displayed in the Table 5 . %Recovery was observed between 99%-101% for both methods for EFD and TEL which shows that no interference are present due to excipients. Table 5 Accuracy, LOD, LOQ and Assay data of EFD and TEL by UV-vis Spectroscopic method and HPLC Accuracy UV-vis Spectroscopic method HPLC Drug Level Amount of sample taken (µg/ml) Amount of standard spiked (µg/ml) Total amount of drug Amount of standard recovery mean % Recovery (n = 3) ± SD Amount of standard recovery mean % Recovery (n = 3) ± SD TEL 80 20 16 36 36.52 101.4 ± 0.406 36.44 101.25 ± 0.232 100 20 20 40 40.45 101.1 ± 0.177 40.43 101.09 ± 0.162 120 20 24 44 44.55 101.2 ± 0.401 43.92 99.83 ± 0.205 EFD 80 20 16 36 35.87 99.6 ± 0.211 35.68 99.12 ± 0.387 100 20 20 40 39.61 99.0 ± 0.202 39.68 99.20 ± 0.490 120 20 24 44 43.72 99.3 ± 0.210 43.92 99.82 ± 0.411 LOD and LOQ UV-vis Spectroscopic method HPLC TEL EFD TEL EFD LOD 0.67 0.73 0.59 1.28 LOQ 2.03 2.21 1.81 3.90 Assay EFNOCAR-T UV-vis Spectroscopic method HPLC Label claim Amount found % assay ± SD Amount found % assay ± SD TEL 40 39.49 98.72 ± 0.300 39.52 98.80 ± 0.604 EFD 40 39.61 99.02 ± 0.149 39.68 99.22 ± 0.490 Limit of Detection (LOD) and Limit of Quantification (LOQ): LOD and LOQ was performed by analysing by three replicates solutions of standard which are containing TEL and EFD. LOD and LOQ results for both methods for TEL and EFD were displayed on Table 5 . LOD value for TEL was observed 0.67 and 0.59 for UV-vis Spectroscopic method and HPLC method, respectively. LOD value for EFD was observed 0.73 and 1.28 for UV-vis Spectroscopic method and HPLC method, respectively. LOQ value for TEL was observed 2.03 and 1.81 for UV-vis Spectroscopic method and HPLC method, respectively. LOQ value for EFD was observed 2.21 and 3.90 for UV-vis Spectroscopic method and HPLC method, respectively. Assay: It is performed on a marketed formulation to check %purity. Assay results for both methods for EFD and TEL displayed on Table 5 . Results of %Assay was found in the range of acceptance criteria 98–102%. Assessment of Greenness by Analytical Greenness Index (AGREE): AGREE is a green model which calculate the greenness based on 12 principles of GAC. Numerical value of AGREE demonstrated that how ecofriendly UV-vis Spectroscopic method and HPLC methods were. Final results are derived from sum of twelve input variables ranging from 0 to 1. A higher score indicates more environment friendly method was. Results for both methods shown in Fig. 7 (A) and 7(B). The greenness attributes of the developed UV-vis Spectroscopic method was 0.74 and HPLC method was 0.73 assessed using the AGREE evaluation. This indicates that the method aligns with the principles of GAC. Higher AGREE score of UV-vis Spectroscopic method shows that method was more environment friendly due to it uses less energy, less waste production, uses minimum solvent while HPLC method less than UV-vis Spectroscopic method due to more waste production and higher usage of solvent. Any score above 0.5 reflects efforts to reduce environmental impact, minimize hazardous substance usage, and enhance safety, demonstrating a commitment to greener analytical practices and the broader goals of sustainable chemistry. Assessment of Whiteness by RGBfast model: RGBfast model is a latest version for calculating whiteness. This model has 6 criteria which parted into 3 Red, 1 Green and 2 Blue. Red colour includes trueness (R1), precision (R2), LOD (R3). For Green, Applying ChlorTox Scale Criterion model. Blue colour includes Usage of electricity estimation (B2) and sample throughout(B1). Higher the whiteness score indicates more ecofriendly method was. Results for both methods shown in Fig. 7 (C) and Fig. 7 (D). UV-vis Spectroscopic method score was found to be 50 and HPLC method score was found to be 51. UV-Vis spectroscopy accomplished a greater greenness score (51.7) because of reduced solvent usage and higher energy efficiency, developing it the more environmentally friendly. Though, its lower trueness and limit of detection (LOD) contributed to a lower whiteness score (50). In contrast, HPLC shows superior analytical performance, with a higher trueness value (76.0) and an overall whiteness score of 51, though it had a lower greenness score (48.4) due to increased solvent usage. Therefore, while UV-Vis Spectroscopic method is the more sustainable method, HPLC offers greater analytical precision at the expense of a higher environmental impact. UV-vis Spectroscopic method seems more greener than HPLC method while HPLC method seems most whiteness than UV-vis Spectroscopic method. Conclusion A validated UV-vis Spectroscopic method and RP-HPLC methods have been effectively developed for simultaneous estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan in their Pharmaceutical dosage form. Simultaneous equation method was used for estimation of UV. Central composite design (CCD) was used to optimize the %ethanol in mobile phase, pH and flow rate for RP-HPLC optimization. All validation parameters like linearity, accuracy, precision, limit of detection, limit of quantification was analysed by ICH Q2(R1) guideline and were determined to meet the requirements for approval. Evaluation of greenness and whiteness scores states that both methods are environmentally sustainable. UV-vis Spectroscopic method seems most green and HPLC seems most white. Both validated methods provide accurate, ecofriendly and cost-effective substitute for the routine analysis of Efonidipine Hydrochloride Ethanolate and Telmisartan in their pharmaceutical dosage forms. Declarations Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Ethical Approval This article does not contain any studies with human participants performed by any of the authors. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Vishwa S. Vyas, Dr. Dilip G. Maheshwari and Dr. Darshil Shah. The first draft of the manuscript was written by Vishwa S. Vyas and Dr. Jignesh Shah and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors would like to express their sincere gratitude to Paweł M. Nowak for developing the RGBfast model, which was instrumental in evaluating the greenness and whiteness of our analytical methods. References Copley JB, Rosario R (2005) Hypertension: a review and rationale of treatment. Dis Mon 51(10–11):548–614. https://doi.org/10.1016/j.disamonth.2005.10.004 Chandran SK (2022) A review on simultaneous determination of anti-hypertensive drug combinations containing amlodipine besylate using chromatographic analysis. Asian J Pharm Res Dev 10(4):86–92. https://doi.org/10.22270/ajprd.v10i4.1149 Darji H, Dedania Z, Dedania R, Jain V (2024) Eco-friendly based HPLC and UV-spectrophotometric methods for simultaneous estimation of Efonidipine hydrochloride Ethanolate and Chlorthalidone in their dosage form. Green Anal Chem. https://doi.org/10.1016/j.greeac.2024.100092 Chohan MS, Attimarad M, Venugopala KN et al. (2022) Sensitivity enhanced ecofriendly UV spectrophotometric methods for quality control of telmisartan and benidipine formulations: Comparison of whiteness and greenness with HPLC methods. Int J Environ Res Public Health. https://doi.org/10.3390/ijerph19127260 Solanki KH, Joshi DN, Shah DA et al. (2024) Simultaneous estimation of novel antihypertensive drug combination efonidipine hydrochloride and telmisartan using liquid chromatographic method. Essential Chem 1:1–11 https://doi.org/10.1080/28378083.2024.2368001 Joshi H, Goyal A (2025) Method development and validation for UV-visible spectroscopy and high-performance liquid chromatography: A review Anastas P, Eghbali N (2010) Green chemistry: principles and practice. Chem Soc Rev 39:301–312 https://doi.org/10.1080/28378083.2024.2368001 Miladinović SM (2024) Green analytical chemistry: integrating sustainability into undergraduate education: S. Miladinović. Anal Bioanal Chem. https://doi.org/10.1007/s00216-024-05680-4 Nowak PM, Wietecha-Posłuszny R, Pawliszyn J (2021) White analytical chemistry: An approach to reconcile the principles of green analytical chemistry and functionality. TrAC Trends Anal Chem. https://doi.org/10.1016/j.trac.2021.116223 Hafez HM, Barghash SS, Soliman MM et al. (2023) Central composite design driven optimization of sustainable stability indicating HPLC method for the determination of tigecycline and greenness assessment. F1000Res 12:341 https://doi.org/10.12688/f1000research.130861.1 Chaudhari SR, Shirkhedkar AA (2020) Application of Plackett-Burman and central composite designs for screening and optimization of factor influencing the chromatographic conditions of HPTLC method for quantification of efonidipine hydrochloride. J Anal Sci Technol. https://doi.org/10.1186/s40543-020-00246-2 Rajput AS, Jha DK, Gurram S et al. (2020) RP-HPLC method development and validation for the quantification of efonidipine hydrochloride in HME processed solid dispersions. Futur J Pharm Sci. https://doi.org/10.1186/s43094-020-00094-2 Bio-analytical method development for efonidipine in human plasma by using RP-HPLC (2023) Int Res J Mod Eng Technol Sci. https://doi.org/10.56726/IRJMETS40755 Liu M, Deng M, Zhang D et al. (2016) A chiral LC-MS/MS method for the stereospecific determination of efonidipine in human plasma. J Pharm Biomed Anal 122:35–41 https://doi.org/10.1016/j.jpba.2016.01.039 Patel GH, Adeshra SD, Meshram DB (2021) RP-HPLC method development and validation for simultaneous estimation of efonidipine hydrochloride ethanolate and telmisartan in their synthetic mixture. Int J Pharm Drug Anal 190–195 https://doi.org/10.47957/ijpda.v9i3.480 Dudhrejiya A, Patel A, Chavda J et al. (2022) Spectrophotometric simultaneous determination of efonidipine hydrochloride ethanolate and telmisartan in synthetic mixture by first order derivative method. J Med Pharm Allied Sci 11:4547–4551 Patel B, Vekaria H (2023) 2 3 factorial design for optimization of HPLC-PDA method for the simultaneous estimation of efonidipine hydrochloride ethanolate and telmisartan in tablet dosage form Damle M, Bidkar SR (2020) Stability indicating telmisartan. Int J Pharm Res Appl. https://doi.org/10.13040/IJPSR.0975-8232.13(1).439-44 Attimarad M, Chohan MS, Elgorashe REE (2020) Smart analysis of a ternary mixture of amlodipine, hydrochlorothiazide and telmisartan by manipulation of UV spectra: Development, validation and application to formulations. J Mol Struct. https://doi.org/10.1016/j.molstruc.2020.128095 Bhat LR, Godge RK, Vora AT, Damle MC (2007) Validated RP-HPLC method for simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical formulation. J Liq Chromatogr Relat Technol 30:3059–3067 Alhazmi HA, Alnami AM, Arishi MAA et al. (2017) A fast and validated reversed-phase HPLC method for simultaneous determination of simvastatin, atorvastatin, telmisartan and irbesartan in bulk drugs and tablet formulations. Sci Pharm. https://doi.org/10.3390/scipharm86010001 Rupareliya RH, Joshi HS, Ram VR et al. (2016) Stability indicating simultaneous validation of telmisartan and cilnidipine with forced degradation behavior study by RP-UPLC in tablet dosage form. Int J Pharm Qual Assur 7:39–45 http://dx.doi.org/10.1155/2013/461461 International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Harmonised Tripartite Guideline (2005) Validation of analytical procedures: text and methodology Q2(R1) Additional Declarations No competing interests reported. Supplementary Files floatimage1.jpeg Graphical abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6433370","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475653911,"identity":"ae185b53-aa5b-49fa-a8f3-a955db49e562","order_by":0,"name":"VISHWA VYAS","email":"","orcid":"","institution":"L J Institute of Pharmacy, L J University","correspondingAuthor":false,"prefix":"","firstName":"VISHWA","middleName":"","lastName":"VYAS","suffix":""},{"id":475653912,"identity":"018fcca1-31df-454d-a03f-9494a7c5ad67","order_by":1,"name":"DR. DILIP MAHESHWARI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDCCAwwMzEAqAcz+UMBmAGJIgBExWg7OMCBVCzOPAQNMC27Ad3514ueCiro8g+NnHx62MeAzNjjAfPA2D4NFHi4tkjfebpaeceZwscGZdIPDOQZsZgYH2JKteRgkinFpMbhxdoM0b9uBxG0H0hhAWmwMDvCYSQO1JDbg1rL5N++/usRt558xHLYAa+H/hl/L+d5t0rwNzInbbgBtYQA7jIcNrxbJG7zbrHmOHU7cf+MZw8EeAzZjycNsxpZzDHBr4Tt/dvNtnpq6xJn9acwfflQcM+w73vzwxpuKOpxaGCQSULjHINEEiR4cgP8ACrcGj9JRMApGwSgYqQAAwahb3c0aFcUAAAAASUVORK5CYII=","orcid":"","institution":"L J Department of Pharmaceutical Sciences, L J University","correspondingAuthor":true,"prefix":"DR.","firstName":"DILIP","middleName":"","lastName":"MAHESHWARI","suffix":""},{"id":475653913,"identity":"d7f8d191-3dc8-404f-a8ed-de7390b9a650","order_by":2,"name":"Dr. Darshil Shah","email":"","orcid":"","institution":"L J Institute of Pharmacy, L J University","correspondingAuthor":false,"prefix":"Dr.","firstName":"Darshil","middleName":"","lastName":"Shah","suffix":""},{"id":475653914,"identity":"f33b351b-5a21-4a26-8cbd-beb6c26f6eec","order_by":3,"name":"Dr. Jignesh Shah","email":"","orcid":"","institution":"L J Institute of Pharmacy, L J University","correspondingAuthor":false,"prefix":"Dr.","firstName":"Jignesh","middleName":"","lastName":"Shah","suffix":""}],"badges":[],"createdAt":"2025-04-12 08:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6433370/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6433370/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85495418,"identity":"5e0c9d77-d46b-4017-903c-6d7843b1dc4b","added_by":"auto","created_at":"2025-06-26 13:45:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28531,"visible":true,"origin":"","legend":"\u003cp\u003ea: Structure of EFD b: Structure of TEL\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/840bfaeb9108d1a67d245958.png"},{"id":85495420,"identity":"978108fc-617a-48cd-b3c4-d9f21e88d32e","added_by":"auto","created_at":"2025-06-26 13:45:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146369,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Perturbation plot of each factor effect on Retention time of TEL (b)Perturbation plot of each factor effect on retention time of EFD\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/40852814096a3eca14413131.jpeg"},{"id":85496054,"identity":"1c869755-115b-47d7-b061-208141a6793f","added_by":"auto","created_at":"2025-06-26 13:53:34","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":864297,"visible":true,"origin":"","legend":"\u003cp\u003ea: Contour plot shows design space for tailing factor of Telmisartan pH vs. flow rate (ml/min)\u003c/p\u003e\n\u003cp\u003eb: Contour plot shows design space for tailing factor of Telmisartan Ethanol vs. pH\u003c/p\u003e\n\u003cp\u003ec: Contour plot shows design space for tailing factor of Telmisartan ethanol vs. pH (ml/min)\u003c/p\u003e\n\u003cp\u003ed: Contour plot shows design space for retention time of Efonidipine Hydrochloride Ethanolate pH vs. flow rate (ml/min)\u003c/p\u003e\n\u003cp\u003ee: Contour plot shows design space for retention time of Efonidipine