A Green Analytical Stability-Indicating RP-HPLC method for Simultaneous Determination of Anti-Hypercholesterolemic Agents and Their related Impurities

A Green Analytical Stability-Indicating RP-HPLC method for Simultaneous Determination of Anti-Hypercholesterolemic Agents and Their related Impurities | 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 A Green Analytical Stability-Indicating RP-HPLC method for Simultaneous Determination of Anti-Hypercholesterolemic Agents and Their related Impurities Sridevi Ranjitha Karanam, Divya Narla, Sai Priyanka Kruthiveni, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9113640/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A straightforward, trustworthy and eco-friendly RP-HPLC-UV stability-indicating technique for separation and quantification of Bempedoic acid(BPA), Ezetimibe(EZM) and their impurities(BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) was developed and validated in accordance with ICH Q2(R2) for accurate determination of BPA & EZM by minimizing the interference from impurities with impurity analysis simultaneously. Chromatographic separation was accomplished on an Inertsil ODS C18 Column (250×4.6mm, 5µm) using gradient elution mode with 0.1% TFA and Acetonitrile at a flow rate of 1.0 ml/min, with detection wavelength of 211nm and total run time is 40mins. Method reliability was assessed through key parameters such as specificity, linearity, accuracy, precision, sensitivity (LOD & LOQ) and robustness. The principles of green analytical chemistry were followed to minimize the usage of solvents. The results of linearity were consistent with R 2 values above 0.999; percent recovery is in the range of 99–101%; RSD% values were below 2.0% for precision. LOD and LOQ values were found to be 0.03 and 0.1 µg/ml for impurities (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B), 5.4 & 18µg/ml for BPAand 0.3 & 0.1µg/ml for EZM. By inducing degradation under extreme conditions, the stability-indicating property of the approach was explored.The approach was found to be effective in differentiating between BPA, EZM and their impurities showcasing the suitability of the method for routine quality control.The results of the devised technique confirm the reliability for quantification of analytes and impurities in their fixed dose combination. The AGREE and GAPI tools used to assess the greenness of the method showed a score of 0.72 and 85 showcasing reasonable balance among environmental sustainability and analytical performance, indicating the eco-friendliness of the developed method. Bempedoic acid Ezetimibe Impurity analysis Forced degradation RP-HPLC-UV Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Bempedoic acid and Ezetimibe is a fixed dose combination (FDC) approved by USFDA for the treatment of hypercholesterolemia with high Low Density Lipoprotein Cholesterol (LDL-C)( 1 ). It is prescribed for people with AtheroSclerotic Cardiovascular Disease (ASCVD)( 2 ) who require additional reduction of LDL-C and for those having statin intolerance( 3 , 4 ). Bempedoic acid(BPA) is a prodrug, belonging to class of ‘Adenosine triphosphate citrate lyase’(ACL) inhibitor( 5 , 6 ), activated in liver by very long chain acyl-CoA synthetase-1 (ACSVL1) to active metabolite ETC-1002-CoA( 5 ).The metabolite reduces serum LDL-C by increasing liver uptake and clearance( 7 , 8 ). Ezetimibe (EZM) lowers LDL-C in serum by inhibiting the absorption of cholesterol in the small intestine, by targeting cholesterol transport protein Niemann-Pick C1-Like 1 (NPC1L1) protein( 9 ). Bempedoic acid and Ezetimibe show complementary action by reducing cholesterol synthesis in liver and intestinal absorption( 1 , 2 , 10 ). Bempedoic acid is white to off-white crystalline powder, a hydroxyalkane diacid, with low aqueous solubility and high solubility in organic solvents( 11 , 12 ). Chemically it is “8-Hydroxy-2,2,14,14-tetramethylpentadecanedioic acid”, with chemical formula C 19 H 36 O 5 having molar mass of 344.49( 11 , 12 ). Ezetimibe is white to off-white crystalline powder, substituted azetidin-2-one,freely soluble in ethanol, methanol, acetone and insoluble in water( 13 ). Chemically Ezetimibe is “(3R,4S)-1-(4-fluorophenyl)-3-{(3S)-3-(4-fluorophenyl)-3-hydroxypropyl}-4-(4-hydroxyphenyl)azetidin-2-one”, with molecular weight of 409.42g/mol and C 24 H 21 F 2 NO 3 is its chemical formula( 14 ). The chemical structures were shown in Fig. 1. Figure 1: Chemical Structure of (a) Bempedoic acid; (b) Ezetemibe The presence of impurities in active pharmaceutical ingredients (APIs) and in pharmaceutical dosage form not only effects purity, safety and efficacy of formulation but also reflects the non compliance of regulatory guidelines( 15 ). Hence international council for harmonization (ICH) provided guidelines for detection, identification, quantification and structure elucidation of impurities in their APIs [Q3A(R2)]( 16 ) or in dosage form [ICH Q3B(R2)]( 17 ) by various analytical techniques, of which HPLC is most widely employed owing to its high sensitivity, selectivity and reproducibility. Green Analytical Chemistry(GAC), a central paradigm in pharmaceutical analysis promotes design of analytical methods that minimize environmental impact, reduce hazard solvent consumption and improve lab safety with non-negiotable analytical performance. Adoption of green analytical principles lowers generation of toxic waste, reduces exposure to harmful reagents, decreases operational cost, thereby enhances both environmental and economic sustainability in pharmaceutical quality control laboratories. Analytical greenness assessment metrics such as “Green Analytical Procedure Index(GAPI) and Analytical GREEnness metric approach (AGREE)” serve as quantitative tools to evaluate the environmental footprint of the developed HPLC method. The review of literature revealed various chromatographic techniques for estimation of BPA and EZM in pure and dosage forms by HPLC( 18 – 23 ), HPLC-PDA method for their determination in rat plasma( 24 ), RP-UPLC( 25 , 26 ); impurity profiling of BPA by HPLC( 27 ), LC-MS/MS method for determination of BPA and its metabolites in human plasma( 28 ), EZM and its metabolite in human plasma( 29 ). However there is no validated specific HPLC method reported for impurity profiling in FDC of BPA & EZM. This gap is addressed by developing and validating a sensitive, rapid & robust high performance liquid chromatographic technique in reverse phase mode for concurrent determination of BPA, EZM and their impurities. MATERIALS AND METHODS The pure forms of Bempedoic acid, Ezetimibe and Impurities (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) were obtained from Glenmark Pharmaceuticals as gift samples with potency ranging from 99.52 to 99.98%. The HPLC-grade solvents Acetonitrile(ACN) and Water were procured form Merck ltd, Mumbai. The development and validation of current method was executed on Alliance 2695 (WATERS HPLC) equipped with PDA detector operated with Empower-2 software. Method Development The present technique was created with the help of Waters HPLC instrument (Alliance 2695), equipped with PDA and quaternary pump. The development of method was initiated with isocratic mode followed by gradient mode of elution with different mobile phase ratios containing Methanol, Acetonitrile, 0.1% Ortho Phosphoric Acid and 0.1% Tri Fluoro Acetic acid (TFA) to achieve better resolution between analytes & impurities, good peak shape and plate count. Finally, mobile phase of 0.1% TFA in water (Buffer) and Acetonitrile in conjunction with Inertsil C18 column (250×4.6mm, 5µm) was opted in gradient mode of elution as on basis of system suitability parameters. The eluted compounds were detected at 211nm when the liquid mobile phase is fed at a flow rate of 1ml/min to column at temperature of 30 ± 2°C. Preparation of standard solution Transfer 180mg of BPA and 10mg of EZM into a 100ml capacity volumetric flask, add 60ml of diluent,sonicated for 10mins, filtered through 0.45µ membrane and adjust the final volume. Preparation of Impurity standard solution 5mg of each impurity (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) were precisely weighed and transferred into 10ml volumetric flask, solubilized in 7ml of diluent, sonicated and volume was made upto the mark.A concentration of 10µg/ml was achieved by further diluting the solutionof each impurity. Preparation of Spiked standard solution Mix 1ml of each standard stock and impurity solution in a 10ml flask and diluted to 10ml with diluent to obtain the concentration of 180µg/ml of BPA, 10µg/ml of EZM and 1µg/ml of each impurity. Preparation of sample/test solution The finely powdered tablet equivalent to 180mg of BPA & 10mg of EZM was channelized into a 100ml volumetric flask, 60ml of diluent is added, sonicated for 10min; adjust the volume and passed through 0.45µm membrane filter. Further, adequate volume isdiluted to attain the concentrations of 180µg/ml of BPA, 10µg/ml of EZM. Method validation ICH Q2 (R2) criteria were adhered to in order to validate the proposed approach. System suitability The suitability of the developed method was ascertained by subsequent injections (n = 6) of standard solution containing 180µg/ml of BPA, 10µg/ml of EZM and 1µg/ml of each impurity (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) into chromatographic system. The recorded chromatograms were assessed for system suitability parameters such as theoretical plates (N), resolution, tailing factor/asymmetry(As) and %RSD of peak areas( 30 ). Specificity The method's capacity to ascertain the desired analyte even in the presence of other potential components is referred as specificity of method. The specificity ensures identification and quantification of target analyte in the presence of other interfering substances. Specificity of developed approach was evaluated by injecting blank and placebo solutions and the recorded chromatograms were assessed for the retention times of standards and impurities to identify the interferences at the respective retention times( 31 ). Linearity Thelinearity estimation for BPA, EZM and their related impurities was studied in the concentration levels of 10% to 150%, 18µg/ml to 270µg/ml for BPA; 0.10µg/ml to 1.50µg/ml for BPA Imp-1 & BPA Imp-2; 1µg/ml to 