Hydrochloride Ethanolate Ethanol vs. flow rate (ml/min)\u003c/p\u003e\n\u003cp\u003ef: Contour plot shows design space for retention time of Efonidipine Hydrochloride Ethanolate ethanol vs. pH\u003c/p\u003e\n\u003cp\u003eg: 3-D model shows design space for tailing factor of Telmisartan pH vs. flow rate (ml/min)\u003c/p\u003e\n\u003cp\u003eh: 3-D model shows design space for tailing factor of Telmisartan Ethanol vs. flow rate (ml/min)\u003c/p\u003e\n\u003cp\u003ei: \u0026nbsp;3-D model shows design space for tailing factor of Telmisartan Ethanol vs. pH\u003c/p\u003e\n\u003cp\u003ej: 3-D model shows design space for retention time of Efonidipine Hydrochloride Ethanolate pH vs flow rate (ml/min)\u003c/p\u003e\n\u003cp\u003ek: 3-D model shows design space for retention time of Efonidipine Hydrochloride Ethanolate Ethanol vs flow rate (ml/min)\u003c/p\u003e\n\u003cp\u003el:3-D model shows design space for retention time of Efonidipine Hydrochloride Ethanolate : Ethanol vs pH\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/220098be5d63f04a3f1a9ee8.jpeg"},{"id":85496051,"identity":"e6ab5318-283e-4c4d-ad81-aeea24801728","added_by":"auto","created_at":"2025-06-26 13:53:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20222,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of mixture of EFD (40 µg/ml) and TEL (40 µg/ml) by HPLC\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/2865d2d46a4bb49ff8e7aece.png"},{"id":85495434,"identity":"259c5cad-614f-4506-82c9-8cc52e4b1d2c","added_by":"auto","created_at":"2025-06-26 13:45:34","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":318548,"visible":true,"origin":"","legend":"\u003cp\u003ea: Overlay spectra of EFD by UV-Vis Spectroscopy at 251 nm b: Overlay spectra of TEL by UV-Vis Spectroscopy at 296 nm c: Overlay spectra of EFD and TEL by HPLC at 250 nm\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/8773065c5873b2bab6613b46.jpeg"},{"id":85496952,"identity":"365f2be0-8d83-4aa1-a67b-e3c7c6925fc7","added_by":"auto","created_at":"2025-06-26 14:01:34","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":339704,"visible":true,"origin":"","legend":"\u003cp\u003ea: Calibration curve of EFD by UV-Vis Spectroscopyb: Calibration curve of TEL by UV-Vis Spectroscopy\u003c/p\u003e\n\u003cp\u003ec: Calibration curve of TEL by HPLC d: Calibration curve of EFD by HPLC\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/0e577aae6c2eb89501638f3b.jpeg"},{"id":85495425,"identity":"ecf8fd09-4f66-4bfc-a378-70d106d47d58","added_by":"auto","created_at":"2025-06-26 13:45:34","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":614564,"visible":true,"origin":"","legend":"\u003cp\u003ea: AGREE score by UV-vis Spectroscopic method b: AGREE score by HPLC c: RGBfast score by UV-vis Spectroscopic method d: RGBfast score by HPLC\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/af7a5dd0e7d17f16802ac55e.jpeg"},{"id":88694244,"identity":"b3c1e252-65c3-447b-8889-c07f0149e479","added_by":"auto","created_at":"2025-08-09 16:01:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3157435,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/5aa912c8-d800-4ec5-9b58-3bb6f3f4e4de.pdf"},{"id":85495429,"identity":"f95b609e-144b-4ab3-9304-352360fc4f7d","added_by":"auto","created_at":"2025-06-26 13:45:34","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":330160,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6433370/v1/8d6bf3907175ff5c25f0344c.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Eco-Friendly UV Spectroscopic and RP-HPLC Methods for Simultaneous Estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan: A DOE-Optimized Approach with Greenness and Whiteness Assessment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertension is a chronic condition in which blood pressure in the arteries remains high. On the ranking of the determinants of the disease burden across life, It is placed third in terms of disability-adjusted life years.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Numerous medications in various forms can be used to treat the hypertension. Calcium channels blockers, Diuretics, Angiotensin II receptor blockers, Angiotensin-converting enzyme inhibitors are classes of drugs which are used to treat the hypertension.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eEfonidipine Hydrochloride Ethanolate (EFD) chemically referred as 2-(\u003cem\u003eN\u003c/em\u003e-benzylanilino)ethyl 5-(5,5-dimethyl-2-oxo-1,3,2λ\u003csup\u003e5\u003c/sup\u003e-dioxaphosphinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylate;ethanol;hydrochloride.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] It is a dihydropyridine (DHP) calcium channel blocker. It shows antihypertensive impact through vasodilatation by blocking T-type and L-type calcium channels.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eTelmisartan (TEL) chemically referred as\u003c/p\u003e \u003cp\u003e2-[4-[[4-methyl-6-(1-methylbenzimidazol-2-yl)-2-propylbenzimidazol-1-yl]methyl]phenyl]benzoic acid.\u003c/p\u003e \u003cp\u003eAngiotensin II produced by a catalytic reaction between Angiotensin I and Angiotensin-converting enzyme (AT1), is the primary presser agent of the renin-angiotensin system, causing vasoconstriction, aldosterone release, cardiac stimulation, and sodium renal reabsorption. Telmisartan selectively blocks its binding to AT1 receptors and exhibit vasodilation.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eChoosing High-Performance liquid chromatography (HPLC) and UV-Visible (UV-Vis) Spectroscopy for research provides a robust platform for analysis. UV-Vis Spectroscopy is a cost-effective, simple and less time consuming. It is perfect for routine analysis while HPLC provides precise and accurate analysis of mixtures. It has high-resolution capabilities. Both techniques are ideal for identify the components in pharmaceutical compositions and improves the depth and reliability of research outcomes.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eGreen Analytical Chemistry (GAC) enhance analytical methods through reducing the amount of waste, Improving the efficiency of energy using, using green solvent which are not harmful to humans and environment and making a method eco-friendly. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eWhite Analytical Chemistry (WAC) builds on the idea of GAC. It is a cost-effective, efficient and reproducible analytical approach. It contains three main aspects: Red (analytical efficiency), Green (environmental impact), Blue (Practicality).[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eDesign of Experiments (DOE) is a methodical approach that searches into the corelation between numerous input factors and its effects on intended output responses. Central Composite Design (CCD) is the significant method in DOE. It is a valuable in developing analytical method, like improving sustainability and stability of HPLC method for determining pharmaceutical compounds.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eAccording to literature survey, a various analytical methods have been reported for the analysis of EFD and TEL. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Nevertheless, there are no UV-vis Spectroscopic and HPLC techniques have been reported with greenness and whiteness assessment of EFD and TEL. The main objective of this research is to develop and validate a precise and eco-friendly UV-vis Spectroscopic and DOE assisted RP-HPLC which can replace the hazardous and costly method for determination of EFD and TEL in their pharmaceutical dosage form.