15µg/ml for EZM; 0.10µg/ml to 1.50µg/ml for EZM Imp-A & EZM Imp-B. The standard solutions of each concentration is injected in triplicate, linearity graph is plotted between concentration (abscissa) and peak area (ordinate) to compute R 2 value( 32 ). Accuracy Accuracy of the present approach was assessed through percentage recovery studies, in which the sample solutions were spiked with impurities at three levels of target concentration (50%, 100% & 150%). Each target concentration is serially injected thrice into the HPLC system and percentage mean recovery is calculated for each impurity in the spiked solutions( 33 ). Precision Method precision of current optimized HPLC technique was assessed by injecting 6 consecutive injectionsof sample solution spiked with impurities and calculating the percentage RSD for impurities( 34 ). Intermediate precision was established by injecting the sample solutions spiked with impurities on second day to check how precisely the method is able to quantify the impurities. The mean value of peak area is used to divide the standard deviation of six measurements, expressed as percentage to yield %RSD. Lower values of %RSD reflect higher precision. Sensitivity (LOD and LOQ) Calibration data derived from calibration curve was used to establish LOD & LOQ for BPA, EZM and their impurities. The following equations were employed to calculate LOD & LOQ, in which standard deviation (σ) and slope (S) values obtained from calibration plot were substituted( 35 ). $$LOD=3.3\sigma/S;LOQ=10\sigma/S$$ The smallest concentration of analyte that can be detected reliably is defined as LOD (Limit of Detection) while the lowest concentration that can be measured accurately is referred to as LOQ (Limit of Quantification). Robustness The optimized chromatographic conditions were intentionally altered in a controlled manner and the system suitability parameters were evaluated to assess the robustness of the developed HPLC method. The key analytical parameters subjected to variation included flow rate (± 0.1 ml/min), composition of mobile phase (± 10% of organic phase) and column temperature (± 5°C)which would assess the procedure reliability and overall robustness of the method indicating the method is least affected by operational factors and environmental factors. Forced Degradation Studies The degradation of sample under certain stress conditions was carried out to establish the method’s stability indicating nature and to identify possible degradants formed. The developed method is evaluated for its capability to separate degradants formed under various stressful conditions from the desired analytes.The sample is subjected to hydrolytic degradation under acidic, basic & neutral conditions; peroxide degradation; photolytic degradation; thermal degradation; reduction degradation. These studies prove the explicity of the method for analytes even in the presence of degradation products( 34 ). Assessment of GREEnness GAC metric tools such as AGREE and GAPI are used to evaluate the greenness of the developed method. The eco friendliness of chromatographic methods is evaluated by the comprehensive tool AGREE, developed to assess analytical greenness based on TWELVE principles of GAC. A twelve segmented circular diagram, each segment representing one green analytical principle, is used to express the method greenness by AGREE( 36 ). The score for each segment is given from 0 to 1, with overall value closer to 1 indicating a greener analytical method. GAPI examines every stage of analytical workflow to evaluate the environmental impact of the analytical method. The assessment is represented through five-section pentagonal pictogram divided into multiple colored fields, with color codes of green, yellow and red indicating low, medium and high impact on the environment( 37 ). RESULTS AND DISCUSSION Optimized method After several experimental runs, a methodology with octa decyl silane (ODS) Inertsil column with dimensions of 250×4.6mm having 5µ particle size, mobile phase comprising of 0.1% TFA (buffer) and Acetonitrile in gradient elution mode pumped at a rate of 1.0ml/min and 211nm as detection wavelength is used for separation of BPA, EZM, BPA Imputity-1, BPA Impurity − 2, EZM Impurity A & EZM Impurity B with a run time of 40 minutes.The gradient program was represented in Table 1 . The retention times were found to be BPA-3.4, BPA Impurity 1-5.4, BPA Impurity 2-7.9, EZM − 11.9, EZM Impurity A-16.7 and EZM Impurity B-17.6 with acceptable resolution. Table 1 Mobile phase gradient elution program for optimized method Time (min) Flow rate (ml/min) Buffer (0.1% TFA) (ml) Acetonitrile (ml) 0 0.1 80 20 10 0.1 50 50 20 0.1 20 80 30 0.1 50 50 40 0.1 50 50 Method validation System suitability and Specificity The chromatographic parameters such as theoretical plates (N > 2000), tailing factor (NMT 2.0), percentage RSD of peak areas (NMT2.0) were assessed to establish the system suitability. The observed values for BPA: N (9076), tailing factor (0.92), % RSD (1.36); for EZM:plate count (N 7820), tailing factor (1.0), % RSD (0.31) substantially prove the suitability of the method proposed. Table 2 depicts all the system suitability parameters including sensitivity and robustness data. Evaluation of method specificity was achieved by injecting standard solutions of BPA, EZM and their impurities separately, standard spiked solution, sample solution, blank and placebo. At the retention times of BPA, EZM &Impurities (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) no interference peaks were observed in placebo, impurities were not eluted at the retention times of BPA & EZM and no additional peaks were observed in standard and sample solutions as shown in Fig. 2. Table 2 System suitability results for Bempedoic acid, Ezetimibe and their Impurities Rt (min) BPA BPA Imp-1 BPA Imp-2 EZM EZM Imp A EZM Imp B 3.4 5.4 7.96 11.92 16.73 17.66 USP plate 7920 8721 15468 19076 22397 24615 USP tailing 0.92 0.78 0.93 1.00 0.76 0.95 % RSD (peak area) 1.36 0.72 0.83 0.31 1.29 1.08 Linearity (µg/ml) 18–270 0.10–1.50 0.10–1.50 1–15 0.10–1.50 0.10–1.50 R 2 0.9997 0.9998 0.9993 0.9994 0.9992 0.9997 LOD (µg/ml) 5.4 0.03 0.03 0.3 0.03 0.03 LOQ (µg/ml) 18 0.1 0.1 1 0.1 0.1 Flow rate Minus(0.9ml/min) %RSD 0.89 0.97 1.19 0.56 1.09 0.93 Flow rate Plus(1.1ml/min) %RSD 0.47 1.23 0.92 1.02 0.73 0.81 Mobile Phase Plus(+ 10%) %RSD 1.05 0.78 0.69 0.23 0.54 0.79 Mobile Phase minus(-10%) %RSD 0.56 0.97 0.85 0.34 1.23 0.83 Assay 99.24 - - 100.21 - - Stability (20–25°C)) stable stable stable stable stable stable Stability (2–8°C) stable stable stable stable stable stable Figure 2: Chromatogram of Blank (A) and Placebo (B) Linearity The correlation between analyte concentrations and the detector response (Peak area) was assessed to establish the linearity of the developed method. The peak areas were recorded from triplicate injections of analytes and impurities at various concentrations within a designated range. The coefficient of determination (R 2 ) is above 0.999 suggesting the responses are in harmony with increasing concentration of analyte/impurity across the stated interval of concentrations. The linearity range and correlation values (R 2 ) were given in Table 3 and plots were presented in Fig. 3. Thus the developed method can quantify the analytes and their impurities over or within the stated range of concentrations. Figure 3: Linearity plots of BPA (a); BPA Imp–1(b); BPA Imp-2 (c); EZM (d); EZM Imp-A(e) and EZM Imp-B (f) Table 3 Linearity data and results obtained from developed HPLC method Conc (µg/ml) Peak Area BPA Conc (µg/ml) Peak Area EZM Conc (µg/ml) Peak Area BPA Imp-1 Peak Area BPA Imp-2 Peak Area EZM Imp-A Peak Area EZM Imp B 18.00 1575421 1.00 690347 0.10 18541 39402 42630 41975 45.00 3506514 2.50 1742365 0.25 50320 105274 112407 107498 90.00 7098754 5.00 3560207 0.50 92621 195402 215698 202017 135.00 10320561 7.50 5412657 0.75 140218 306528 320505 299845 180.00 14102364 10.00 6823479 1.00 192145 386547 402517 413659 225.00 17874157 12.50 8596741 1.25 238651 473248 523064 512439 270.00 20889585 15.00 10123647 1.50 284156 567814 603251 603247 Slope 77845.56 678833.93 189987.83 377674.74 404372.27 404641.13 Intercept 50260.25 78978.47 27.14 6706.89 7085.04 1981.24 R 2 0.9997 0.9994 0.9998 0.9993 0.9992 0.9997 Accuracy From the recovery studies, method accuracy is determined at three concentration levels (50%, 100% & 150%) of test solution spiked with impurities. The acceptable percentage recovery for BPA, EZM and their impurities confirms the possibility of the method for estimation of content uniformity where the % recovery values were within the range of 100.10 to 100.8% as depicted in Table 4 . Table 4 Percentage recovery results of Bempedoic acid, Ezetimibe and their Impurities. % Level Percentage Mean Recovery ± RSD BPA BPA Imp-1 BPA Imp-2 EZM EZM Imp-A EZM Imp B 50 100.2 ± 0.17 100.1 ± 1.7 100.3 ± 0.11 100.2 ± 0.25 100.1 ± 0.25 100.3 ± 0.17 100 100.8 ± 0.2 100.4 ± 0.05 100.3 ± 0.37 100.6 ± 0.49 100.3 ± 0.25 100.3 ± 0.32 150 100.1 ± 0.199 100.5 ± 0.25 100.4 ± 0.3 100.2 ± 0.15 100.5 ± 0.26 100.2 ± 0.09 Precision Repeatability or Intraday precision reflects the degree of consistency among a series of measurements obtained under identical experimental conditions on same day. The test solution (n = 6) was injected into HPLC system under the same set of conditions to evaluate method precision and is conveyed as percentage relative standard deviation (%RSD). Lower values of %RSD(less than 2.0) demonstrate reproducibility of the developed method with more consistency. Tables 5 and 6 show the data for method precision and intermediate precision. Table 5 Method Precision data of Bempedoic acid, Ezetimibe and their impurities Injection No BPA BPA Imp-1 BPA Imp-2 EZM EZM Imp A EZM Imp B 1 13,924,520 6,801,220 190,020 381,220 398,420 410,225 2 14,157,900 6,874,930 191,875 384,755 400,785 412,480 3 14,206,700 6,912,445 193,640 386,740 401,960 414,905 4 14,279,200 6,745,600 194,280 388,615 404,215 416,330 5 14,027,160 6,990,150 195,955 389,782 406,380 418,720 6 14,018,706 6,616,529 187,100 388,470 403,342 409,294 M ean 14,102,364 6,823,479 192,145 386,597 402,517 413,659 SD 122150.19 120920.28 2925.46 2888.2007 2535.8128 3330.068 %RSD 0.8661681 1.7721206 1.522527 0.7470831 0.629989 0.805027 Table 6 Intermediate precision data for Bempedoic acid Impurities and Ezetimibe impurities S.No % Purity of related substances BPA EZM Day 1 Day 2 Day 1 Day 2 1 99.76 99.77 99.46 99.48 2 99.73 99.74 99.43 99.45 3 99.75 99.78 99.45 99.46 4 99.78 99.75 99.49 99.44 5 99.77 99.77 99.51 99.45 6 99.74 99.75 99.48 99.46 Average 99.755 99.76 99.47 99.45667 SD 0.017078 0.0141421 0.0264575 0.012472 % RSD 0.01712 0.0141762 0.0265985 0.01254 Robustness The intentional changes in experimental conditions have no influence/impact on performance of the developed method. The detection of impurities was not influenced by changes in flow rate, mobile phase composition and column temperature, which showed only minor deviation of retention time and/or resolution as shown in Table 2 . As these minor variations in chromatographic conditions had not affected the detectable results noticeably, it strongly suggests the robustness of the method. Forced degradation studies The spiked sample solution was exposed to various stress conditions in the laboratory to induce degradation of analytes and the recorded chromatograms were evaluated whether the degradant peaks were interfering with analyte peaks. Lower values of purity angle than purity threshold suggests quantification of analytes even when degradants reside. The degradation data and the chromatograms were presented in Table 7 . Table 7 Forced Degradation Studies data of Bempedoic acid and Ezetimibe Stress condition BPA EZM % P % D % P % D Acid degradation 94.36 5.64 92.14 7.86 Alkali degradation 93.17 6.83 90.54 9.46 Oxidative degradation 92.44 7.56 89.17 10.83 Reduction degradation 95.21 4.79 94.36 5.64 Thermal degradation 94.18 5.82 92.77 7.23 Photolytic degradation 96.16 3.84 95.36 4.64 Hydrolysis degradation 98.24 1.76 97.68 2.32 % P: Percentage Purity; % D: Percentage Degradation Application of proposed method The proposed and developed method was applied for the assay of fixed dose combination, Bempesta EZ (Torrent Pharmaceuticals Ltd). The percentage purity of BPA and EZM were computed from the peak areas and the satisfactory result confirms the intended suitability of the method. The assay values and the chromatograms were represented in Table 2 and Fig. 4 . Assessment of GREEnness AGREE and GAPI tools were used for thorough evaluation of proposed analytical methodology. The experimental procedures followed were given as input into the software and the pictograms were presented in Fig. 5. The AGREE & GAPI yielded a score of 0.77 and 85 confirming the high level of environmental compatibility and strong alignment with GAC principles. The assessment reveals most aspects of methodology such as sample amount, number of preparation steps, automation, avoidance of derivatization, waste control, analytical throughput and energy efficiency are rated green demonstrating minimal sample consumption and efficient chromatographic operation. Figure 5: Greenness evaluation (a) AGREE- Analytical GREEnness Metric Approach (b) GAPI-Green Analytical Procedure Index CONCLUSION An efficient and sensitive reverse phase HPLC method for computation of Bempedoic acid, BPA Impurity-1, BPA Impurity-2, Ezetimibe, EZM Impurity-A and EZM Impurity-B in bulk and fixed dose combination tablets was successfully improved and validated complying the specifications as per ICH guidelines. With the optimized method parameters, the developed method is simple, specific, accurate, precise and robust. The prominent features of the method include high sensitivity and reproducible specificity. The felicity of the method for stability testing was confirmed through FDS studies. The scores of green metric tools confirmed the environmental friendliness of the proposed RP-HPLC method balancing the analytical performance. Hence, the present HPLC method is highly suitable for routine impurity profiling of Bempedoic acid(BPA) and Ezetimibe(EZM) in bulk and fixed dose combination. Declarations Conflicts of Interest: The authors declare no conflicts of interest. Author Contribution Sridevi ranjitha, Divya Narla and SrinivasRao Yarraguntla : Conceptualization, supervision, methodology design, validation strategy, critical review of manuscript and final approval Sai Priyanka, Rohan Ratnakar, Neelima, Satvik : Method development, Chromatographic analysis, validation experiments, preparation of tables and figures.Divya and Sridevi Ranjitha : Forced Degradation studies, validation experiments, Interpretation of analytical results, statistical analysis.Divya Narla : Green analytical assessment – AGREE and GAPI evaluation, data curation and preparation of graphical representations.All authors reviewed and accepted the manuscript. References Ballantyne CM, Laufs U, Ray KK, Leiter LA, Bays HE, Goldberg AC, et al. Bempedoic acid plus ezetimibe fixed-dose combination in patients with hypercholesterolemia and high CVD risk treated with maximally tolerated statin therapy. 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Front Pharmacol. 2024;15. doi: 10.3389/fphar.2024.1406493 Bhagavathula AS, Al Matrooshi NO, Clark CCT, Rahmani J. Bempedoic Acid and Ezetimibe for the Treatment of Hypercholesterolemia: A Systematic Review and Meta-Analysis of Randomized Phase II/III trials. Clin Drug Investig. 2021;41(1):19–28. doi: 10.1007/s40261-020-00989-1 Bempedoic acid [Internet]. [cited 2025 Sep 25]. Available from: https://go.drugbank.com/drugs/DB11936 American Chemical Society [Internet]. [cited 2025 Sep 25]. Bempedoic acid. Available from: https://www.acs.org/molecule-of-the-week/archive/b/bempedoic-acid.html Ezetimibe [Internet]. [cited 2025 Nov 15]. Available from: https://go.drugbank.com/drugs/DB00973 PubChem. Ezetimibe [Internet]. [cited 2025 Nov 15]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/150311 Finotti Cordeiro C, Lopardi Franco L, Teixeira Carvalho D, Bonfilio R. Impurities in Active Pharmaceutical Ingredients and Drug Products: A Critical Review. Critical Reviews in Analytical Chemistry. 0(0):1–21. doi:10.1080/10408347.2024.2384046 PubMed PMID: 39058576. Abraham J. International Conference On Harmonisation Of Technical Requirements For Registration Of Pharmaceuticals For Human Use. In: Tietje C, Brouder A, editors. Handbook of Transnational Economic Governance Regimes [Internet]. Brill | Nijhoff; 2010 [cited 2025 Nov 15]. p. 1041–53. Available from: https://brill.com/view/book/edcoll/9789004181564/Bej.9789004163300.i-1081_085.xml doi: 10.1163/ej.9789004163300.i-1081.897 Abraham J. International Conference On Harmonisation Of Technical Requirements For Registration Of Pharmaceuticals For Human Use. In: Brouder A, editor. Handbook of Transnational Economic Governance Regimes [Internet]. Brill | Nijhoff; 2010 [cited 2025 Nov 15]. p. 1041–53. Available from: https://brill.com/view/book/edcoll/9789004181564/Bej.9789004163300.i-1081_085.xml doi: 10.1163/ej.9789004163300.i-1081.897 Bairam R, Navya G, S.y M, Balakrishanaiah P. A New Stability Indicating RP-HPLC Method Development and Validation for the Simultaneous Estimation of Bempedoic Acid and Ezetimibe in Pharmaceutical Dosage Form. IJSAT - International Journal on Science and Technology. 2025;16(1). doi: 10.71097/IJSAT.v16.i1.1202 Bhuvanagiri SK, Kumar J. DEVELOPMENT AND VALIDATION OF A RP - HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF BEMPEDOIC ACID & EZETIMIBE IN PURE AND PHARMACEUTICAL DOSAGE FORM. Vol. 12. 2022;12(01). Maheshwari K, Rani SS. Validated method for the simultaneous estimation of bempedoic acid and ezetimibein bulk and tablet formulation by RP-HPLC method: https://doi.org/10.54037/WJPS.2022.100903 . World Journal of Pharmaceutical Sciences. 2022;33–41. Deepthi N. A Novel Rp-Hplc Method Development and Validation for Determination and Estimation of Bempedoic Acid and Ezetimibe drug with its Bulk form and Tablet Formulation. World Journal of Pharmaceutical Sciences [Internet]. 2025 Aug 29 [cited 2025 Sep 15]. Available from: https://wjpsonline.com/index.php/wjps/article/view/1915 Priya KC. DEVELOPMENT AND VALIDATION OF A ROBUST RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF BEMPEDOIC ACID AND EZETIMIBE IN PHARMACEUTICAL DOSAGE FORMS. World Journal of Pharmaceutical Sciences [Internet]. 2025 Jun 5 [cited 2025 Sep 15]. Available from: https://www.wjpsonline.com/index.php/wjps/article/view/1839 Suresh A, Balakrishnan A, Ramaswamy V, Natesan S. Analytical method development and validation for simultaneous estimation of Bempedoic acid and Ezetimibe in pure and its pharmaceutical dosage form by RP-HPLC. Biomedical Chromatography. 2024;38(9):e5938. doi: 10.1002/bmc.5938 Karla VR, Raghasudha M, Chitta R. Simultaneous Determination of Bempedoic Acid and Ezetimibe in Rat Plasma Using HPLC–PDA and Its Applications to a Pharmacokinetic Study. Chemistry Africa. 2022;5(4):917–27. doi: 10.1007/s42250-022-00392-7 Yarra UST, Gummadi S. Stability indicating RP-UPLC method for simultaneous quantification of bempedoic acid and ezetimibe in bulk and pharmaceutical formulations. Futur J Pharm Sci. 2021;7(1):209. doi: 10.1186/s43094-021-00363-8 Reddemma M, Valligatla P, Bojan S, Ellampati SK, Tummalapalli M, Salva C. Quantification of Bempedoic Acid and Ezetimibe with Comprehensive Stability Assessment in Drug Substance and Drug Product by UPLC. Biosciences Biotechnology Research Asia [Internet]. 2025 Sep 18 [cited 2025 Oct 6];22(3). Available from: https://www.biotech-asia.org/vol22no3/quantification-of-bempedoic-acid-and-ezetimibe-with-comprehensive-stability-assessment-in-drug-substance-and-drug-product-by-uplc/ Ibrahim MA, Algohary AM, A.Babaker M, Ibrahim AM. Design of Experiment and green analytical chemistry principles for developing a robust and sustainable Stability-Indicating HPLC method for Bempedoic acid impurity profiling. Microchemical Journal. 2024;207:112133. doi: 10.1016/j.microc.2024.112133 Engel BJ, Preusch K, Brown C, Cramer CT, Shoup R. Measurement of bempedoic acid and its keto metabolite in human plasma and urine using solid phase extraction and electrospray LC-MS/MS. Journal of Chromatography B. 2020;1154:122291. doi: 10.1016/j.jchromb.2020.122291 Mahesh M, Devanna N. Development and validation of a QuEChERS-Based LC-MS/MS method for simultaneous quantification of Ezetimibe and its active metabolite in human plasma. Analytical Chemistry Letters. 