\u003c/p\u003e "},{"header":"Materials and Reagents","content":"\u003cp\u003eA gift sample of EFD was provided by Zuventus healthcare ltd., Pune. TEL gift sample was provided by Zydus lifesciences, Ahmedabad. Combination product of EFD and TEL (Label claim: 40 mg EFD and 40 mg TEL) EFNOCAR-T Manufactured by Zuventus healthcare ltd was bought by local pharmacy. Acetonitrile, Ethanol, methanol and water (HPLC grade) acquired from Thermo Fisher Scientific India Pvt. Ltd. The supplier of Orthophosphoric acid was ACS Chemicals in India.\u003c/p\u003e\u003cp\u003eApparatus and Software:\u003c/p\u003e\u003cp\u003eThe study was carried out by UV- Visible spectrophotometer (Shimadzu model 1800 (Japan)) with software UV Probe, HPLC was completed with a Shimandzu HPLC system with model LC-2010 CHT fitted with the UV detector and serial dual plugger pump with software Labsolution version 5.52. Chromatographic separations were done by Shimadzu C \u003csub\u003e18\u003c/sub\u003e (250 × 4.6 mm, 5 µm) column. For Design of experiments (Central Composite Design) Design-Expert v13.0.12.0 by Stat Ease was used. For weighing purpose, analytical weight balance (Wensar electronic balance) was used. Prior to use, every piece of glasswares were calibrated.\u003c/p\u003e\u003cp\u003ePreparation of Mobile phase:\u003c/p\u003e\u003cp\u003e70 ml of ethanol, 20 ml of acetonitrile and 10 ml of water were taken and mixed with the use of sonicator. pH 4 adjusted with the 1% Orthophosphoric acid.\u003c/p\u003e\u003cp\u003ePreparation of 1% Orthophosphoric acid:\u003c/p\u003e\u003cp\u003e1 ml of orthophophoric acid dissolved in 100 ml of water.\u003c/p\u003e\u003cp\u003ePreparation of Standard Stock Solution:\u003c/p\u003e\u003cp\u003eFor UV-vis Spectroscopic technique, 40 mg of TEL and EFD dissolved in 100 ml of Methanol to make standard stock solution of TEL (400 µg/ml) and EFD (400 µg/ml), respectively. After that, Methanol was added in the standard solution to make a various concentrations 10–60 µg/ml for TEL and 10–60 µg/ml for EFD.\u003c/p\u003e\u003cp\u003eFor HPLC technique, 40 mg of TEL and EFD dissolved in 100 ml of Ethanol to make standard stock solution of TEL (400 µg/ml) and EFD (400 µg/ml), respectively. After that, the solution of standard was diluted with Mobile phase to make a various concentrations 10–60 µg/ml for TEL and 10–60 µg/ml for EFD.\u003c/p\u003e\u003cp\u003eUV-Visible Spectroscopic method:\u003c/p\u003e\u003cp\u003eSimultaneous equation approach: TEL and EFD mixture is present in ratio of 1:1, respectively. Using methanol as a solvent, spectra of absorption of API and their mixture were measured in range 200 to 400 nm. Absorbance of TEL and EFD at 296 nm and 251 nm respectively. This method is evaluated by below formula:[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Cx=\\left(\\frac{{A}_{2}a{y}_{1}-{A}_{1}a{y}_{2}}{a{x}_{2}a{y}_{1}-a{x}_{1}a{y}_{2}}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:{C}_{y}=\\left(\\frac{{A}_{1}a{x}_{2}-{A}_{2}a{x}_{1}}{a{y}_{1}a{x}_{2}-a{y}_{2}a{x}_{1}}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003ewhere, Cx = Concentration of TEL, Cy = Concentration of EFD, A\u003csub\u003e1\u003c/sub\u003e = Absorbance of test at λ\u003csub\u003e1\u003c/sub\u003e (λ\u003csub\u003emax\u003c/sub\u003e of TEL), A\u003csub\u003e2\u003c/sub\u003e = Absorbance of test at λ\u003csub\u003e2\u003c/sub\u003e (λ\u003csub\u003emax\u003c/sub\u003e of EFD), ax1 = Absorptivity of x drug (TEL) at λ1, ax2 = Absorptivity of x drug (TEL) at λ2, ay1 = Absorptivity of y drug (EFD) at λ1, ay2 = Absorptivity of y drug (EFD) at λ2.\u003c/p\u003e\u003cp\u003eOptimized Chromatographic condition for HPLC:\u003c/p\u003e\u003cp\u003eThe Reverse phase technique was used for developing RP-HPLC method with a Shimadzu C \u003csub\u003e18\u003c/sub\u003e (250 × 4.6 mm, 5 µm) column. Mobile phase comprised of Ethanol: Acetonitrile: Water (70:20:10%v/v) with pH 4 adjusted with orthophosphoric acid. Detection was carried out at 250nm wavelength with a flow rate 1 ml/min. Run time was 10 min and injection volume was 10 µl.\u003c/p\u003e\u003cp\u003eExperimental Design:\u003c/p\u003e\u003cp\u003eFaced central composite design (FCCD) was used for the optimization of pH, % ethanol and flow rate for the effective separation. FCCD included five replicates including axial and center points. Using Derringer’s desirability, we evaluated the R2 coefficient for determining polynomial models to access the optimize condition.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eMethod development and optimization:\u003c/p\u003e \u003cp\u003eRP-HPLC method was developed by application of DOE to explore the three independent variables: pH, Flow rate and %ethanol in the mobile phase. 250 nm of wavelength was selected by the analysis of UV\u0026ndash;vis overlay spectra of EFD and TEL drugs. Chromatographic separation of compounds was done by using a composite ratio of ethanol (70%) at a pH 4 and flow rate 1 ml/min. The present study emphasizes on studying tailing factor and retention time in 15 experiments. The effective range for flow rate between 0.8 to 1.2 ml/min, pH between 3.5 to 4.5 and % ratio of ethanol is 65 to 75%v/v. Applying CCD finds optimal conditions and gives model\u0026rsquo;s suitability.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows indicate the regression model and statistical parameters obtained by ANOVA which shows that factors affecting the tailing factor and retention time for TEL and EFD respectively. P-value is less than 0.5 which indicates that models were significant. Adjusted R\u003csup\u003e2\u003c/sup\u003e for both drugs was greater than 0.8 which shows that drugs both drugs provide linear response.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHPLC independent variables\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLevel (-)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLevel (0)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLevel (+)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlow\u003c/p\u003e \u003cp\u003erate(ml/min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConditions and responses of HPLC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDesign\u003c/p\u003e \u003cp\u003ePoints\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFactor Level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eResponses\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFactor 1\u003c/p\u003e \u003cp\u003eFlow rate(ml/min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFactor2\u003c/p\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFactor 3\u003c/p\u003e \u003cp\u003eConcof\u003c/p\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTailing Factor of TEL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRetention time of EFD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.671\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.264\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical paramters and regression model which are based on ANOVA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRes pons e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegression Model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdjusted R2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModel P-Value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e% C.V\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAdequa te Precisio\u003c/p\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePredicte d R2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF\u003c/p\u003e \u003cp\u003evalu e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;1.32-0.059A-0.0935B-0.0239C-0.0075AB-0.0068AC\u0026thinsp;+\u0026thinsp;0.010BC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e˂0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.9782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.9035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e46.