2025;15(3):492–503. doi: 10.1080/22297928.2025.2505626 Pappula N, Narla D. Development and Validation of Stability Indicating UHPLC Method for Simultaneous Estimation of Ombitasvir, Paritaprevir and Ritonavir in Pharmaceutical Dosage Forms. RJPT. 2022;1307–12. doi: 10.52711/0974-360X.2022.00218 Koppisetty BRB, Kollabathula VR, Challa GN, Yarraguntla SR. Quality by Design-Assisted RP-HPLC Method for Determination of Ritonavir and Darunavir in Pharmaceutical Formulation Using Central Composite Design. SEPARATION SCIENCE PLUS. 2024;7(10):e202300210. doi: 10.1002/sscp.202300210 Panda M, Dadi V, Yarraguntla SR, K VPR. RP-HPLC Method for Determination of Azelnidipine and Telmisartan in Pharmaceutical Dosage Form. Research Journal of Pharmacy and Technology. 2023;16(2):509–13. doi: 10.52711/0974-360X.2023.00086 Swathi S, Kumar HT, Rao PK. Validated RP-HPLC method for simultaneous determination of rosuvastatin calcium and ezetimibe in pharmaceutical dosage form. Int J Pharm And Pharm sci. 2015;7(4):209–13. Narla D, Pappula N, Sarella PNK. Development and validation of a versatile ultra performance liquid chromatography method for simultaneous estimation of selected antiviral drugs in bulk and dosage form. actapharm. 2025;63(3):611. doi: 10.23893/1307-2080.APS6339 Rajendra Prasad Y, Karanam SR. DEVELOPMENT AND VALIDATION OF NEW METHOD FOR THE SIMULTANEOUS ESTIMATION OF CEFTRIAXONE AND TAZOBACTAM USING RP-HPLC. YMER. 2022;21(03):45–57. doi: 10.37896/YMER21.03/06 Pena-Pereira F, Wojnowski W, Tobiszewski M. AGREE—Analytical GREEnness Metric Approach and Software. Anal Chem. 2020;92(14):10076–82. doi: 10.1021/acs.analchem.0c01887 Mansour FR, Płotka-Wasylka J, Locatelli M. Modified GAPI (MoGAPI) Tool and Software for the Assessment of Method Greenness: Case Studies and Applications. Analytica. 2024;5(3):451–7. doi: 10.3390/analytica5030030 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9113640","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607673703,"identity":"9ca0e0f0-451d-493a-876e-613a644c2ea5","order_by":0,"name":"Sridevi Ranjitha Karanam","email":"","orcid":"","institution":"Vignan Institute of Pharmaceutical Technology , Duvvada,","correspondingAuthor":false,"prefix":"","firstName":"Sridevi","middleName":"Ranjitha","lastName":"Karanam","suffix":""},{"id":607673708,"identity":"42a6b2bb-715e-4f69-94ab-da31fa0cff6f","order_by":1,"name":"Divya Narla","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYHACgwNAQg7KYQYjorQYk6YFRCQ2wLUQAuYMzBsP87bZpfe3Hz72gKHCOrGBnfcAXi2WDWwFQC3JuTPOpKUbMJxJT2xg5kvA76oDPAYHZ7Yx5zbc4DGTYGw7DNTCY0CMlvp0ebCWf0RqOfCx7XCCAVhLAzFaDrMVHPhw7rjhRpBfEo6lG7cR1HK8efOHhLJqebnjwBD7UGMt289/Br8WcEQwsoGZbAwJYJIo8AeqZRSMglEwCkYBNgAAPBBC8otBxiUAAAAASUVORK5CYII=","orcid":"","institution":"Aditya College of Pharmacy, Surampalem","correspondingAuthor":true,"prefix":"","firstName":"Divya","middleName":"","lastName":"Narla","suffix":""},{"id":607673711,"identity":"2472ada5-5a9d-46f7-9d5c-a7f8f57e7b93","order_by":2,"name":"Sai Priyanka Kruthiveni","email":"","orcid":"","institution":"Aditya College of Pharmacy, Surampalem","correspondingAuthor":false,"prefix":"","firstName":"Sai","middleName":"Priyanka","lastName":"Kruthiveni","suffix":""},{"id":607673714,"identity":"fce17497-0997-4abf-aca8-a39264aa7e38","order_by":3,"name":"Rohan Ratnakar Peela","email":"","orcid":"","institution":"Vignan Institute of Pharmaceutical Technology , Duvvada,","correspondingAuthor":false,"prefix":"","firstName":"Rohan","middleName":"Ratnakar","lastName":"Peela","suffix":""},{"id":607673715,"identity":"36211687-68f1-4274-a92c-1576cfe05027","order_by":4,"name":"Neelima Mummuluri","email":"","orcid":"","institution":"Vignan Institute of Pharmaceutical Technology , Duvvada,","correspondingAuthor":false,"prefix":"","firstName":"Neelima","middleName":"","lastName":"Mummuluri","suffix":""},{"id":607673718,"identity":"50caf0a0-e839-4a23-969c-beefffd88407","order_by":5,"name":"Satvik Mudili","email":"","orcid":"","institution":"Vignan Institute of Pharmaceutical Technology , Duvvada,","correspondingAuthor":false,"prefix":"","firstName":"Satvik","middleName":"","lastName":"Mudili","suffix":""},{"id":607673719,"identity":"5edc0036-887e-4a75-a7c1-2bbcfe3cba0c","order_by":6,"name":"Srinivasa Rao Yarraguntla","email":"","orcid":"","institution":"Vignan Institute of Pharmaceutical Technology , Duvvada,","correspondingAuthor":false,"prefix":"","firstName":"Srinivasa","middleName":"Rao","lastName":"Yarraguntla","suffix":""}],"badges":[],"createdAt":"2026-03-13 10:28:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9113640/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9113640/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105034281,"identity":"477041c4-f392-4e49-a190-92f3fe3af137","added_by":"auto","created_at":"2026-03-20 07:23:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChemical Structure of (a) Bempedoic acid; (b) Ezetemibe\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9113640/v1/c9842345e542649f95885e98.jpg"},{"id":104885950,"identity":"b8ba2876-389a-41a9-8ba1-c698b37256ec","added_by":"auto","created_at":"2026-03-18 10:02:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatogram of Blank (A) and Placebo (B)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9113640/v1/c875577f9963e39fc8df0715.jpg"},{"id":104885951,"identity":"97f50f25-0538-4b04-b4ed-97fd55bb0a84","added_by":"auto","created_at":"2026-03-18 10:02:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":65671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinearity plots of BPA (a); BPA Imp–1(b); BPA Imp-2 (c); EZM (d); EZM Imp-A(e) and EZM Imp-B (f)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9113640/v1/596674a182a574277760966e.jpg"},{"id":104885954,"identity":"43fa3682-3d24-4f21-9ba6-1f5c0abdb977","added_by":"auto","created_at":"2026-03-18 10:03:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":31506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatogram of Standard (a) and Test (b)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9113640/v1/ccb511bcfa22f1935482e460.jpg"},{"id":104885953,"identity":"61f8ae68-7170-41d1-84bc-7ed04dc71b59","added_by":"auto","created_at":"2026-03-18 10:02:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGreenness evaluation (a) AGREE- Analytical GREEnness Metric Approach (b) GAPI-Green Analytical Procedure Index\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9113640/v1/3f625b8810d7b0fe48fe490c.jpg"},{"id":105495014,"identity":"f8259103-c5e5-4647-8fe2-00e2dcad49e3","added_by":"auto","created_at":"2026-03-26 16:11:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1662203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9113640/v1/d061e8dc-f062-4f89-b2a6-62cf8f53982e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Green Analytical Stability-Indicating RP-HPLC method for Simultaneous Determination of Anti-Hypercholesterolemic Agents and Their related Impurities","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBempedoic acid and Ezetimibe is a fixed dose combination (FDC) approved by USFDA for the treatment of hypercholesterolemia with high Low Density Lipoprotein Cholesterol (LDL-C)(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is prescribed for people with AtheroSclerotic Cardiovascular Disease (ASCVD)(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) who require additional reduction of LDL-C and for those having statin intolerance(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBempedoic acid(BPA) is a prodrug, belonging to class of \u0026lsquo;Adenosine triphosphate citrate lyase\u0026rsquo;(ACL) inhibitor(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), activated in liver by very long chain acyl-CoA synthetase-1 (ACSVL1) to active metabolite ETC-1002-CoA(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).The metabolite reduces serum LDL-C by increasing liver uptake and clearance(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Ezetimibe (EZM) lowers LDL-C in serum by inhibiting the absorption of cholesterol in the small intestine, by targeting cholesterol transport protein Niemann-Pick C1-Like 1 (NPC1L1) protein(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Bempedoic acid and Ezetimibe show complementary action by reducing cholesterol synthesis in liver and intestinal absorption(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Bempedoic acid is white to off-white crystalline powder, a hydroxyalkane diacid, with low aqueous solubility and high solubility in organic solvents(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Chemically it is \u0026ldquo;8-Hydroxy-2,2,14,14-tetramethylpentadecanedioic acid\u0026rdquo;, with chemical formula C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e having molar mass of 344.49(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Ezetimibe is white to off-white crystalline powder, substituted azetidin-2-one,freely soluble in ethanol, methanol, acetone and insoluble in water(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Chemically Ezetimibe is \u0026ldquo;(3R,4S)-1-(4-fluorophenyl)-3-{(3S)-3-(4-fluorophenyl)-3-hydroxypropyl}-4-(4-hydroxyphenyl)azetidin-2-one\u0026rdquo;, with molecular weight of 409.42g/mol and C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003eis its chemical formula(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The chemical structures were shown in Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1: Chemical Structure of (a) Bempedoic acid; (b) Ezetemibe\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe presence of impurities in active pharmaceutical ingredients (APIs) and in pharmaceutical dosage form not only effects purity, safety and efficacy of formulation but also reflects the non compliance of regulatory guidelines(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Hence international council for harmonization (ICH) provided guidelines for detection, identification, quantification and structure elucidation of impurities in their APIs [Q3A(R2)](\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) or in dosage form [ICH Q3B(R2)](\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) by various analytical techniques, of which HPLC is most widely employed owing to its high sensitivity, selectivity and reproducibility.