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;6.20-0.1037A\u0026thinsp;+\u0026thinsp;0.0251B-0.5609C\u0026thinsp;+\u0026thinsp;0.0536AB\u0026thinsp;+\u0026thinsp;0.0386AC\u0026thinsp;+\u0026thinsp;0.0514BC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e˂0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27.6001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.8596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e82.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDerringer\u0026rsquo;s Desirability Function (D):\u003c/p\u003e \u003cp\u003eIt is a technique which optimize multiple objectives with multiple targets and responses. Design Expert 13 was used to optimize standards which focuses on retention time while method development. By utilizing a pH 4, flow rate 1 ml/min and %ethanol concentration (v/v), Total desirability value found to be 1 which indicates that these coordinates are the most appropriate parameters for recommended method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSystem suitability studies:\u003c/p\u003e \u003cp\u003eThe system suitability was assessed by six replicate analyses of EFD and TEL at concentration of 40 \u0026micro;g/ml. The column resolution, tailing factor, theoretical plates were analysed for standard solutions. Peak area for TEL was found to be 2435985 and Peak area for EFD was found to be 1106705. Retention time for TEL and EFD was found to be 2.288 and 6.264, respectively. Plate count for TEL and EFD was found to be 2094 and 8827 and tailing factor TEL and EFD was found to be 1.322 and 1.643, respectively. Resolution between both drugs were found to be 17.039.\u003c/p\u003e \u003cp\u003eMethod Validation:\u003c/p\u003e \u003cp\u003eThe suggested method was validated with regards to linearity, accuracy, limit of detection, limit of quantification, repeatability, intermediate precision based on ICH Q2(R1) guideline.\u003c/p\u003e \u003cp\u003eLinearity:\u003c/p\u003e \u003cp\u003eThe linearity of TEL and EFD was established by scanning different concentrations at six levels. The solution of concentration in range of 10\u0026ndash;60 \u0026micro;g/ml for both drugs for both UV-vis Spectroscopic and HPLC method. A graph of Concentration vs Absorbance and Concentration vs Peak area was plotted for UV-vis Spectroscopic and HPLC method respectively and regression equation was acquired. Results of TEL and EFD for both methods was displayed in Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. UV-vis Spectroscopic overlay chromatograph was displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A) and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B) of both drugs and it was found to be linear over 10\u0026ndash;60 \u0026micro;g/ml with regression line equation y\u0026thinsp;=\u0026thinsp;0.0361x\u0026thinsp;+\u0026thinsp;0.0362 with r2\u0026thinsp;=\u0026thinsp;0.998 for EFD and y\u0026thinsp;=\u0026thinsp;0.0437x\u0026thinsp;+\u0026thinsp;0.2356 with r2\u0026thinsp;=\u0026thinsp;0.998 for TEL. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(A) and 6(B)). HPLC overlay chromatogram was displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(C)10\u0026ndash;60 \u0026micro;g/ml with retention time 2.28 min and 6.264 min for TEL and EFD, respectively. Regression line equation y\u0026thinsp;=\u0026thinsp;63083x-157627 with r2\u0026thinsp;=\u0026thinsp;0.9981 for TEL and y\u0026thinsp;=\u0026thinsp;29313x-80692 with r2\u0026thinsp;=\u0026thinsp;0.9979 for EFD(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(C) and 6(D))..\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLinearity and Precision data of EFD and TEL by UV-vis Spectroscopy and HPLC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eLinearity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eSimultaneous equation UV-vis Spectroscopic Method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsorbance\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsorbance\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.672\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.394\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.762\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.082\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.771\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.772\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.540\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.114\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.520\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.021\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.467\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.465\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.463\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.554\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.842\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.872\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.805\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.208\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.539\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeak area\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePeak area\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e492981\u0026thinsp;\u0026plusmn;\u0026thinsp;3388\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e193544\u0026thinsp;\u0026plusmn;\u0026thinsp;1084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.560\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1154859\u0026thinsp;\u0026plusmn;\u0026thinsp;10758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e525975\u0026thinsp;\u0026plusmn;\u0026thinsp;3189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.606\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1718879\u0026thinsp;\u0026plusmn;\u0026thinsp;11703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e803897\u0026thinsp;\u0026plusmn;\u0026thinsp;7516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.935\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2404176\u0026thinsp;\u0026plusmn;\u0026thinsp;23026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.957\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1111288\u0026thinsp;\u0026plusmn;\u0026thinsp;5390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.485\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2915219\u0026thinsp;\u0026plusmn;\u0026thinsp;15452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1343184\u0026thinsp;\u0026plusmn;\u0026thinsp;12496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.930\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3565496\u0026thinsp;\u0026plusmn;\u0026thinsp;28028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1693629\u0026thinsp;\u0026plusmn;\u0026thinsp;13287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.784\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrecision\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eUV-vis Spectroscopic method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eHPLC method\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean absorbance\u003c/p\u003e \u003cp\u003e\u0026plusmn; SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean peak area\u003c/p\u003e \u003cp\u003e\u0026plusmn; SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eIntraday precision (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.673\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e497429\u0026thinsp;\u0026plusmn;\u0026thinsp;2182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.487\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.084\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1165358\u0026thinsp;\u0026plusmn;\u0026thinsp;6929\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.594\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1729854\u0026thinsp;\u0026plusmn;\u0026thinsp;9950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.575\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.393\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.817\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e193079\u0026thinsp;\u0026plusmn;\u0026thinsp;1044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.540\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.775\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.733\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e526882\u0026thinsp;\u0026plusmn;\u0026thinsp;3288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.624\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.122\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e802822\u0026thinsp;\u0026plusmn;\u0026thinsp;5568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.693\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eInterday precision (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.676\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e493564\u0026thinsp;\u0026plusmn;\u0026thinsp;2986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.673\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.086\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1157278\u0026thinsp;\u0026plusmn;\u0026thinsp;9669\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.835\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.552\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.759\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1716061\u0026thinsp;\u0026plusmn;\u0026thinsp;15861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.924\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.394\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e193247\u0026thinsp;\u0026plusmn;\u0026thinsp;1537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.795\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.776\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0060\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.783\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e523821\u0026thinsp;\u0026plusmn;\u0026thinsp;3413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.651\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.128\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.934\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e802646\u0026thinsp;\u0026plusmn;\u0026thinsp;6098\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.759\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eRepeatability (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1160386\u0026thinsp;\u0026plusmn;\u0026thinsp;8463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.729\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.771\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e525233\u0026thinsp;\u0026plusmn;\u0026thinsp;3843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.731\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003ePrecision:\u003c/p\u003e \u003cp\u003eIntraday precision and intermediate precision (interday precision) were performed by estimating SD and %RSD for three times determinations of 3 concentrations within the range of linearity. Repeatability was performed by calculating SD and %RSD for the six times determinations of 1 concentration. Results of both methods for EFD and TEL were displayed in Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. %RSD value of EFD and TEL for both methods were found to be less than 2 which indicates that method was precise.\u003c/p\u003e \u003cp\u003eAccuracy:\u003c/p\u003e \u003cp\u003eAccuracy was calculated by determining the % recovery of TEL and EFD at 80%, 100%, 120% in placebo through spiking API. Accuracy results for both methods were displayed in the Table \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. %Recovery was observed between 99%-101% for both methods for EFD and TEL which shows that no interference are present due to excipients.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAccuracy, LOD, LOQ and Assay data of EFD and TEL by UV-vis Spectroscopic method and HPLC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e \u003cp\u003eAccuracy\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eUV-vis Spectroscopic method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLevel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmount of sample taken (\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmount of standard spiked\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal amount of drug\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAmount of standard recovery mean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e% Recovery (n\u0026thinsp;=\u0026thinsp;3)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAmount of standard recovery mean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e% Recovery (n\u0026thinsp;=\u0026thinsp;3)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e101.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e36.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e101.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.232\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e101.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e101.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e101.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e43.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e99.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.205\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e35.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e99.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.387\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e39.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e99.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.490\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e43.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e99.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.411\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLOD and LOQ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eUV-vis Spectroscopic method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eLOD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eLOQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e3.