\u003c/p\u003e \u003cp\u003eGreen Analytical Chemistry(GAC), a central paradigm in pharmaceutical analysis promotes design of analytical methods that minimize environmental impact, reduce hazard solvent consumption and improve lab safety with non-negiotable analytical performance. Adoption of green analytical principles lowers generation of toxic waste, reduces exposure to harmful reagents, decreases operational cost, thereby enhances both environmental and economic sustainability in pharmaceutical quality control laboratories. Analytical greenness assessment metrics such as \u0026ldquo;Green Analytical Procedure Index(GAPI) and Analytical GREEnness metric approach (AGREE)\u0026rdquo; serve as quantitative tools to evaluate the environmental footprint of the developed HPLC method. The review of literature revealed various chromatographic techniques for estimation of BPA and EZM in pure and dosage forms by HPLC(\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), HPLC-PDA method for their determination in rat plasma(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), RP-UPLC(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e); impurity profiling of BPA by HPLC(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), LC-MS/MS method for determination of BPA and its metabolites in human plasma(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), EZM and its metabolite in human plasma(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However there is no validated specific HPLC method reported for impurity profiling in FDC of BPA \u0026amp; EZM. This gap is addressed by developing and validating a sensitive, rapid \u0026amp; robust high performance liquid chromatographic technique in reverse phase mode for concurrent determination of BPA, EZM and their impurities.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eThe pure forms of Bempedoic acid, Ezetimibe and Impurities (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) were obtained from Glenmark Pharmaceuticals as gift samples with potency ranging from 99.52 to 99.98%. The HPLC-grade solvents Acetonitrile(ACN) and Water were procured form Merck ltd, Mumbai. The development and validation of current method was executed on Alliance 2695 (WATERS HPLC) equipped with PDA detector operated with Empower-2 software.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMethod Development\u003c/h2\u003e \u003cp\u003eThe present technique was created with the help of Waters HPLC instrument (Alliance 2695), equipped with PDA and quaternary pump. The development of method was initiated with isocratic mode followed by gradient mode of elution with different mobile phase ratios containing Methanol, Acetonitrile, 0.1% Ortho Phosphoric Acid and 0.1% Tri Fluoro Acetic acid (TFA) to achieve better resolution between analytes \u0026amp; impurities, good peak shape and plate count. Finally, mobile phase of 0.1% TFA in water (Buffer) and Acetonitrile in conjunction with Inertsil C18 column (250\u0026times;4.6mm, 5\u0026micro;m) was opted in gradient mode of elution as on basis of system suitability parameters. The eluted compounds were detected at 211nm when the liquid mobile phase is fed at a flow rate of 1ml/min to column at temperature of 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of standard solution\u003c/h3\u003e\n\u003cp\u003eTransfer 180mg of BPA and 10mg of EZM into a 100ml capacity volumetric flask, add 60ml of diluent,sonicated for 10mins, filtered through 0.45\u0026micro; membrane and adjust the final volume.\u003c/p\u003e\n\u003ch3\u003ePreparation of Impurity standard solution\u003c/h3\u003e\n\u003cp\u003e5mg of each impurity (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) were precisely weighed and transferred into 10ml volumetric flask, solubilized in 7ml of diluent, sonicated and volume was made upto the mark.A concentration of 10\u0026micro;g/ml was achieved by further diluting the solutionof each impurity.\u003c/p\u003e\n\u003ch3\u003ePreparation of Spiked standard solution\u003c/h3\u003e\n\u003cp\u003eMix 1ml of each standard stock and impurity solution in a 10ml flask and diluted to 10ml with diluent to obtain the concentration of 180\u0026micro;g/ml of BPA, 10\u0026micro;g/ml of EZM and 1\u0026micro;g/ml of each impurity.\u003c/p\u003e\n\u003ch3\u003ePreparation of sample/test solution\u003c/h3\u003e\n\u003cp\u003eThe finely powdered tablet equivalent to 180mg of BPA \u0026amp; 10mg of EZM was channelized into a 100ml volumetric flask, 60ml of diluent is added, sonicated for 10min; adjust the volume and passed through 0.45\u0026micro;m membrane filter. Further, adequate volume isdiluted to attain the concentrations of 180\u0026micro;g/ml of BPA, 10\u0026micro;g/ml of EZM.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eICH Q2 (R2) criteria were adhered to in order to validate the proposed approach.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSystem suitability\u003c/h3\u003e\n\u003cp\u003eThe suitability of the developed method was ascertained by subsequent injections (n\u0026thinsp;=\u0026thinsp;6) of standard solution containing 180\u0026micro;g/ml of BPA, 10\u0026micro;g/ml of EZM and 1\u0026micro;g/ml of each impurity (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) into chromatographic system. The recorded chromatograms were assessed for system suitability parameters such as theoretical plates (N), resolution, tailing factor/asymmetry(As) and %RSD of peak areas(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSpecificity\u003c/h3\u003e\n\u003cp\u003eThe method's capacity to ascertain the desired analyte even in the presence of other potential components is referred as specificity of method. The specificity ensures identification and quantification of target analyte in the presence of other interfering substances. Specificity of developed approach was evaluated by injecting blank and placebo solutions and the recorded chromatograms were assessed for the retention times of standards and impurities to identify the interferences at the respective retention times(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLinearity\u003c/h2\u003e \u003cp\u003eThelinearity estimation for BPA, EZM and their related impurities was studied in the concentration levels of 10% to 150%, 18\u0026micro;g/ml to 270\u0026micro;g/ml for BPA; 0.10\u0026micro;g/ml to 1.50\u0026micro;g/ml for BPA Imp-1 \u0026amp; BPA Imp-2; 1\u0026micro;g/ml to 15\u0026micro;g/ml for EZM; 0.10\u0026micro;g/ml to 1.50\u0026micro;g/ml for EZM Imp-A \u0026amp; EZM Imp-B. The standard solutions of each concentration is injected in triplicate, linearity graph is plotted between concentration (abscissa) and peak area (ordinate) to compute R\u003csup\u003e2\u003c/sup\u003e value(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAccuracy\u003c/h2\u003e \u003cp\u003eAccuracy of the present approach was assessed through percentage recovery studies, in which the sample solutions were spiked with impurities at three levels of target concentration (50%, 100% \u0026amp; 150%). Each target concentration is serially injected thrice into the HPLC system and percentage mean recovery is calculated for each impurity in the spiked solutions(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrecision\u003c/h2\u003e \u003cp\u003eMethod precision of current optimized HPLC technique was assessed by injecting 6 consecutive injectionsof sample solution spiked with impurities and calculating the percentage RSD for impurities(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Intermediate precision was established by injecting the sample solutions spiked with impurities on second day to check how precisely the method is able to quantify the impurities. The mean value of peak area is used to divide the standard deviation of six measurements, expressed as percentage to yield %RSD. Lower values of %RSD reflect higher precision.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSensitivity (LOD and LOQ)\u003c/h2\u003e \u003cp\u003eCalibration data derived from calibration curve was used to establish LOD \u0026amp; LOQ for BPA, EZM and their impurities. The following equations were employed to calculate LOD \u0026amp; LOQ, in which standard deviation (σ) and slope (S) values obtained from calibration plot were substituted(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$LOD=3.3\\sigma/S;LOQ=10\\sigma/S$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe smallest concentration of analyte that can be detected reliably is defined as LOD (Limit of Detection) while the lowest concentration that can be measured accurately is referred to as LOQ (Limit of Quantification).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRobustness\u003c/h2\u003e \u003cp\u003eThe optimized chromatographic conditions were intentionally altered in a controlled manner and the system suitability parameters were evaluated to assess the robustness of the developed HPLC method. The key analytical parameters subjected to variation included flow rate (\u0026plusmn;\u0026thinsp;0.1 ml/min), composition of mobile phase (\u0026plusmn;\u0026thinsp;10% of organic phase) and column temperature (\u0026plusmn;\u0026thinsp;5\u0026deg;C)which would assess the procedure reliability and overall robustness of the method indicating the method is least affected by operational factors and environmental factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eForced Degradation Studies\u003c/h2\u003e \u003cp\u003eThe degradation of sample under certain stress conditions was carried out to establish the method\u0026rsquo;s stability indicating nature and to identify possible degradants formed. The developed method is evaluated for its capability to separate degradants formed under various stressful conditions from the desired analytes.The sample is subjected to hydrolytic degradation under acidic, basic \u0026amp; neutral conditions; peroxide degradation; photolytic degradation; thermal degradation; reduction degradation. These studies prove the explicity of the method for analytes even in the presence of degradation products(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of GREEnness\u003c/h2\u003e \u003cp\u003eGAC metric tools such as AGREE and GAPI are used to evaluate the greenness of the developed method. The eco friendliness of chromatographic methods is evaluated by the comprehensive tool AGREE, developed to assess analytical greenness based on TWELVE principles of GAC. A twelve segmented circular diagram, each segment representing one green analytical principle, is used to express the method greenness by AGREE(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The score for each segment is given from 0 to 1, with overall value closer to 1 indicating a greener analytical method. GAPI examines every stage of analytical workflow to evaluate the environmental impact of the analytical method. The assessment is represented through five-section pentagonal pictogram divided into multiple colored fields, with color codes of green, yellow and red indicating low, medium and high impact on the environment(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eOptimized method\u003c/h2\u003e \u003cp\u003eAfter several experimental runs, a methodology with octa decyl silane (ODS) Inertsil column with dimensions of 250\u0026times;4.6mm having 5\u0026micro; particle size, mobile phase comprising of 0.1% TFA (buffer) and Acetonitrile in gradient elution mode pumped at a rate of 1.0ml/min and 211nm as detection wavelength is used for separation of BPA, EZM, BPA Imputity-1, BPA Impurity\u0026thinsp;\u0026minus;\u0026thinsp;2, EZM Impurity A \u0026amp; EZM Impurity B with a run time of 40 minutes.The gradient program was represented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The retention times were found to be BPA-3.4, BPA Impurity 1-5.4, BPA Impurity 2-7.9, EZM\u0026thinsp;\u0026minus;\u0026thinsp;11.9, EZM Impurity A-16.7 and EZM Impurity B-17.6 with acceptable resolution.