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAssay\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEFNOCAR-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eUV-vis Spectroscopic method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLabel claim\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eAmount found\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e% assay\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eAmount found\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e% assay\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTEL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e39.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e39.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e98.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.604\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEFD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e39.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e39.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e99.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.490\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLimit of Detection (LOD) and Limit of Quantification (LOQ):\u003c/p\u003e \u003cp\u003eLOD and LOQ was performed by analysing by three replicates solutions of standard which are containing TEL and EFD. LOD and LOQ results for both methods for TEL and EFD were displayed on Table \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. LOD value for TEL was observed 0.67 and 0.59 for UV-vis Spectroscopic method and HPLC method, respectively. LOD value for EFD was observed 0.73 and 1.28 for UV-vis Spectroscopic method and HPLC method, respectively. LOQ value for TEL was observed 2.03 and 1.81 for UV-vis Spectroscopic method and HPLC method, respectively. LOQ value for EFD was observed 2.21 and 3.90 for UV-vis Spectroscopic method and HPLC method, respectively.\u003c/p\u003e \u003cp\u003eAssay:\u003c/p\u003e \u003cp\u003eIt is performed on a marketed formulation to check %purity. Assay results for both methods for EFD and TEL displayed on Table \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Results of %Assay was found in the range of acceptance criteria 98\u0026ndash;102%.\u003c/p\u003e \u003cp\u003eAssessment of Greenness by Analytical Greenness Index (AGREE):\u003c/p\u003e \u003cp\u003eAGREE is a green model which calculate the greenness based on 12 principles of GAC. Numerical value of AGREE demonstrated that how ecofriendly UV-vis Spectroscopic method and HPLC methods were. Final results are derived from sum of twelve input variables ranging from 0 to 1. A higher score indicates more environment friendly method was. Results for both methods shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(A) and 7(B). The greenness attributes of the developed UV-vis Spectroscopic method was 0.74 and HPLC method was 0.73 assessed using the AGREE evaluation. This indicates that the method aligns with the principles of GAC. Higher AGREE score of UV-vis Spectroscopic method shows that method was more environment friendly due to it uses less energy, less waste production, uses minimum solvent while HPLC method less than UV-vis Spectroscopic method due to more waste production and higher usage of solvent. Any score above 0.5 reflects efforts to reduce environmental impact, minimize hazardous substance usage, and enhance safety, demonstrating a commitment to greener analytical practices and the broader goals of sustainable chemistry.\u003c/p\u003e \u003cp\u003eAssessment of Whiteness by RGBfast model:\u003c/p\u003e \u003cp\u003eRGBfast model is a latest version for calculating whiteness. This model has 6 criteria which parted into 3 Red, 1 Green and 2 Blue. Red colour includes trueness (R1), precision (R2), LOD (R3). For Green, Applying ChlorTox Scale Criterion model. Blue colour includes Usage of electricity estimation (B2) and sample throughout(B1). Higher the whiteness score indicates more ecofriendly method was. Results for both methods shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(C) and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e(D). UV-vis Spectroscopic method score was found to be 50 and HPLC method score was found to be 51. UV-Vis spectroscopy accomplished a greater greenness score (51.7) because of reduced solvent usage and higher energy efficiency, developing it the more environmentally friendly. Though, its lower trueness and limit of detection (LOD) contributed to a lower whiteness score (50). In contrast, HPLC shows superior analytical performance, with a higher trueness value (76.0) and an overall whiteness score of 51, though it had a lower greenness score (48.4) due to increased solvent usage. Therefore, while UV-Vis Spectroscopic method is the more sustainable method, HPLC offers greater analytical precision at the expense of a higher environmental impact. UV-vis Spectroscopic method seems more greener than HPLC method while HPLC method seems most whiteness than UV-vis Spectroscopic method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA validated UV-vis Spectroscopic method and RP-HPLC methods have been effectively developed for simultaneous estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan in their Pharmaceutical dosage form. Simultaneous equation method was used for estimation of UV. Central composite design (CCD) was used to optimize the %ethanol in mobile phase, pH and flow rate for RP-HPLC optimization. All validation parameters like linearity, accuracy, precision, limit of detection, limit of quantification was analysed by ICH Q2(R1) guideline and were determined to meet the requirements for approval. Evaluation of greenness and whiteness scores states that both methods are environmentally sustainable. UV-vis Spectroscopic method seems most green and HPLC seems most white. Both validated methods provide accurate, ecofriendly and cost-effective substitute for the routine analysis of Efonidipine Hydrochloride Ethanolate and Telmisartan in their pharmaceutical dosage forms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e \u003cp\u003eCompliance with Ethical Standards\u003c/p\u003e \u003cp\u003e \u003cb\u003eConflict of Interest\u003c/b\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthical Approval\u003c/b\u003e This article does not contain any studies with human participants performed by any of the authors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Vishwa S. Vyas, Dr. Dilip G. Maheshwari and Dr. Darshil Shah. The first draft of the manuscript was written by Vishwa S. Vyas and Dr. Jignesh Shah and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to express their sincere gratitude to Paweł M. Nowak for developing the RGBfast model, which was instrumental in evaluating the greenness and whiteness of our analytical methods.