\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\u003eMobile phase gradient elution program for optimized method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlow rate (ml/min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBuffer (0.1% TFA) (ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcetonitrile (ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e80\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003eSystem suitability and Specificity\u003c/h2\u003e \u003cp\u003eThe chromatographic parameters such as theoretical plates (N\u0026thinsp;\u0026gt;\u0026thinsp;2000), tailing factor (NMT 2.0), percentage RSD of peak areas (NMT2.0) were assessed to establish the system suitability. The observed values for BPA: N (9076), tailing factor (0.92), % RSD (1.36); for EZM:plate count (N 7820), tailing factor (1.0), % RSD (0.31) substantially prove the suitability of the method proposed. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts all the system suitability parameters including sensitivity and robustness data. Evaluation of method specificity was achieved by injecting standard solutions of BPA, EZM and their impurities separately, standard spiked solution, sample solution, blank and placebo. At the retention times of BPA, EZM \u0026amp;Impurities (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) no interference peaks were observed in placebo, impurities were not eluted at the retention times of BPA \u0026amp; EZM and no additional peaks were observed in standard and sample solutions as shown in Fig.\u0026nbsp;2.\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\u003eSystem suitability results for Bempedoic acid, Ezetimibe and their Impurities\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eRt (min)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBPA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBPA Imp-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBPA Imp-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEZM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEZM Imp A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEZM Imp B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.96\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.92\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.73\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.66\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUSP plate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8721\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15468\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22397\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24615\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUSP tailing\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e% RSD (peak area)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLinearity (\u0026micro;g/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u0026ndash;270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u0026ndash;1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.10\u0026ndash;1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u0026ndash;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.10\u0026ndash;1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.10\u0026ndash;1.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9997\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLOD (\u0026micro;g/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLOQ (\u0026micro;g/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlow rate Minus(0.9ml/min) %RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlow rate Plus(1.1ml/min) %RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMobile Phase Plus(+\u0026thinsp;10%) %RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMobile Phase minus(-10%) %RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAssay\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStability (20\u0026ndash;25\u0026deg;C))\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStability (2\u0026ndash;8\u0026deg;C)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003estable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2: Chromatogram of Blank (A) and Placebo (B)\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLinearity\u003c/h2\u003e \u003cp\u003eThe correlation between analyte concentrations and the detector response (Peak area) was assessed to establish the linearity of the developed method. The peak areas were recorded from triplicate injections of analytes and impurities at various concentrations within a designated range. The coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) is above 0.999 suggesting the responses are in harmony with increasing concentration of analyte/impurity across the stated interval of concentrations. The linearity range and correlation values (R\u003csup\u003e2\u003c/sup\u003e) were given in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and plots were presented in Fig.\u0026nbsp;3. Thus the developed method can quantify the analytes and their impurities over or within the stated range of concentrations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3: Linearity plots of BPA (a); BPA Imp\u0026ndash;1(b); BPA Imp-2 (c); EZM (d); EZM Imp-A(e) and EZM Imp-B (f)\u003c/b\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\u003eLinearity data and results obtained from developed HPLC method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\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=\"left\" 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=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConc (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeak Area BPA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConc (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeak Area EZM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eConc (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePeak Area BPA Imp-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePeak Area BPA Imp-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePeak Area EZM Imp-A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePeak Area EZM Imp B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1575421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e690347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e39402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e42630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e41975\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3506514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1742365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e105274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e112407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e107498\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7098754\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3560207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92621\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e195402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e215698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e202017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e135.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10320561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5412657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e140218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e306528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e320505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e299845\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e180.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14102364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6823479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e192145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e386547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e402517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e413659\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e225.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17874157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8596741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e238651\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e473248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e523064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e512439\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e270.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20889585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10123647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e284156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e567814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e603251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e603247\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSlope\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77845.