\u003c/p\u003e"},{"header":"References","content":"\u003col start=\"1\" type=\"1\"\u003e\n\u003cli\u003eCopley JB, Rosario R (2005) Hypertension: a review and rationale of treatment. Dis Mon 51(10\u0026ndash;11):548\u0026ndash;614. https://doi.org/10.1016/j.disamonth.2005.10.004\u003c/li\u003e\n\u003cli\u003eChandran SK (2022) A review on simultaneous determination of anti-hypertensive drug combinations containing amlodipine besylate using chromatographic analysis. Asian J Pharm Res Dev 10(4):86\u0026ndash;92. https://doi.org/10.22270/ajprd.v10i4.1149\u003c/li\u003e\n\u003cli\u003eDarji H, Dedania Z, Dedania R, Jain V (2024) Eco-friendly based HPLC and UV-spectrophotometric methods for simultaneous estimation of Efonidipine hydrochloride Ethanolate and Chlorthalidone in their dosage form. Green Anal Chem. https://doi.org/10.1016/j.greeac.2024.100092\u003c/li\u003e\n\u003cli\u003eChohan MS, Attimarad M, Venugopala KN et al. (2022) Sensitivity enhanced ecofriendly UV spectrophotometric methods for quality control of telmisartan and benidipine formulations: Comparison of whiteness and greenness with HPLC methods. Int J Environ Res Public Health. https://doi.org/10.3390/ijerph19127260\u003c/li\u003e\n\u003cli\u003eSolanki KH, Joshi DN, Shah DA et al. (2024) Simultaneous estimation of novel antihypertensive drug combination efonidipine hydrochloride and telmisartan using liquid chromatographic method. Essential Chem 1:1\u0026ndash;11 https://doi.org/10.1080/28378083.2024.2368001\u003c/li\u003e\n\u003cli\u003eJoshi H, Goyal A (2025) Method development and validation for UV-visible spectroscopy and high-performance liquid chromatography: A review\u003c/li\u003e\n\u003cli\u003eAnastas P, Eghbali N (2010) Green chemistry: principles and practice. Chem Soc Rev 39:301\u0026ndash;312 https://doi.org/10.1080/28378083.2024.2368001\u003c/li\u003e\n\u003cli\u003eMiladinović SM (2024) Green analytical chemistry: integrating sustainability into undergraduate education: S. Miladinović. Anal Bioanal Chem. https://doi.org/10.1007/s00216-024-05680-4\u003c/li\u003e\n\u003cli\u003eNowak PM, Wietecha-Posłuszny R, Pawliszyn J (2021) White analytical chemistry: An approach to reconcile the principles of green analytical chemistry and functionality. TrAC Trends Anal Chem. https://doi.org/10.1016/j.trac.2021.116223\u003c/li\u003e\n\u003cli\u003eHafez HM, Barghash SS, Soliman MM et al. (2023) Central composite design driven optimization of sustainable stability indicating HPLC method for the determination of tigecycline and greenness assessment. F1000Res 12:341 https://doi.org/10.12688/f1000research.130861.1\u003c/li\u003e\n\u003cli\u003eChaudhari SR, Shirkhedkar AA (2020) Application of Plackett-Burman and central composite designs for screening and optimization of factor influencing the chromatographic conditions of HPTLC method for quantification of efonidipine hydrochloride. J Anal Sci Technol. https://doi.org/10.1186/s40543-020-00246-2\u003c/li\u003e\n\u003cli\u003eRajput AS, Jha DK, Gurram S et al. (2020) RP-HPLC method development and validation for the quantification of efonidipine hydrochloride in HME processed solid dispersions. Futur J Pharm Sci. https://doi.org/10.1186/s43094-020-00094-2\u003c/li\u003e\n\u003cli\u003eBio-analytical method development for efonidipine in human plasma by using RP-HPLC (2023) Int Res J Mod Eng Technol Sci. https://doi.org/10.56726/IRJMETS40755\u003c/li\u003e\n\u003cli\u003eLiu M, Deng M, Zhang D et al. (2016) A chiral LC-MS/MS method for the stereospecific determination of efonidipine in human plasma. J Pharm Biomed Anal 122:35\u0026ndash;41 https://doi.org/10.1016/j.jpba.2016.01.039\u003c/li\u003e\n\u003cli\u003ePatel GH, Adeshra SD, Meshram DB (2021) RP-HPLC method development and validation for simultaneous estimation of efonidipine hydrochloride ethanolate and telmisartan in their synthetic mixture. Int J Pharm Drug Anal 190\u0026ndash;195 https://doi.org/10.47957/ijpda.v9i3.480\u003c/li\u003e\n\u003cli\u003eDudhrejiya A, Patel A, Chavda J et al. (2022) Spectrophotometric simultaneous determination of efonidipine hydrochloride ethanolate and telmisartan in synthetic mixture by first order derivative method. J Med Pharm Allied Sci 11:4547\u0026ndash;4551\u003c/li\u003e\n\u003cli\u003ePatel B, Vekaria H (2023) 2 3 factorial design for optimization of HPLC-PDA method for the simultaneous estimation of efonidipine hydrochloride ethanolate and telmisartan in tablet dosage form\u003c/li\u003e\n\u003cli\u003eDamle M, Bidkar SR (2020) Stability indicating telmisartan. Int J Pharm Res Appl. https://doi.org/10.13040/IJPSR.0975-8232.13(1).439-44\u003c/li\u003e\n\u003cli\u003eAttimarad M, Chohan MS, Elgorashe REE (2020) Smart analysis of a ternary mixture of amlodipine, hydrochlorothiazide and telmisartan by manipulation of UV spectra: Development, validation and application to formulations. J Mol Struct. https://doi.org/10.1016/j.molstruc.2020.128095\u003c/li\u003e\n\u003cli\u003eBhat LR, Godge RK, Vora AT, Damle MC (2007) Validated RP-HPLC method for simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical formulation. J Liq Chromatogr Relat Technol 30:3059\u0026ndash;3067\u003c/li\u003e\n\u003cli\u003eAlhazmi HA, Alnami AM, Arishi MAA et al. (2017) A fast and validated reversed-phase HPLC method for simultaneous determination of simvastatin, atorvastatin, telmisartan and irbesartan in bulk drugs and tablet formulations. Sci Pharm. https://doi.org/10.3390/scipharm86010001\u003c/li\u003e\n\u003cli\u003eRupareliya RH, Joshi HS, Ram VR et al. (2016) Stability indicating simultaneous validation of telmisartan and cilnidipine with forced degradation behavior study by RP-UPLC in tablet dosage form. Int J Pharm Qual Assur 7:39\u0026ndash;45 http://dx.doi.org/10.1155/2013/461461\u003c/li\u003e\n\u003cli\u003eInternational Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Harmonised Tripartite Guideline (2005) Validation of analytical procedures: text and methodology Q2(R1)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Telmisartan, Efonidipine Hydrochloride Ethanolate, RP-HPLC, UV, Central Composite Design, Greenness and Whiteness Assessment","lastPublishedDoi":"10.21203/rs.3.rs-6433370/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6433370/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn accurate, simple and environment friendly Ultraviolet-visible spectroscopic method and Reverse phase High performance liquid chromatography method was established for simultaneous estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan in their pharmaceutical dosage form. Ultraviolet-visible spectroscopic method utilized the simultaneous equation method using 251 nm and 296 nm wavelengths for Efonidipine Hydrochloride Ethanolate and Telmisartan, respectively. Reverse phase high performance liquid chromatography was optimizing with the help of Central Composite Design which is a Design of Experiment approach, to accomplish optimal separation of both drugs with using minimum solvent. Optimal chromatographic separation was obtained by Shimandzu C18 column (250 \u0026times; 4.6 mm, 5 \u0026micro;m) with Mobile phase which comprising Ethanol: Acetonitrile: Water (70:20:10%v/v) at pH 4, flow rate 1 ml/min at detection wavelength 250 nm. Both methods were validated as per ICH Q2 (R1) guideline, showing linearity, precision, accuracy, limit of detection, limit of quantification. Assessment of greenness and whiteness score, ensures that methods have environmental sustainability and analytical efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Eco-Friendly UV Spectroscopic and RP-HPLC Methods for Simultaneous Estimation of Efonidipine Hydrochloride Ethanolate and Telmisartan: A DOE-Optimized Approach with Greenness and Whiteness Assessment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 13:45:29","doi":"10.21203/rs.3.rs-6433370/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"24fd3226-fa92-440a-be1c-fd876b16af65","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-09T15:53:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 13:45:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6433370","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6433370","identity":"rs-6433370","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}