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e678833.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e189987.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e377674.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e404372.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e404641.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIntercept\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50260.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78978.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6706.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7085.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1981.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.9992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.9997\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eAccuracy\u003c/h2\u003e \u003cp\u003eFrom the recovery studies, method accuracy is determined at three concentration levels (50%, 100% \u0026amp; 150%) of test solution spiked with impurities. The acceptable percentage recovery for BPA, EZM and their impurities confirms the possibility of the method for estimation of content uniformity where the % recovery values were within the range of 100.10 to 100.8% as depicted in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\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\u003ePercentage recovery results of Bempedoic acid, Ezetimibe and their Impurities.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e% Level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003ePercentage Mean Recovery\u0026thinsp;\u0026plusmn;\u0026thinsp;RSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBPA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBPA Imp-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBPA Imp-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEZM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEZM Imp-A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEZM Imp B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e100.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e100.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e100.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e100.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e100.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e100.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e100.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e100.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e100.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e100.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ePrecision\u003c/h2\u003e \u003cp\u003eRepeatability or Intraday precision reflects the degree of consistency among a series of measurements obtained under identical experimental conditions on same day. The test solution (n\u0026thinsp;=\u0026thinsp;6) was injected into HPLC system under the same set of conditions to evaluate method precision and is conveyed as percentage relative standard deviation (%RSD). Lower values of %RSD(less than 2.0) demonstrate reproducibility of the developed method with more consistency. Tables\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e show the data for method precision and intermediate precision.\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\u003eMethod Precision data of Bempedoic acid, Ezetimibe and their impurities\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInjection No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBPA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBPA Imp-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBPA Imp-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEZM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEZM Imp A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEZM Imp B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13,924,520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,801,220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e190,020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e381,220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e398,420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e410,225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14,157,900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,874,930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e191,875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e384,755\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e400,785\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e412,480\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14,206,700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,912,445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e193,640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e386,740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e401,960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e414,905\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14,279,200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,745,600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e194,280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e388,615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e404,215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e416,330\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14,027,160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,990,150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e195,955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e389,782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e406,380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e418,720\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14,018,706\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,616,529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e187,100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e388,470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e403,342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e409,294\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eM ean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14,102,364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,823,479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e192,145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e386,597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e402,517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e413,659\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e122150.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e120920.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2925.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2888.2007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2535.8128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3330.068\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e%RSD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8661681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7721206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.522527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7470831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.629989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.805027\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=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIntermediate precision data for Bempedoic acid Impurities and Ezetimibe impurities\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=\"char\" char=\".\" 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 \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"3\"\u003e \u003cp\u003eS.No\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e% Purity of related substances\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBPA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eEZM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.48\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.45\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.46\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.44\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.45\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.755\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e99.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.45667\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.017078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0141421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0264575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.012472\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e% RSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.01712\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0141762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0265985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.01254\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eRobustness\u003c/h2\u003e \u003cp\u003eThe intentional changes in experimental conditions have no influence/impact on performance of the developed method. The detection of impurities was not influenced by changes in flow rate, mobile phase composition and column temperature, which showed only minor deviation of retention time and/or resolution as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As these minor variations in chromatographic conditions had not affected the detectable results noticeably, it strongly suggests the robustness of the method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eForced degradation studies\u003c/h2\u003e \u003cp\u003eThe spiked sample solution was exposed to various stress conditions in the laboratory to induce degradation of analytes and the recorded chromatograms were evaluated whether the degradant peaks were interfering with analyte peaks. Lower values of purity angle than purity threshold suggests quantification of analytes even when degradants reside. The degradation data and the chromatograms were presented in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eForced Degradation Studies data of Bempedoic acid and Ezetimibe\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStress condition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBPA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eEZM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% P\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% P\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e% D\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcid degradation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e94.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlkali degradation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e93.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOxidative degradation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e92.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eReduction degradation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThermal degradation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e94.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhotolytic degradation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydrolysis degradation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e98.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e% P: Percentage Purity; % D: Percentage Degradation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eApplication of proposed method\u003c/h2\u003e \u003cp\u003eThe proposed and developed method was applied for the assay of fixed dose combination, Bempesta EZ (Torrent Pharmaceuticals Ltd). The percentage purity of BPA and EZM were computed from the peak areas and the satisfactory result confirms the intended suitability of the method. The assay values and the chromatograms were represented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of GREEnness\u003c/h2\u003e \u003cp\u003eAGREE and GAPI tools were used for thorough evaluation of proposed analytical methodology. The experimental procedures followed were given as input into the software and the pictograms were presented in Fig.\u0026nbsp;5. The AGREE \u0026amp; GAPI yielded a score of 0.77 and 85 confirming the high level of environmental compatibility and strong alignment with GAC principles. The assessment reveals most aspects of methodology such as sample amount, number of preparation steps, automation, avoidance of derivatization, waste control, analytical throughput and energy efficiency are rated green demonstrating minimal sample consumption and efficient chromatographic operation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 5: Greenness evaluation (a) AGREE- Analytical GREEnness Metric Approach (b) GAPI-Green Analytical Procedure Index\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eAn efficient and sensitive reverse phase HPLC method for computation of Bempedoic acid, BPA Impurity-1, BPA Impurity-2, Ezetimibe, EZM Impurity-A and EZM Impurity-B in bulk and fixed dose combination tablets was successfully improved and validated complying the specifications as per ICH guidelines. With the optimized method parameters, the developed method is simple, specific, accurate, precise and robust. The prominent features of the method include high sensitivity and reproducible specificity. The felicity of the method for stability testing was confirmed through FDS studies. The scores of green metric tools confirmed the environmental friendliness of the proposed RP-HPLC method balancing the analytical performance. Hence, the present HPLC method is highly suitable for routine impurity profiling of Bempedoic acid(BPA) and Ezetimibe(EZM) in bulk and fixed dose combination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSridevi ranjitha, Divya Narla and SrinivasRao Yarraguntla : Conceptualization, supervision, methodology design, validation strategy, critical review of manuscript and final approval Sai Priyanka, Rohan Ratnakar, Neelima, Satvik : Method development, Chromatographic analysis, validation experiments, preparation of tables and figures.Divya and Sridevi Ranjitha : Forced Degradation studies, validation experiments, Interpretation of analytical results, statistical analysis.Divya Narla : Green analytical assessment \u0026ndash; AGREE and GAPI evaluation, data curation and preparation of graphical representations.All authors reviewed and accepted the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBallantyne CM, Laufs U, Ray KK, Leiter LA, Bays HE, Goldberg AC, et al. Bempedoic acid plus ezetimibe fixed-dose combination in patients with hypercholesterolemia and high CVD risk treated with maximally tolerated statin therapy. Eur J Prev Cardiol. 2020;27(6):593\u0026ndash;603. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/2047487319864671\u003c/span\u003e\u003cspan address=\"10.1177/2047487319864671\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerri N, Ruscica M, Santos RD, Corsini A. Fixed Combination for the Treatment of Dyslipidaemia. Curr Atheroscler Rep. 2023;25(10):691\u0026ndash;9. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11883-023-01142-x\u003c/span\u003e\u003cspan address=\"10.1007/s11883-023-01142-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYarrarapu SNS, Goyal A, Venkata VS, Panchal V, Sivasubramanian BP, Du DT, et al. Comprehensive review of statin-intolerance and the practical application of Bempedoic Acid. Journal of Cardiology. 2024;84(1):22\u0026ndash;9. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jjcc.2024.03.006\u003c/span\u003e\u003cspan address=\"10.1016/j.jjcc.2024.03.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaudhari MV, Chaudhari U, Sahu JK, Srivastava SK, Sharma S, Sahu A. Bempedoic Acid\u0026rsquo;s Chemistry, Pharmacological Characteristics and Bioanalytical Techniques: An Updated Review [Internet]. 2024 Nov 22. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/0115734110335673241119051406\u003c/span\u003e\u003cspan address=\"10.2174/0115734110335673241119051406\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZagelbaum NK, Yandrapalli S, Nabors C, Frishman WH. Bempedoic Acid (ETC-1002): ATP Citrate Lyase Inhibitor: Review of a First-in-Class Medication with Potential Benefit in Statin-Refractory Cases. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wjpsonline.com/index.php/wjps/article/view/1915\u003c/span\u003e\u003cspan address=\"https://wjpsonline.com/index.php/wjps/article/view/1915\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePriya KC. DEVELOPMENT AND VALIDATION OF A ROBUST RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF BEMPEDOIC ACID AND EZETIMIBE IN PHARMACEUTICAL DOSAGE FORMS. World Journal of Pharmaceutical Sciences [Internet]. 2025 Jun 5 [cited 2025 Sep 15]. 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YMER. 2022;21(03):45\u0026ndash;57. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.37896/YMER21.03/06\u003c/span\u003e\u003cspan address=\"10.37896/YMER21.03/06\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePena-Pereira F, Wojnowski W, Tobiszewski M. AGREE\u0026mdash;Analytical GREEnness Metric Approach and Software. Anal Chem. 2020;92(14):10076\u0026ndash;82. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.analchem.0c01887\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.0c01887\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMansour FR, Płotka-Wasylka J, Locatelli M. Modified GAPI (MoGAPI) Tool and Software for the Assessment of Method Greenness: Case Studies and Applications. Analytica. 2024;5(3):451\u0026ndash;7. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/analytica5030030\u003c/span\u003e\u003cspan address=\"10.3390/analytica5030030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bempedoic acid, Ezetimibe, Impurity analysis, Forced degradation, RP-HPLC-UV","lastPublishedDoi":"10.21203/rs.3.rs-9113640/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9113640/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA straightforward, trustworthy and eco-friendly RP-HPLC-UV stability-indicating technique for separation and quantification of Bempedoic acid(BPA), Ezetimibe(EZM) and their impurities(BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B) was developed and validated in accordance with ICH Q2(R2) for accurate determination of BPA \u0026amp; EZM by minimizing the interference from impurities with impurity analysis simultaneously. Chromatographic separation was accomplished on an Inertsil ODS C18 Column (250\u0026times;4.6mm, 5\u0026micro;m) using gradient elution mode with 0.1% TFA and Acetonitrile at a flow rate of 1.0 ml/min, with detection wavelength of 211nm and total run time is 40mins. Method reliability was assessed through key parameters such as specificity, linearity, accuracy, precision, sensitivity (LOD \u0026amp; LOQ) and robustness. The principles of green analytical chemistry were followed to minimize the usage of solvents. The results of linearity were consistent with R\u003csup\u003e2\u003c/sup\u003e values above 0.999; percent recovery is in the range of 99\u0026ndash;101%; RSD% values were below 2.0% for precision. LOD and LOQ values were found to be 0.03 and 0.1 \u0026micro;g/ml for impurities (BPA Imp-1, BPA Imp-2, EZM Imp-A, EZM Imp-B), 5.4 \u0026amp; 18\u0026micro;g/ml for BPAand 0.3 \u0026amp; 0.1\u0026micro;g/ml for EZM. By inducing degradation under extreme conditions, the stability-indicating property of the approach was explored.The approach was found to be effective in differentiating between BPA, EZM and their impurities showcasing the suitability of the method for routine quality control.The results of the devised technique confirm the reliability for quantification of analytes and impurities in their fixed dose combination. The AGREE and GAPI tools used to assess the greenness of the method showed a score of 0.72 and 85 showcasing reasonable balance among environmental sustainability and analytical performance, indicating the eco-friendliness of the developed method.\u003c/p\u003e","manuscriptTitle":"A Green Analytical Stability-Indicating RP-HPLC method for Simultaneous Determination of Anti-Hypercholesterolemic Agents and Their related Impurities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 10:02:54","doi":"10.21203/rs.3.rs-9113640/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":"d72ef137-ed84-450c-b4f3-4871b9b6128d","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T16:09:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 10:02:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9113640","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9113640","identity":"rs-9113640","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}